KR20130087014A - Supplementation of fatty acids for improving alcohol productivity - Google Patents

Abstract

발효 과정에서의 단계에서 바이오매스로부터 유래되는 지방산은 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 배지에 첨가될 수 있다. 미생물의 성장 속도 및 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 미생물의 성장 속도 및 발효성 탄소 소비량보다 더 크다. 지방산의 첨가는 글루코스 소비를 증가시킬 수 있으며, 미생물의 바이오매스 생성 (세포 성장/밀도) 및 성장 속도를 향상시키고 이럼으로써 생성 시간을 감소시키고 발효 과정의 생산성을 증가시킬 수 있다.Fatty acids derived from the biomass at the stage in the fermentation process may be added to the fermentation medium containing the recombinant microorganism that produces the product alcohol. At least one of the growth rate and fermentable carbon consumption of the microorganism is greater than the growth rate and fermentable carbon consumption of the microorganism in the presence of the fatty acid, in the absence of the fatty acid. The addition of fatty acids can increase glucose consumption and improve microbial biomass production (cell growth / density) and growth rates, thereby reducing production time and increasing fermentation process productivity.

Description

알코올 생산성 향상을 위한 지방산의 보충{SUPPLEMENTATION OF FATTY ACIDS FOR IMPROVING ALCOHOL PRODUCTIVITY}SUPPLEMENTATION OF FATTY ACIDS FOR IMPROVING ALCOHOL PRODUCTIVITY

본 출원은 2010년 6월 18일자로 출원된 미국 가특허 출원 제61/356,290호; 2010년 7월 28일자로 출원된 미국 가특허 출원 제61/368,451호; 2010년 7월 28일자로 출원된 미국 가특허 출원 제61/368,436호; 2010년 7월 28일자로 출원된, 미국 가특허 출원 제61/368,444호; 2010년 7월 28일자로 출원된 미국 가특허 출원 제61/368,429호; 2010년 9월 2일자로 출원된 미국 가특허 출원 제61/379,546호; 및 2011년 2월 7일자로 출원된 미국 가특허 출원 제61/440,034호; 2011년 6월 15일자로 출원된 미국 특허 출원 제13/160,766호의 이익을 청구하며, 이들의 전체 내용은 전부가 본 명세서에 참고로 포함된다.This application discloses US Provisional Patent Application 61 / 356,290, filed June 18, 2010; United States Provisional Patent Application 61 / 368,451, filed July 28, 2010; United States Provisional Patent Application 61 / 368,436, filed July 28, 2010; United States Provisional Patent Application 61 / 368,444, filed July 28, 2010; United States Provisional Patent Application 61 / 368,429, filed July 28, 2010; United States Provisional Patent Application 61 / 379,546, filed September 2, 2010; And US Provisional Patent Application 61 / 440,034, filed February 7, 2011; Claims benefit of US patent application Ser. No. 13 / 160,766, filed June 15, 2011, the entire contents of which are incorporated herein by reference in their entirety.

본 출원과 관련된 서열 목록은 EFS-Web을 통하여 전자 형태로 출원되며, 본 발명에서 전체적으로 본 명세서 내에 참고로 포함된다.The Sequence Listing associated with this application is filed in electronic form via EFS-Web, which is incorporated herein by reference in its entirety.

본 발명은 발효 알코올, 예를 들어 부탄올의 생성, 그리고 특히 향상된 알코올 생산성을 성취하기 위한 알코올 발효 공정에 관한 것이며, 여기서 재조합 미생물의 발효 성장은 발효 과정 중 단계에서 바이오매스(biomass)로부터 유래된 지방산의 존재 하에 있다.The present invention relates to the production of fermented alcohols, such as butanol, and in particular to alcohol fermentation processes to achieve improved alcohol productivity, wherein the fermentation growth of recombinant microorganisms is a fatty acid derived from biomass at a stage during the fermentation process. Is in the presence of.

알코올은 산업계 및 과학에서 음료(즉, 에탄올), 연료, 시약, 용매 및 소독제와 같은 다양한 응용을 갖는다. 예를 들어, 부탄올은 연료 첨가제로서의 용도, 플라스틱 산업에서 공급재료(feedstock) 화학물질로서의 용도, 및 식품 및 향미료 산업에서 식품 등급 추출제로서의 용도를 비롯하여 다양한 응용을 갖는 중요한 산업용 화학물질인 알코올이다. 따라서, 효율적이고 환경 친화적인 생성 방법에 대한 것뿐만 아니라 부탄올과 같은 알코올에 대한 큰 수요도 있다.Alcohols have a variety of applications in industry and science, such as beverages (ie ethanol), fuels, reagents, solvents and disinfectants. For example, butanol is an alcohol that is an important industrial chemical with a variety of applications, including as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavoring industry. Thus, there is a great demand for alcohols such as butanol as well as for efficient and environmentally friendly production methods.

미생물에 의한 발효를 이용한 부탄올과 같은 알코올의 생성은 환경 친화적인 하나의 생성 방법이다. 효모와 같은 미생물은 알코올 생성물의 생성에 사용되어 왔으며, 여기서 자연적으로 생성된 피루베이트는 알코올 생성물의 생합성 경로에서 출발 기질로서 사용된다. 부탄올은 효모 발효의 부산물로서 생물학적으로 생성될 수 있지만, 그 수율은 전형적으로 매우 낮을 수 있다. 부탄올과 같은 원하는 생성물의 생성을 향상시키기 위하여, 효모는 내인성 생합성 경로를 변경시키거나 또는 새로운 경로를 도입하는 효소를 발현시키기 위하여, 및/또는 내인성 효소의 발현을 파괴하여 대사산물 흐름을 변경시킴으로써 조작되었다. 세포 피루베이트를 기질로 사용하는 도입된 경로는 예를 들어 부탄올의 이성체의 생성 경로를 포함한다. 피루베이트 데카르복실라아제의 파괴는 부탄올과 같은 원하는 생성물을 생성하는 경로에 있어서의 피루베이트 이용성을 증가시키기 위하여 사용되어 왔다. 부가적으로, 1-부탄올 생합성 경로 (미국 특허 공개 제2008/0182308A1호), 2-부탄올 생합성 경로 (미국 특허 공개 제2007/0259410A1호 및 미국 특허 공개 제2007/0292927호), 및 아이소부탄올 생합성 경로 (미국 특허 공개 제2007/0092957호)를 발현하는 재조합 미생물 생산 숙주가 개시되었다.Production of alcohols such as butanol using fermentation by microorganisms is one environmentally friendly production method. Microorganisms such as yeast have been used for the production of alcohol products, where naturally produced pyruvate is used as a starting substrate in the biosynthetic pathway of alcohol products. Butanol can be produced biologically as a byproduct of yeast fermentation, but its yield can typically be very low. In order to enhance the production of the desired product, such as butanol, yeast is manipulated by altering endogenous biosynthetic pathways or by expressing enzymes that introduce new pathways, and / or by altering metabolite flow by disrupting the expression of endogenous enzymes. It became. Introduced pathways using cellular pyruvate as a substrate include, for example, pathways for the generation of isomers of butanol. Breakdown of pyruvate decarboxylase has been used to increase pyruvate availability in the pathways that produce the desired product, such as butanol. Additionally, the 1-butanol biosynthesis pathway (US Patent Publication No. 2008 / 0182308A1), the 2-butanol biosynthesis pathway (US Patent Publication No. 2007 / 0259410A1 and US Patent Publication No. 2007/0292927), and the isobutanol biosynthesis pathway A recombinant microbial production host is disclosed that expresses (US Patent Publication No. 2007/0092957).

예를 들어, 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 효모는 2개의 주요 피루베이트 데카르복실라아제(pyruvate decarboxylase; PDC) 유전자에서의 파괴 돌연변이에 의해 대사적으로 조작될 수 있다. PDC1 및 PDC5로 일반적으로 언급되는 이들 유전자는 에탄올 생성에 직접적으로 관계된 효소를 생성하며, 이들 유전자의 파괴는 성장에 부정적인 영향을 준다. 피루베이트 데카르복실라아제 및 알코올 데하이드로게나아제(alcohol dehydrogenase; ADH)의 넉아웃(knock-out)은 생합성 경로를 변경시켜 지방산이 덜 생성되게 한다. 지방산은 세포벽 형성에 필요하며, 따라서 세포 성장에 필요하다. 세포 성장에 있어서의 지방산의 중요성은 예를 들어 문헌[Otoguro, et al., J. Biochem. 89:523-529, 1981]에 입증되어 있으며, 상기 문헌에는 지방산 합성의 공지된 저해제인 항생제 세룰레닌의 세포 성장에 대한 영향이 기재되어 있다. 세룰레닌은 에스. 세레비지애 배양물에 첨가되어 성장의 저해를 야기하였지만, 올레산을 소정의 포화 지방산 (구체적으로, 미리스트산, 팔미트산 또는 펜타데칸산)과 함께 첨가했을 때 상기 성장은 복구되었다.For example, Saccharomyces cerevisiae ) Yeast can be metabolically manipulated by disruptive mutations in two major pyruvate decarboxylase (PDC) genes. These genes, commonly referred to as PDC1 and PDC5, produce enzymes that are directly involved in ethanol production, and destruction of these genes negatively affects growth. Knock-out of pyruvate decarboxylase and alcohol dehydrogenase (ADH) alters the biosynthetic pathway to produce less fatty acids. Fatty acids are required for cell wall formation and therefore for cell growth. The importance of fatty acids in cell growth is described, for example, in Otoguro, et al., J. Biochem. 89: 523-529, 1981, which describes the effect on the cell growth of the antibiotic cerulenin, a known inhibitor of fatty acid synthesis. Cerulenin s. Although it was added to the cerevisiae culture, causing growth inhibition, the growth was restored when oleic acid was added with certain saturated fatty acids (specifically, myristic acid, palmitic acid or pentadecanoic acid).

일반적으로 효모에서의 글루코스 대사는 글루코스를 피루베이트로 전환시켜 아세틸-CoA로 전환시켜 세포 매스(mass)로 전환시키는 경로를 따른다. 이에 상응하여, 피루베이트를 아세트알데히드로 전환시켜 에탄올로 전환시키는 것 또는 아세트알데히드를 아세틸-CoA로 전환시켜 지방산 합성을 하는 것이 존재할 수 있다. 재조합 미생물과 관련하여, 단일 PDC 결실은 최대 성장을 감소시키지만 훨씬 더 적은 정도로 감소시킨다. 단지 하나의 PDC 유전자가 파괴될 때, 다른 하나의 PDC 유전자는 아세트알데히드로의 그리고 후속적으로 에탄올 및 아세테이트로의 탄소 흐름을 허용하기에 충분한 활성을 갖는다. 그러나, 부탄올 생성물이 요구될 때, 에탄올 생성은 기질에서의 부탄올 생성물 수율을 감소시킨다. PDC 유전자는 피루베이트를 아세트알데히드로 데려가는 데 책임이 있으며, PDC1 및 PDC5의 이중 돌연변이는 아세트알데히드의 생성을 방지하여, 지방산 생합성으로의 경로를 변경시키고 이럼으로써 세포 성장을 저해하게 된다.In general, glucose metabolism in yeast follows the route of converting glucose to pyruvate to acetyl-CoA to cell mass. Correspondingly, there may be conversion of pyruvate to acetaldehyde to ethanol or acetaldehyde to acetyl-CoA for fatty acid synthesis. In the context of recombinant microorganisms, a single PDC deletion reduces maximal growth but decreases to a much lesser extent. When only one PDC gene is destroyed, the other PDC gene has sufficient activity to allow carbon flow of acetaldehyde and subsequently to ethanol and acetate. However, when butanol product is desired, ethanol production reduces the butanol product yield in the substrate. The PDC gene is responsible for taking pyruvate to acetaldehyde, and double mutations of PDC1 and PDC5 prevent the production of acetaldehyde, altering and thus inhibiting cell growth to fatty acid biosynthesis.

따라서, 미생물의 성장 속도 및/또는 바이오매스 생성이 미생물에 의한 지방산 생합성의 감소 또는 제거에도 불구하고 향상될 수 있는, 재조합 미생물을 사용한 발효 알코올 생성 방법에 대한 계속적인 필요성이 존재한다. 본 발명은 하기의 실시 형태들의 설명에 의해 명백해지는 바와 같이 추가의 관련 이점을 제공한다.Thus, there is a continuing need for methods of producing fermentation alcohol using recombinant microorganisms in which the growth rate and / or biomass production of microorganisms can be improved despite the reduction or elimination of fatty acid biosynthesis by the microorganisms. The present invention provides further related advantages as will be apparent from the description of the following embodiments.

본 발명은 (a) 발효성 탄소 공급원으로부터 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 브로쓰를 제공하는 단계 - 상기 재조합 미생물은 피루베이트 데카르복실라아제(피루베이트 데카르복실라아제; PDC) 활성의 감소 또는 제거를 포함함 - 와; (b) 발효 브로쓰를 발효성 탄소 공급원과 접촉시키고, 이에 의해 재조합 미생물이 발효성 탄소 공급원을 소비하고 생성물 알코올을 생성하게 하는 단계와; (c) 발효 브로쓰를, 상기 발효 과정에서의 단계에서 바이오매스로부터 유래된 지방산과 접촉시키는 단계를 포함하는 방법에 관한 것이며, 여기서 재조합 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 크다. 추가의 실시 형태에서, 단계 (b) 및 (c)는 실질적으로 동시에 일어난다. 일 실시 형태에서, 지방산은 올레산, 팔미트산, 미리스트산 및 그 혼합물로부터 선택되며, 다른 실시 형태에서, 바이오매스는 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀, 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스(switchgrass), 폐지, 사탕수수 바가스, 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물로부터 유래된다. 추가의 실시 형태에서, 발효성 탄소 공급원은 바이오매스로부터 유래된다. 일 실시 형태에서, 생성물 알코올은 부탄올이며, 다른 실시 형태에서, 발효 브로쓰는 에탄올을 추가로 포함한다. 다른 실시 형태에서, 재조합 미생물은 하나 이상의 피루베이트 데카르복실라아제(PDC) 유전자 결실을 갖는다.The present invention provides a fermentation broth comprising a recombinant microorganism that produces a product alcohol from a fermentable carbon source, wherein the recombinant microorganism is pyruvate decarboxylase (pyruvate decarboxylase; PDC) activity. Including reduction or elimination of-and; (b) contacting the fermentation broth with a fermentable carbon source, thereby causing the recombinant microorganism to consume the fermentable carbon source and produce product alcohol; (c) contacting the fermentation broth with fatty acids derived from biomass in the step of the fermentation process, wherein the (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism At least one of which is greater than the growth rate and / or fermentable carbon consumption of the recombinant microorganism in the presence of fatty acids, in the absence of fatty acids. In further embodiments, steps (b) and (c) occur substantially simultaneously. In one embodiment, the fatty acid is selected from oleic acid, palmitic acid, myristic acid and mixtures thereof, and in other embodiments, the biomass is corn grain, corncob, crop residues, for example corn husk, corn cob, grass. , Wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane vargas, sorghum, sugar cane, soybean, components obtained by milling of cereals, cellulosic material, ligno Cellulosic materials, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure and mixtures thereof. In further embodiments, the fermentable carbon source is derived from biomass. In one embodiment, the product alcohol is butanol and in another embodiment the fermentation broth further comprises ethanol. In other embodiments, the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.

또한 본 발명은 (a) 발효성 탄소 공급원 및 오일을 포함하는 바이오매스를 제공하는 단계와; (b) 오일의 적어도 일부분을 지방산으로 전환시켜 지방산을 포함하는 바이오매스를 형성하는 단계와; (c) 바이오매스를, 발효성 탄소 공급원으로부터 생성물 알코올을 생성할 수 있는 재조합 미생물을 포함하는 발효 브로쓰와 접촉시키는 단계 - 상기 재조합 미생물은 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함함 - 와; (d) 발효 브로쓰를 지방산과 접촉시키는 단계를 포함하는 생성물 알코올의 생성 방법에 관한 것이며, 여기서 재조합 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 크다. 추가의 실시 형태에서, 오일의 적어도 일부분을 지방산으로 전환시키는 단계 (b)는 오일을, 오일의 일부를 지방산으로 가수분해시킬 수 있는 하나 이상의 물질과 접촉시키는 것을 포함한다. 일 실시 형태에서, 상기 하나 이상의 물질은 하나 이상의 효소를 포함하며, 다른 실시 형태에서, 상기 하나 이상의 효소는 리파아제 효소를 포함한다. 추가의 실시 형태에서, 단계 (c) 전에, 오일의 적어도 일부분이 가수분해된 후에 상기 하나 이상의 효소를 불활성화시킬 수 있다. 일 실시 형태에서, 단계 (b), (c), 및 (d) 중 하나 이상은 발효 용기에서 일어나며, 다른 실시 형태에서, 단계 (b), (c), 및 (d) 중 하나 이상은 실질적으로 동시에 일어난다. 일 실시 형태에서, 본 방법은 오일의 적어도 일부분을 지방산으로 전환시키는 단계 (b) 전에 오일을 바이오매스로부터 분리시키는 단계를 추가로 포함한다. 다른 실시 형태에서, 본 방법은 바이오매스를 액화시켜 액화된 바이오매스를 생성하는 단계 - 상기 액화된 바이오매스는 올리고당류를 포함함 - 와; 액화된 바이오매스를, 올리고당류를 발효성 당으로 전환시킬 수 있는 당화 효소와 접촉시켜 당화된 바이오매스를 형성하는 단계를 추가로 포함하며, 여기서 단계 (c)는 당화된 바이오매스를, 재조합 미생물을 포함하는 발효 브로쓰와 접촉시키는 것을 포함한다. 일 실시 형태에서, 지방산은 올레산, 팔미트산, 미리스트산 및 그 혼합물로부터 선택되며, 다른 실시 형태에서, 바이오매스는 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀, 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스, 폐지, 사탕수수 바가스, 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물로부터 유래된다. 일 실시 형태에서, 본 방법은 발효성 탄소 공급원을 발효시켜 생성물 알코올을 생성하는 단계를 추가로 포함한다. 일 실시 형태에서, 생성물 알코올은 부탄올이며, 다른 실시 형태에서, 발효 브로쓰는 에탄올을 추가로 포함한다. 다른 실시 형태에서, 재조합 미생물은 하나 이상의 피루베이트 데카르복실라아제(PDC) 유전자 결실을 갖는다.The present invention also provides a method of preparing a biomass comprising: (a) providing a biomass comprising a fermentable carbon source and an oil; (b) converting at least a portion of the oil to fatty acids to form a biomass comprising fatty acids; (c) contacting the biomass with a fermentation broth comprising recombinant microorganisms capable of producing product alcohols from the fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity. - Wow; (d) contacting the fermentation broth with a fatty acid, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is present in the presence of fatty acid, Greater than the rate of growth and / or fermentable carbon consumption of the recombinant microorganism in the absence of fatty acids. In a further embodiment, step (b) of converting at least a portion of the oil to a fatty acid comprises contacting the oil with one or more substances capable of hydrolyzing a portion of the oil to the fatty acid. In one embodiment, the one or more substances comprise one or more enzymes, and in other embodiments, the one or more enzymes comprise a lipase enzyme. In a further embodiment, prior to step (c), the at least one enzyme may be inactivated after at least a portion of the oil has been hydrolyzed. In one embodiment, one or more of steps (b), (c), and (d) occur in a fermentation vessel, and in another embodiment, one or more of steps (b), (c), and (d) are substantially Happens simultaneously. In one embodiment, the method further comprises separating the oil from the biomass before step (b) of converting at least a portion of the oil into fatty acids. In another embodiment, the method includes liquefying biomass to produce liquefied biomass, wherein the liquefied biomass comprises oligosaccharides; Contacting the liquefied biomass with a glycosylating enzyme capable of converting oligosaccharides into fermentable sugars to form a glycosylated biomass, wherein step (c) comprises the glycosylated biomass, a recombinant microorganism. It includes contacting the fermentation broth comprising a. In one embodiment, the fatty acid is selected from oleic acid, palmitic acid, myristic acid and mixtures thereof, and in other embodiments, the biomass is corn grain, corncob, crop residues, for example corn husk, corn cob, grass. , Wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane vargas, sorghum, sugar cane, soybean, components obtained by milling of grain, cellulosic material, lignocellulosic material , Trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure and mixtures thereof. In one embodiment, the method further comprises fermenting the fermentable carbon source to produce product alcohol. In one embodiment, the product alcohol is butanol and in another embodiment the fermentation broth further comprises ethanol. In other embodiments, the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.

본 발명의 다른 방법은 (a) 공급재료를 제공하는 단계와; (b) 상기 공급재료를 액화시켜 공급재료 슬러리를 생성하는 단계와; (c) 공급재료 슬러리를 분리시켜 (i) 발효성 탄소 공급원을 포함하는 수성 층, (ii) 오일 층, 및 (iii) 고형물 층을 포함하는 생성물을 생성하는 단계와; (d) 오일 층으로부터 오일을 수득하고 오일의 적어도 일부분을 지방산으로 전환시키는 단계와; (e) (c)의 수성 층을 발효성 탄소 공급원으로부터 생성물 알코올을 생성할 수 있는 재조합 미생물을 포함하는 발효 브로쓰가 들어 있는 발효 용기에 공급하는 단계 - 상기 재조합 미생물은 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함함 - 와; (f) 수성 층의 발효성 탄소 공급원을 발효시켜 생성물 알코올을 생성하는 단계와; (g) 발효 브로쓰를 지방산과 접촉시키는 단계를 포함하는 생성물 알코올의 생성 방법을 포함하며, 여기서 재조합 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 크다. 추가의 실시 형태에서, 오일의 적어도 일부분을 지방산으로 전환시키는 단계 (d)는 오일을, 오일의 일부를 지방산으로 가수분해시킬 수 있는 하나 이상의 물질과 접촉시키는 것을 포함한다. 일 실시 형태에서, 상기 하나 이상의 물질은 하나 이상의 효소를 포함하며, 다른 실시 형태에서, 상기 하나 이상의 효소는 리파아제 효소를 포함한다. 일 실시 형태에서, 지방산은 올레산, 팔미트산, 미리스트산 및 그 혼합물로부터 선택되며, 다른 실시 형태에서, 바이오매스는 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀, 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스, 폐지, 사탕수수 바가스, 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물로부터 유래된다. 일 실시 형태에서, 생성물 알코올은 부탄올이다. 다른 실시 형태에서, 재조합 미생물은 하나 이상의 피루베이트 데카르복실라아제(PDC) 유전자 결실을 갖는다.Another method of the present invention comprises the steps of (a) providing a feedstock; (b) liquefying the feedstock to produce a feedstock slurry; (c) separating the feed slurry to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer, and (iii) a solids layer; (d) obtaining an oil from the oil layer and converting at least a portion of the oil to a fatty acid; (e) feeding the aqueous layer of (c) to a fermentation vessel containing a fermentation broth comprising recombinant microorganisms capable of producing product alcohols from the fermentable carbon source, wherein the recombinant microorganism is pyruvate decarboxylase. Including reducing or eliminating activity; (f) fermenting the fermentable carbon source of the aqueous layer to produce product alcohol; (g) contacting the fermentation broth with a fatty acid, wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is present in the presence of fatty acid, Greater than the rate of growth and / or fermentable carbon consumption of the recombinant microorganism in the absence of fatty acids. In a further embodiment, step (d) of converting at least a portion of the oil to a fatty acid comprises contacting the oil with one or more substances capable of hydrolyzing a portion of the oil to the fatty acid. In one embodiment, the one or more substances comprise one or more enzymes, and in other embodiments, the one or more enzymes comprise a lipase enzyme. In one embodiment, the fatty acid is selected from oleic acid, palmitic acid, myristic acid and mixtures thereof, and in other embodiments, the biomass is corn grain, corncob, crop residues, for example corn husk, corn cob, grass. , Wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane vargas, sorghum, sugar cane, soybean, components obtained by milling of grain, cellulosic material, lignocellulosic material , Trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure and mixtures thereof. In one embodiment, the product alcohol is butanol. In other embodiments, the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions.

또한 본 발명은 지방산 및 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함하는 재조합 미생물을 포함하는 조성물에 관한 것이다.The invention also relates to a composition comprising a recombinant microorganism comprising reducing or eliminating fatty acid and pyruvate decarboxylase activity.

발효 과정에서의 단계에서 바이오매스로부터 유래되는 지방산 (예를 들어, 올레산, 팔미트산, 및 그 혼합물)은 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 배지에 첨가될 수 있다. 미생물은 효모 또는 다른 알코올 생성 미생물일 수 있다. 또한, 미생물은 하나 이상의 PDC 유전자 결실을 갖고/갖거나 감소된 또는 제거된 피루베이트 데카르복실라아제 활성을 가질 수 있다. 지방산의 첨가는 글루코스 소비를 증가시킬 수 있으며, 미생물의 바이오매스 생성(세포 성장) 및 성장 속도를 향상시킬 수 있다. 성장 속도 향상은 생성 시간을 감소시키고 이럼으로써 알코올 발효 과정의 생산성을 증가시킬 수 있다.Fatty acids derived from the biomass (eg, oleic acid, palmitic acid, and mixtures thereof) at the stage in the fermentation process may be added to the fermentation medium comprising recombinant microorganisms that produce the product alcohol. The microorganism may be a yeast or other alcohol producing microorganism. In addition, the microorganism may have one or more PDC gene deletions and / or have reduced or eliminated pyruvate decarboxylase activity. The addition of fatty acids can increase glucose consumption and improve microbial biomass production (cell growth) and growth rate. Improving growth rates can reduce production time and thereby increase the productivity of the alcoholic fermentation process.

일부 실시 형태에서, 발효 과정에서 생성물 알코올을 생성하는 방법은 (a) 발효성 탄소 공급원으로부터 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 브로쓰를 제공하는 단계와; (b) 발효 브로쓰를 발효성 탄소 공급원과 접촉시키고, 이에 의해 미생물이 발효성 탄소 공급원을 소비하고 생성물 알코올을 생성하게 하는 단계와; (c) 발효 브로쓰를, 상기 발효 과정에서의 단계에서 바이오매스로부터 유래된 지방산과 접촉시키는 단계를 포함하며, 여기서 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 크다.In some embodiments, a method of producing a product alcohol in a fermentation process comprises: (a) providing a fermentation broth comprising recombinant microorganisms that produce a product alcohol from a fermentable carbon source; (b) contacting the fermentation broth with a fermentable carbon source, thereby causing the microorganism to consume the fermentable carbon source and produce product alcohol; (c) contacting the fermentation broth with fatty acids derived from the biomass in the step of the fermentation process, wherein at least one of (i) the growth rate of the microorganism and (ii) the fermentable carbon consumption is a fatty acid. In the presence of greater than the growth rate and / or fermentable carbon consumption of the recombinant microorganism in the absence of fatty acids.

일부 실시 형태에서, 지방산은 유리 지방산(free fatty acid; FFA)이다. 일부 실시 형태에서, 지방산은 올레산을 포함한다. 일부 실시 형태에서, 지방산은 포화 지방산을 포함한다. 일부 실시 형태에서, 지방산은 팔미트산을 포함한다. 일부 실시 형태에서, 지방산은 미리스트산을 포함한다.In some embodiments, the fatty acid is free fatty acid (FFA). In some embodiments, the fatty acid comprises oleic acid. In some embodiments, the fatty acid comprises saturated fatty acid. In some embodiments, the fatty acid comprises palmitic acid. In some embodiments, the fatty acid comprises myristic acid.

일부 실시 형태에서, 생성물 알코올은 부탄올이다.In some embodiments, the product alcohol is butanol.

일부 실시 형태에서, 발효성 탄소 공급원은 바이오매스로부터 유래된다. 일부 실시 형태에서, 바이오매스는 옥수수를 포함하며, 지방산은 옥수수유 지방산이다.In some embodiments, the fermentable carbon source is from biomass. In some embodiments, the biomass comprises corn and the fatty acid is corn oil fatty acid.

일부 실시 형태에서, 발효 브로쓰는 에탄올을 추가로 포함한다. 일부 실시 형태에서, 본 방법은 발효 브로쓰를 에탄올과 접촉시키는 단계를 추가로 포함한다.In some embodiments, the fermentation broth further comprises ethanol. In some embodiments, the method further comprises contacting the fermentation broth with ethanol.

일부 실시 형태에서, 발효 브로쓰를 지방산과 접촉시키는 단계는 바이오매스로부터 유래된 트라이글리세라이드를, 트라이글리세라이드를 유리 지방산으로 가수분해시킬 수 있는 하나 이상의 효소와 접촉시키고 이에 의해 트라이글리세라이드를 유리 지방산으로 가수분해시키는 것과; 발효 브로쓰를 유리 지방산과 접촉시키는 것을 포함하며, 여기서 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 유리 지방산의 부재 하에서보다 유리 지방산의 존재 하에서 더 크다. 일부 실시 형태에서, 상기 하나 이상의 효소는 리파아제 효소를 포함한다.In some embodiments, contacting the fermentation broth with fatty acids contacts triglycerides derived from biomass with one or more enzymes capable of hydrolyzing triglycerides to free fatty acids, thereby freeing the triglycerides. Hydrolyzing with fatty acids; Contacting the fermentation broth with free fatty acids, wherein at least one of the (i) growth rate and (ii) fermentable carbon consumption of the microorganism is greater in the presence of free fatty acids than in the absence of free fatty acids. In some embodiments, the one or more enzymes comprise a lipase enzyme.

일부 실시 형태에서, 재조합 미생물은 단일 피루베이트 데카르복실라아제(PDC) 유전자 결실을 갖는다. 일부 실시 형태에서, 재조합 미생물은 이중 PDC 유전자 결실을 갖는다. 일부 실시 형태에서, 재조합 미생물은 감소된 또는 제거된 피루베이트 데카르복실라아제 활성을 갖는다.In some embodiments, the recombinant microorganism has a single pyruvate decarboxylase (PDC) gene deletion. In some embodiments, the recombinant microorganism has a double PDC gene deletion. In some embodiments, the recombinant microorganism has reduced or eliminated pyruvate decarboxylase activity.

일부 실시 형태에서, 발효 브로쓰 중 지방산의 농도는 약 0.8 g/L 이하이다.In some embodiments, the concentration of fatty acids in the fermentation broth is about 0.8 g / L or less.

일부 실시 형태에서, 바이오매스의 발효에 의해 생성물 알코올을 생성하는 방법은 (a) 물, 발효성 탄소 공급원 및 소정량의 오일 - 상기 발효성 탄소 공급원 및 오일은 둘 모두 상기 바이오매스로부터 유래됨 - 을 포함하는 수성 바이오매스 공급스트림(feedstream)을 제공하는 단계와; (b) 오일의 적어도 일부분을 지방산으로 가수분해시켜 유리 지방산을 포함하는 바이오매스 공급스트림을 형성하는 단계와; (c) 발효 용기 내에서, 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 배지를 바이오매스 공급스트림과 접촉시키는 단계와; (d) 발효 용기 내에서 발효성 탄소 공급원을 발효시켜 상기 생성물 알코올을 생성하는 단계를 포함하며, 여기서 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 유리 지방산의 존재 하에서, 유리 지방산의 부재 하에서의 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 크다.In some embodiments, the method of producing product alcohol by fermentation of biomass comprises (a) water, fermentable carbon source and a predetermined amount of oil, wherein both the fermentable carbon source and oil are derived from the biomass. Providing an aqueous biomass feedstream comprising a; (b) hydrolyzing at least a portion of the oil into fatty acids to form a biomass feedstream comprising free fatty acids; (c) in a fermentation vessel, contacting a fermentation medium comprising a recombinant microorganism that produces a product alcohol with a biomass feedstream; (d) fermenting the fermentable carbon source in a fermentation vessel to produce the product alcohol, wherein at least one of (i) the growth rate of the microorganism and (ii) the fermentable carbon consumption is in the presence of free fatty acids. Greater than the rate of growth of microorganisms and / or fermentable carbon consumption in the absence of free fatty acids.

일부 실시 형태에서, 오일의 적어도 일부분을 유리 지방산으로 가수분해시키는 단계 (b)는 오일을, 상기 오일 부분을 유리 지방산으로 가수분해시킬 수 있는 하나 이상의 효소를 포함하는 조성물과 접촉시키는 것을 포함한다. 일부 실시 형태에서, 본 방법은 단계 (c) 전에, 오일의 적어도 일부분을 가수분해시킨 후에 상기 하나 이상의 효소를 불활성화시키는 단계를 추가로 포함한다.In some embodiments, step (b) hydrolyzing at least a portion of the oil to free fatty acids comprises contacting the oil with a composition comprising one or more enzymes capable of hydrolyzing the oil portion to free fatty acids. In some embodiments, the method further comprises inactivating said one or more enzymes after hydrolyzing at least a portion of the oil before step (c).

일부 실시 형태에서, 수성 바이오매스 공급스트림은 제분된 비분획화 곡물로부터 형성된 액화 매시(mash)이다. 일부 실시 형태에서, 제분된 비분획화 곡물은 옥수수이며, 오일은 옥수수유이다.In some embodiments, the aqueous biomass feedstream is a liquefied mash formed from milled unfractionated grain. In some embodiments, the milled unfractionated grain is corn and the oil is corn oil.

일부 실시 형태에서, 생성물 알코올을 생성하는 방법은 (a) 글루코스 및 소정량의 트라이글리세라이드를 포함하는 오일을 포함하는 바이오매스를 제공하는 단계와; (b) 오일을, 트라이글리세라이드를 유리 지방산으로 전환시킬 수 있는 하나 이상의 물질을 포함하는 조성물과 접촉시키고 이에 의해 오일 중 트라이글리세라이드의 적어도 일부분을 유리 지방산으로 전환시키는 단계와; (c) 바이오매스를, 글루코스를 생성물 알코올로 전환시킬 수 있는 미생물을 포함하는 발효 브로쓰와 접촉시키고 이에 의해 생성물 알코올을 생성하는 단계와; (d) 유리 지방산을 발효 브로쓰와 접촉시키는 단계를 포함하며, 여기서 미생물의 (i) 성장 속도 및 글루코스 소비량 중 적어도 하나는 유리 지방산의 존재 하에서, 유리 지방산의 부재 하에서의 미생물의 성장 속도 및/또는 글루코스 소비량보다 더 크다.In some embodiments, a method of producing a product alcohol comprises (a) providing a biomass comprising an oil comprising glucose and an amount of triglycerides; (b) contacting the oil with a composition comprising one or more substances capable of converting the triglycerides into the free fatty acids, thereby converting at least a portion of the triglycerides in the oil into the free fatty acids; (c) contacting the biomass with a fermentation broth comprising microorganisms capable of converting glucose into product alcohol, thereby producing a product alcohol; (d) contacting the free fatty acid with the fermentation broth, wherein at least one of (i) the growth rate and glucose consumption of the microorganism is in the presence of the free fatty acid, and / or the growth rate of the microorganism in the absence of the free fatty acid, and / or Greater than glucose consumption.

일부 실시 형태에서, 본 방법은 오일을 상기 하나 이상의 물질과 접촉시키는 단계 (b) 전에 바이오매스로부터 (a)의 오일을 분리하는 것을 추가로 포함한다.In some embodiments, the method further comprises separating the oil of (a) from the biomass prior to (b) contacting the oil with the at least one substance.

일부 실시 형태에서, 오일을 하나 이상의 물질을 포함하는 조성물과 접촉시키는 단계 (b)는 오일을, 트라이글리세라이드를 유리 지방산으로 가수분해시킬 수 있는 하나 이상의 촉매와 접촉시키는 것을 포함한다.In some embodiments, contacting the oil with a composition comprising one or more materials (b) comprises contacting the oil with one or more catalysts capable of hydrolyzing triglycerides to free fatty acids.

일부 실시 형태에서, 오일을, 하나 이상의 물질을 포함하는 조성물과 접촉시키는 단계 (b)는 오일을, 트라이글리세라이드와 화학 반응하여 유리 지방산을 포함하는 반응 생성물이 수득되게 할 수 있는 하나 이상의 반응물 또는 용매와 접촉시키는 것을 포함한다.In some embodiments, step (b) of contacting the oil with a composition comprising one or more materials comprises one or more reactants that can chemically react the oil with triglycerides to obtain a reaction product comprising free fatty acids, or Contact with a solvent.

일부 실시 형태에서, 부탄올을 생성하는 방법은 (a) 전분 및 오일 - 상기 오일은 소정량의 글리세라이드를 포함함 - 을 포함하는 바이오매스를 제공하는 단계와; (b) 바이오매스를 액화시켜 액화된 바이오매스를 생성하는 단계 - 상기 액화된 바이오매스는 전분으로부터 가수분해된 올리고당류를 포함함 - 와; (c) 단계 (a)의 바이오매스 또는 단계 (b)의 액화된 바이오매스를, 글리세라이드를 유리 지방산으로 전환시킬 수 있는 하나 이상의 효소를 포함하는 조성물과 접촉시키고 이에 의해 오일 중 글리세라이드의 적어도 일부분을 유리 지방산으로 전환시키는 단계와; (d) 액화된 바이오매스를, 올리고당류를 단량체성 글루코스를 포함하는 발효성 당으로 전환시킬 수 있는 당화 효소와 접촉시키는 단계와; (e) 액화된 바이오매스를, 발효성 당을 부탄올로 전환시킬 수 있는 재조합 미생물과 접촉시키고 이에 의해 부탄올을 생성하는 단계와; (f) 유리 지방산을 재조합 미생물과 접촉시키는 단계를 포함하며, 여기서 재조합 미생물의 (i) 성장 속도 및 글루코스 소비량 중 적어도 하나는 유리 지방산의 존재 하에서, 유리 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 글루코스 소비량보다 더 크다.In some embodiments, a method of producing butanol comprises the steps of: (a) providing a biomass comprising starch and oil, the oil comprising a predetermined amount of glycerides; (b) liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass comprises oligosaccharides hydrolyzed from starch; (c) contacting the biomass of step (a) or the liquefied biomass of step (b) with a composition comprising one or more enzymes capable of converting glycerides into free fatty acids, thereby at least the glycerides in oil Converting a portion to free fatty acid; (d) contacting the liquefied biomass with a glycosylating enzyme capable of converting oligosaccharides into fermentable sugars comprising monomeric glucose; (e) contacting the liquefied biomass with a recombinant microorganism capable of converting fermentable sugars to butanol, thereby producing butanol; (f) contacting the free fatty acid with the recombinant microorganism, wherein at least one of (i) the growth rate and glucose consumption of the recombinant microorganism is in the presence of the free fatty acid, in the absence of the free fatty acid, and / or Or greater than glucose consumption.

일부 실시 형태에서, 공급재료로부터 생성물 알코올을 생성하는 발효 과정은 (a) 상기 공급재료를 액화시켜 공급재료 슬러리를 생성하는 단계와; (b) 공급재료 슬러리를 원심분리하여 (i) 글루코스를 포함하는 수성 층, (ii) 글리세라이드를 포함하는 오일 층, 및 (iii) 고형물 층을 포함하는 원심분리 생성물을 생성하는 단계와; (c) 글리세라이드의 적어도 일부분을 유리 지방산으로 가수분해시키는 단계와; (d) (b)의 수성 층을, 글루코스로부터 생성물 알코올을 생성할 수 있는 재조합 미생물을 포함하는 발효 브로쓰를 함유하는 발효 용기에 공급하는 단계와; (e) 수성 층의 글루코스를 발효시켜 생성물 알코올을 생성하는 단계와; (f) 유리 지방산을 발효 브로쓰와 접촉시키는 단계를 포함하며, 여기서 미생물의 (i) 성장 속도 및 글루코스 소비량 중 적어도 하나는 유리 지방산의 존재 하에서, 유리 지방산의 부재 하에서의 미생물의 성장 속도 및/또는 글루코스 소비량보다 더 크다.In some embodiments, the fermentation process of producing product alcohol from the feedstock comprises: (a) liquefying the feedstock to produce a feedstock slurry; (b) centrifuging the feed slurry to produce a centrifugation product comprising (i) an aqueous layer comprising glucose, (ii) an oil layer comprising glycerides, and (iii) a solid layer; (c) hydrolyzing at least a portion of the glycerides with free fatty acids; (d) feeding the aqueous layer of (b) to a fermentation vessel containing a fermentation broth comprising recombinant microorganisms capable of producing product alcohol from glucose; (e) fermenting glucose in the aqueous layer to produce product alcohol; (f) contacting the free fatty acid with the fermentation broth, wherein at least one of (i) the growth rate and glucose consumption of the microorganism is in the presence of the free fatty acid, and / or the growth rate of the microorganism in the absence of the free fatty acid. Greater than glucose consumption.

일부 실시 형태에서, 공급재료로부터 생성물 알코올을 생성하는 방법은 글리세라이드를 가수분해시키는 단계 전에 글리세라이드를 발효 용기에 공급하는 단계를 추가로 포함한다.In some embodiments, the method of producing the product alcohol from the feedstock further comprises feeding the glycerides to the fermentation vessel prior to hydrolyzing the glycerides.

일부 실시 형태에서, 발효 과정은 (a) 발효성 탄소 공급원으로부터 생성물 알코올을 생성하는 재조합 미생물, 발효성 탄소 공급원, 생성물 알코올 및 바이오매스로부터 유래된 오일 - 상기 오일은 글리세라이드를 포함함 - 을 포함하는 발효 브로쓰를 제공하는 단계와; (b) 발효 브로쓰를 제1 추출제와 접촉시켜 수성 상 및 유기 상을 포함하는 2상 혼합물을 형성하는 단계 - 생성물 알코올 및 오일은 유기 상 내로 분배되어 생성물 알코올 함유 유기 상을 형성함 - 와; (c) 생성물 알코올 함유 유기 상을 수성 상으로부터 분리하는 단계와; (d) 생성물 알코올을 유기 상으로부터 분리하여 린(lean) 유기 상을 생성하는 단계와; (e) 린 유기 상을, 글리세라이드를 유리 지방산으로 가수분해시킬 수 있는 하나 이상의 촉매를 포함하는 조성물과 접촉시켜 적어도 일부분의 제1 추출제 및 유리 지방산을 포함하는 제2 추출제를 생성하는 단계와; (f) 발효 브로쓰를 단계 (e)의 제2 추출제와 접촉시킴으로써 단계 (b)를 반복하는 단계를 포함하며, 여기서 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 유리 지방산의 존재 하에서, 유리 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 크다.In some embodiments, the fermentation process comprises (a) oils derived from recombinant microorganisms, fermentable carbon sources, product alcohols and biomass that produce product alcohols from the fermentable carbon source, wherein the oil comprises glycerides. Providing a fermentation broth; (b) contacting the fermentation broth with a first extractant to form a biphasic mixture comprising an aqueous phase and an organic phase, wherein the product alcohol and oil are partitioned into an organic phase to form a product alcohol containing organic phase; and ; (c) separating the product alcohol containing organic phase from the aqueous phase; (d) separating the product alcohol from the organic phase to produce a lean organic phase; (e) contacting the lean organic phase with a composition comprising at least one catalyst capable of hydrolyzing glycerides to free fatty acids to produce at least a portion of a first extractant and a second extractant comprising free fatty acids Wow; (f) repeating step (b) by contacting the fermentation broth with the second extractant of step (e), wherein at least one of (i) growth rate and (ii) fermentable carbon consumption of the microorganism Is greater than the growth rate and / or fermentable carbon consumption of the recombinant microorganism in the presence of free fatty acids.

본 명세서에 포함되고 본 명세서의 일부를 형성하는 첨부된 도면은 본 발명을 예시하고, 설명과 함께, 추가로 본 발명의 원리를 설명하는 역할 및 관련 분야의 숙련자가 본 발명을 만들고 사용할 수 있게 하는 역할을 한다.
<도 1>
도 1은 액화된 바이오매스를 지질 가수분해를 위한 하나 이상의 물질과 접촉시키고 발효 용기에 공급하는 본 발명의 예시적인 방법 및 시스템을 개략적으로 도시한다.
<도 2>
도 2는 액화되고 당화된 바이오매스를 지질 가수분해를 위한 하나 이상의 물질과 접촉시키고 발효 용기에 공급하는 본 발명의 예시적인 방법 및 시스템을 개략적으로 도시한다.
<도 3>
도 3은 비용해 고형물 및 지질을 발효 전에 액화 바이오매스로부터 제거하고, 제거된 지질을 하나 이상의 물질을 사용하여 유리 지방산으로 가수분해시키고, 상기 유리 지방산을 발효 용기에 공급하는 본 발명의 예시적인 방법 및 시스템을 개략적으로 도시한다.
<도 4>
도 4는 천연 오일로부터 유래된 지질을 하나 이상의 물질을 사용하여 유리 지방산으로 가수분해시키고, 상기 유리 지방산을 발효 용기에 공급하는 본 발명의 예시적인 방법 및 시스템을 개략적으로 도시한다.
<도 5>
도 5는 발효 용기에서 배출되는 추출제에 존재하는 바이오매스 지질을 유리 지방산으로 전환시키고 이를 발효 용기에 공급하는 본 발명의 예시적인 방법 및 시스템을 개략적으로 도시한다.
<도 6>
도 6은 발효 용기 내에서의 지방산의 존재가 부탄올 생성 스트레인(strain) NGCI-047의 글루코스 소비량에 미치는 영향을 예시하는 그래프이다.
<도 7>
도 7은 발효 용기 내에서의 지방산의 존재가 부탄올 생성 스트레인 NGCI-049의 글루코스 소비량에 미치는 영향을 예시하는 그래프이다.
<도 8>
도 8은 발효 용기 내에서의 지방산의 존재가 부탄올 생성 스트레인 NYLA84의 글루코스 소비량에 미치는 영향을 예시하는 그래프이다.The accompanying drawings, which are incorporated in and form a part of this specification, illustrate the invention, and together with the description further serve to explain the principles of the invention and enable those skilled in the art to make and use the invention. Play a role.
&Lt; 1 >
1 schematically depicts an exemplary method and system of the present invention for contacting a liquefied biomass with one or more materials for lipid hydrolysis and feeding it to a fermentation vessel.
2,
2 schematically illustrates an exemplary method and system of the present invention for contacting liquefied and glycated biomass with one or more materials for lipid hydrolysis and feeding the fermentation vessel.
3,
FIG. 3 is an exemplary method of the present invention in which the costly solids and lipids are removed from the liquefied biomass prior to fermentation, the removed lipids are hydrolyzed to free fatty acids using one or more materials, and the free fatty acids are fed to the fermentation vessel. And a system schematically.
<Fig. 4>
4 schematically depicts an exemplary method and system of the invention for hydrolyzing lipids derived from natural oils into free fatty acids using one or more materials and feeding the free fatty acids to fermentation vessels.
5,
FIG. 5 schematically illustrates an exemplary method and system of the present invention for converting biomass lipids present in extractant exiting a fermentation vessel into free fatty acids and feeding them to the fermentation vessel.
6,
6 is a graph illustrating the effect of the presence of fatty acids in fermentation vessels on glucose consumption of butanol producing strain NGCI-047.
7,
7 is a graph illustrating the effect of the presence of fatty acids in fermentation vessels on glucose consumption of butanol producing strain NGCI-049.
8,
8 is a graph illustrating the effect of the presence of fatty acids in fermentation vessels on glucose consumption of butanol producing strain NYLA84.

달리 정의되지 않으면, 본 명세서에서 사용된 모든 기술 및 과학 용어는 본 발명이 속하는 기술 분야의 숙련자가 일반적으로 이해하는 것과 동일한 의미를 갖는다. 상충될 경우, 정의를 비롯한 본 명세서가 좌우할 것이다. 또한, 문맥에 의해 다르게 요구되지 않으면, 단수형 용어는 복수형을 포함할 것이며, 복수형 용어는 단수형을 포함할 것이다. 본 명세서에 언급된 모든 간행물, 특허 및 기타 참조문헌은 모든 목적을 위해 전체적으로 참고로 포함된다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Also, unless otherwise required by context, the singular terms shall include the plural, and the plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

본 발명을 추가로 정의하기 위하여, 하기의 용어 및 정의가 본 명세서에 제공된다.To further define the present invention, the following terms and definitions are provided herein.

본 명세서에 사용되는 바와 같이, 용어 "포함하다", "포함하는", "포함하고 있다", "포함하고 있는", "갖다", "갖는", "함유하다" 또는 "함유하는" 또는 임의의 다른 그 변이형은 기술된 정수 또는 정수들의 군의 포함을 암시하며 임의의 다른 정수 또는 정수들의 군의 배제를 암시하는 것은 아닌 것으로 이해될 것이다. 예를 들어, 요소들의 목록을 포함하는 조성물, 혼합물, 공정, 방법, 용품, 또는 장치는 반드시 그러한 요소만으로 제한되지는 않고, 명확하게 열거되지 않거나 그러한 조성물, 혼합물, 공정, 방법, 용품, 또는 장치에 내재적인 다른 요소를 포함할 수 있다. 더욱이, 명백히 반대로 기술되지 않는다면, "또는"은 포괄적인 '또는'을 말하며 배타적인 '또는'을 말하는 것은 아니다. 예를 들어, 조건 A 또는 B는 하기 중 어느 하나에 의해 만족된다: A는 참 (또는 존재함)이고 B는 거짓 (또는 존재하지 않음), A는 거짓 (또는 존재하지 않음)이고 B는 참 (또는 존재함), A 및 B가 모두가 참 (또는 존재함).As used herein, the terms "comprise", "comprising", "comprising", "comprising", "have", "having", "contains" or "contains" or any It is to be understood that other variations of are indicative of the inclusion of the described integers or groups of integers and do not imply the exclusion of any other integers or groups of integers. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to such elements, Lt; RTI ID = 0.0 &gt; element. Moreover, unless expressly stated to the contrary, "or" does not mean &quot; comprehensive &quot; or &quot; exclusive &quot; For example, the condition A or B is satisfied by either: A is true (or exists), B is false (or not present), A is false (or nonexistent) (Or present), A and B are both true (or present).

또한, 본 발명의 요소 또는 성분 앞의 부정 관사("a" 및 "an")는 요소 또는 성분의 경우의 수, 즉 발생의 수에 관해서는 비제한적인 것으로 의도된다. 따라서, 부정관사 ("a"" 또는 "an")는 하나 또는 적어도 하나를 포함하는 것으로 파악되어야 하며, 당해 요소 또는 성분의 단수형은 그 수가 명백하게 단수임을 의미하는 것이 아니라면 복수형을 또한 포함한다.Also, the indefinite articles "a" and "an" before an element or component of the present invention are intended to be non-limiting with regard to the number of cases of the element or component, ie the number of occurrences. Thus, the indefinite article "a" or "an" should be understood to include one or at least one, and the singular form of the element or component also includes the plural unless the number is obviously meant to be singular.

본 명세서에 사용되는 바와 같이, 용어 "발명" 또는 "본 발명"은 비제한적인 용어이며, 특정 발명의 임의의 단일 실시 형태를 언급하는 것으로 의도되지 않고, 본원에 기재된 바와 같은 모든 가능한 실시 형태를 포함한다.As used herein, the term “invention” or “invention” is a non-limiting term and is not intended to refer to any single embodiment of a particular invention, and refers to all possible embodiments as described herein. Include.

본 명세서에서 사용된 바와 같이, 이용되는 발명의 성분 또는 반응물의 양을 수식하는 용어 "약"은, 예를 들어, 현실에서 농축물 또는 용액을 제조하는 데 사용되는 통상적인 측정 및 액체 취급 절차; 이들 절차에서의 우발적인 오차; 조성물을 제조하거나 방법을 실시하기 위해 이용된 성분의 제조, 공급원 또는 순도의 차이 등을 통해 발생할 수 있는 수치적 양의 변이를 말한다. 용어 "약"은 또한 특정 초기 혼합물에서 생긴 조성물에 있어서 상이한 평형 조건으로 인해 달라지는 양을 포함한다. 용어 "약"으로 수식되든지 수식되지 않든지 간에, 특허청구범위는 당해 양에 상당하는 양을 포함한다. 일 실시 형태에서, 용어 "약"은 보고된 수치값의 10% 이내, 대안적으로는 보고된 수치값의 5% 이내를 의미한다.As used herein, the term "about," which modifies the amount of a component or reactant of the invention employed, includes, for example, conventional measurement and liquid handling procedures used to prepare concentrates or solutions in practice; Accidental errors in these procedures; Numerical amounts of variation that can occur through the manufacture of the ingredients used to prepare the composition or to carry out the method, differences in source or purity, and the like. The term "about" also includes amounts that vary due to different equilibrium conditions in the composition resulting from the particular initial mixture. Whether modified by the term "about" or not, the claims include amounts corresponding to those amounts. In one embodiment, the term “about” means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value.

본 명세서에 사용되는 바와 같이, "바이오매스"는 발효성 당을 제공하는 가수분해성 다당류를 함유하는 천연 생성물을 말하며, 상기 발효성 당은 천연 공급원, 예를 들어 옥수수, 사탕수수, 밀, 셀룰로오스계 또는 리그노셀룰로오스계 물질 및 셀룰로오스, 헤미셀룰로오스, 리그닌, 전분, 다당류, 이당류 및/또는 단당류를 포함하는 물질, 및 그 혼합물로부터 유래되는 임의의 당 및 전분을 포함한다. 또한 바이오매스는 추가의 성분, 예를 들어 단백질 및/또는 지질을 포함할 수 있다. 바이오매스는 단일 공급원으로부터 유래할 수 있거나, 또는 바이오매스는 1가지 초과의 공급원으로부터 유래된 혼합물을 포함할 수 있다. 예를 들어, 바이오매스는 옥수수 속대 및 옥수수 대의 혼합물, 또는 풀과 잎의 혼합물을 포함할 수 있다. 바이오매스는 바이오에너지 작물, 농업 잔사, 도시 고형 폐기물(municipal solid waste), 산업 고형 폐기물, 제지로부터의 슬러지, 마당 폐기물(yard waste), 나무 및 삼림지 폐기물을 포함하지만, 이에 한정되지 않는다. 바이오매스의 예에는, 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀, 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스, 폐지, 사탕수수 바가스, 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물이 포함되지만, 이에 한정되지 않는다. 예를 들어, 매시, 즙, 당밀, 또는 가수분해물은 제분, 처리 및/또는 액화에 의한 것과 같이 발효 목적을 위한 바이오매스의 프로세싱에 대하여 당업계에 공지된 임의의 프로세싱에 의해 바이오매스로부터 형성될 수 있으며, 발효성 당을 포함할 수 있고 물을 포함할 수 있다. 예를 들어, 셀룰로오스계 및/또는 리그노셀룰로오스계 바이오매스는 당업자에게 공지된 임의의 방법에 의해 발효성 당을 함유하는 가수분해물이 수득되도록 프로세싱될 수 있다. 저 암모니아 전처리가 본 명세서에 참고로 포함된 미국 특허 공개 제2007/0031918A1호에 개시되어 있다. 전형적으로 셀룰로오스계 및/또는 리그노셀룰로오스계 바이오매스의 효소적 당화는 글루코스, 자일로스 및 아라비노스를 포함하는 당을 함유하는 가수분해물을 생성하기 위하여 셀룰로오스 및 헤미셀룰로오스를 분해함에 있어서 효소 컨소시엄(consortium)을 활용한다. (셀룰로오스계 및/또는 리그노셀룰로오스계 바이오매스에 적합한 당화 효소는 문헌[Lynd, et al., Microbiol. Mol. Biol. Rev. 66:506-577, 2002]에 개관되어 있다).As used herein, "biomass" refers to natural products containing hydrolyzable polysaccharides that provide fermentable sugars, which fermentable sugars are natural sources such as corn, sugar cane, wheat, cellulose based Or lignocellulosic materials and materials including cellulose, hemicellulose, lignin, starch, polysaccharides, disaccharides and / or monosaccharides, and any sugars and starches derived from mixtures thereof. Biomass may also include additional components such as proteins and / or lipids. The biomass may be derived from a single source or the biomass may comprise a mixture derived from more than one source. For example, the biomass may comprise corncobs and mixtures of corncobs, or mixtures of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper, yard waste, wood and woodland waste. Examples of biomass include corn grains, corncobs, crop residues, for example corn husks, corn stalks, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane vargas, Include, but are not limited to, sorghum, sugar cane, soybeans, ingredients obtained by milling grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure and mixtures thereof. It is not limited. For example, mash, juice, molasses, or hydrolyzate may be formed from biomass by any processing known in the art for processing of biomass for fermentation purposes, such as by milling, treating, and / or liquefying. May comprise fermentable sugars and may comprise water. For example, cellulosic and / or lignocellulosic biomass may be processed to yield hydrolysates containing fermentable sugars by any method known to those skilled in the art. Low ammonia pretreatment is disclosed in US Patent Publication No. 2007 / 0031918A1, which is incorporated herein by reference. Typically enzymatic glycosylation of cellulosic and / or lignocellulosic biomass is an enzymatic consortium in degrading cellulose and hemicellulose to produce hydrolysates containing sugars including glucose, xylose and arabinose. To utilize. (Saccharifying enzymes suitable for cellulosic and / or lignocellulosic biomass are reviewed in Lynd, et al., Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

매시, 즙, 당밀 또는 가수분해물은 공급재료(12) 및 공급재료 슬러리(16)를 포함할 수 있으며, 이는 본 명세서에 기재된 바와 같다. 수성 피드스트림은 제분, 처리 및/또는 액화에 의한 것과 같이 발효 목적을 위한 바이오매스의 프로세싱에 대하여 당업계에 공지된 임의의 프로세싱에 의해 바이오매스로부터 유래되거나 또는 형성될 수 있으며, 발효성 탄소 기질 (예를 들어, 당)을 포함할 수 있고, 물을 포함할 수 있다. 수성 피드스트림은 공급재료(12) 및 공급재료 슬러리(16)를 포함할 수 있으며, 이는 본 명세서에 기재된 바와 같다.The mash, juice, molasses or hydrolyzate may comprise feedstock 12 and feedstock slurry 16, as described herein. The aqueous feedstream may be derived from or formed from the biomass by any processing known in the art for processing of biomass for fermentation purposes, such as by milling, treating and / or liquefaction, and fermentable carbon substrates. (Eg, sugars), and may include water. The aqueous feedstream may comprise feedstock 12 and feedstock slurry 16, as described herein.

본 명세서에 사용되는 바와 같이, "바이오매스 생성"은 미생물 예비 발효물의 배양 동안 또는 미생물의 발효 성장 동안과 같은 미생물 바이오매스 생성(즉, 세포 바이오매스 생성 또는 세포 성장)을 말한다.As used herein, "biomass production" refers to microbial biomass production (ie, cell biomass production or cell growth), such as during the cultivation of microbial pre-fermentation or during fermentation growth of microorganisms.

본 명세서에 사용되는 바와 같이, "공급재료"는 발효 과정에서의 공급물을 의미하며, 상기 공급물은 비용해 고형물이 있거나 또는 없는 발효성 탄소 공급원을 함유하고, 적용가능할 경우, 상기 공급물은 발효성 탄소 공급원이 액화, 당화 또는 다른 공정에 의한 것과 같은 추가의 프로세싱에 의해 복합 당의 분해에 의해 수득되거나 또는 전분으로부터 유리되기 전 또는 그 후의 발효성 탄소 공급원을 함유한다. 공급재료는 바이오매스를 포함하거나 또는 바이오매스로부터 유래된다. 적합한 공급재료는 호밀, 밀, 옥수수, 사탕수수, 보리, 셀룰로오스계 물질, 리그노셀룰로오스계 물질 또는 그 혼합물을 포함하지만, 이에 한정되지 않는다.As used herein, "feedstock" means a feed during the fermentation process, which feed contains a fermentable carbon source with or without costly solids, where applicable, The fermentable carbon source contains the fermentable carbon source before or after it is obtained by the decomposition of the complex sugar or further liberated from starch by further processing such as by liquefaction, saccharification or other processes. The feedstock includes or is derived from biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, sugar cane, barley, cellulosic material, lignocellulosic material, or mixtures thereof.

본 명세서에 사용되는 바와 같이, "발효 브로쓰"는 물, 당류, 용해된 고형물, 선택적으로, 알코올을 생성하는 미생물, 생성물 알코올, 및 발효 용기에 담긴 물질의 모든 다른 구성성분의 혼합물을 의미하며, 여기서, 생성물 알코올은 존재하는 미생물에 의한 알코올, 물 및 이산화탄소(CO₂)로의 당의 반응에 의해 제조된다. 가끔, 본 명세서에 사용되는 바와 같이, 용어 "발효 배지" 및 "발효된 혼합물"은 "발효 브로쓰"와 동의어로 사용될 수 있다."As used herein, "fermentation broth" means a mixture of water, sugars, dissolved solids, optionally microorganisms that produce alcohol, product alcohols, and all other components of materials contained in fermentation vessels; Where the product alcohol is prepared by the reaction of sugars with alcohol, water and carbon dioxide (CO ₂ ) by the microorganisms present. Sometimes, as used herein, the terms "fermentation medium" and "fermented mixture" may be used synonymously with "fermentation broth."

본 명세서에 사용되는 바와 같이, "발효성 탄소 공급원" 또는 "발효성 탄소 기질"은 발효 알코올의 생성을 위하여 본 명세서에 개시된 미생물에 의해 대사(또는 "소비")될 수 있는 탄소 공급원을 의미한다. 적합한 발효성 탄소 공급원은 단당류, 예를 들어 글루코스 또는 프룩토스; 이당류, 예를 들어 락토스 또는 수크로스; 올리고당류; 다당류, 예를 들어 전분 또는 셀룰로오스; C5 당류, 예를 들어 자일로스 및 아라비노스; 메탄을 포함하는 C1 탄소 기질; 및 그 혼합물을 포함하지만, 이에 한정되지 않는다. 본 명세서에 사용되는 바와 같이, 용어 "소비되는"은 화합물, 예를 들어 글루코스와 같은 유기 화합물이 세포 유래의 효소의 작용에 의해 분해되고 그에 의해 세포가 사용할 수 있는 에너지가 생성되는 과정을 포함한다.As used herein, "fermentable carbon source" or "fermentable carbon substrate" means a carbon source that can be metabolized (or "consumed") by the microorganisms disclosed herein for the production of fermentation alcohol. . Suitable fermentable carbon sources include monosaccharides such as glucose or fructose; Disaccharides such as lactose or sucrose; Oligosaccharides; Polysaccharides such as starch or cellulose; C5 sugars such as xylose and arabinose; C1 carbon substrate comprising methane; And mixtures thereof. As used herein, the term "consumed" includes the process by which a compound, for example an organic compound, such as glucose, is broken down by the action of an enzyme derived from a cell, thereby producing energy available for the cell. .

본 명세서에 사용되는 바와 같이, "발효성 당"은 발효 알코올의 생성을 위하여 본 명세서에 개시된 미생물에 의해 대사(또는 "소비")될 수 있는 하나 이상의 당을 말한다.As used herein, "fermentable sugar" refers to one or more sugars that can be metabolized (or "consumed") by the microorganisms disclosed herein for the production of fermentation alcohol.

본 명세서에 사용되는 바와 같이, "발효 용기"는 발효 반응이 수행되고 이에 의해 부탄올과 같은 생성물 알코올이 당으로부터 만들어지는 용기를 의미한다.As used herein, “fermentation vessel” means a vessel in which a fermentation reaction is carried out whereby a product alcohol such as butanol is made from sugars.

본 명세서에 사용되는 바와 같이, "액화 용기"는 액화가 수행되는 용기를 의미한다. 액화는 올리고당류를 공급재료로부터 유리시키는 과정이다. 공급재료가 옥수수인 일부 실시 형태에서, 올리고당류는 액화 동안 옥수수 전분 내용물로부터 유리된다.As used herein, "liquefaction container" means a container in which liquefaction is performed. Liquefaction is the process of liberating oligosaccharides from the feedstock. In some embodiments where the feedstock is corn, oligosaccharides are liberated from the corn starch contents during liquefaction.

본 명세서에 사용되는 바와 같이, "당화 용기"는 당화(즉, 올리고당류의 단당류로의 분해)가 수행되는 용기를 의미한다. 발효 및 당화가 동시에 일어나는 경우, 당화 용기 및 발효 용기는 동일 용기 내의 것일 수 있다.As used herein, “glycosylation vessel” means a vessel in which saccharification (ie, decomposition of oligosaccharides into monosaccharides) is performed. When fermentation and saccharification occur simultaneously, the saccharification vessel and the fermentation vessel may be in the same vessel.

본 명세서에 사용되는 바와 같이, "당"은 올리고당류, 이당류, 단당류, 및/또는 그 혼합물을 말한다. 또한, "당류"라는 용어는 당뿐만 아니라 전분, 덱스트란, 글라이코겐, 셀룰로오스, 펜토산을 포함하는 탄수화물도 포함한다.As used herein, "sugar" refers to oligosaccharides, disaccharides, monosaccharides, and / or mixtures thereof. The term "saccharides" also includes sugars as well as carbohydrates including starch, dextran, glycogen, cellulose, pentosan.

본 명세서에 사용되는 바와 같이, "당화 효소"는 다당류 및/또는 올리고당류, 예를 들어, 글라이코겐의 알파-1,4-글루코시딕 결합, 또는 전분을 가수분해할 수 있는 하나 이상의 효소를 의미한다. 당화 효소는 셀룰로오스계 또는 리그노셀룰로오스계 물질을 가수분해할 수 있는 효소를 또한 포함할 수 있다.As used herein, “glycosylation enzyme” refers to one or more enzymes capable of hydrolyzing alpha-1,4-glucosidic bonds, or starches of polysaccharides and / or oligosaccharides, eg, glycogen. Means. Glycosylating enzymes may also include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials.

본 명세서에 사용되는 바와 같이, "비용해 고형물"은 공급재료의 비-발효성 부분, 예를 들어 배아(germ), 섬유 및 글루텐을 의미한다.As used herein, "non-hazardous solids" means non-fermentable portions of the feedstock, such as germs, fibers and gluten.

본 명세서에 사용되는 바와 같이, "생성물 알코올"은 발효성 탄소 기질의 공급원으로서 바이오매스를 이용하는, 발효 과정에서의 미생물에 의해 생성될 수 있는 임의의 알코올을 말한다. 생성물 알코올은 C₁ 내지 C₈ 알킬 알코올을 포함하지만, 이에 한정되지 않는다. 일부 실시 형태에서, 생성물 알코올은 C₂ 내지 C₈ 알킬 알코올이다. 다른 실시 형태에서, 생성물 알코올은 C₂ 내지 C₅ 알킬 알코올이다. C₁ 내지 C₈ 알킬 알코올은 메탄올, 에탄올, 프로판올, 부탄올, 및 펜탄올을 포함하지만 이에 한정되지 않음이 인식될 것이다. 마찬가지로, C₂ 내지 C₈ 알킬 알코올은 에탄올, 프로판올, 부탄올, 및 펜탄올을 포함하지만, 이에 한정되지 않는다. 또한 "알코올"은 생성물 알코올과 관련하여 본 명세서에서 사용된다.As used herein, "product alcohol" refers to any alcohol that can be produced by a microorganism in a fermentation process, using biomass as a source of fermentable carbon substrate. Product alcohols include, but are not limited to, C ₁ to C ₈ alkyl alcohols. In some embodiments, the product alcohol is C ₂ to C ₈ alkyl alcohol. In another embodiment, the product alcohol is C ₂ to C ₅ alkyl alcohol. It will be appreciated that C ₁ to C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, and pentanol. Likewise, C ₂ to C ₈ alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, and pentanol. "Alcohol" is also used herein in the context of product alcohols.

본 명세서에 사용되는 바와 같이, "부탄올"은 구체적으로, 개별적인 또는 혼합물로서의 부탄올 이성체들, 1-부탄올 (1-BuOH), 2-부탄올 (2-BuOH), tert-부탄올, 및/또는 아이소부탄올 (iBuOH 또는 i-BuOH 또는 I-BUOH, 2-메틸-1-프로판올로도 공지됨)을 말한다. 가끔, 부탄올의 에스테르를 말할 때, 용어 "부틸 에스테르" 및 "부탄올 에스테르"는 상호교환가능하게 사용될 수 있다.As used herein, "butanol" specifically refers to butanol isomers, individually or as a mixture, 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol, and / or isobutanol (also known as iBuOH or i-BuOH or I-BUOH, 2-methyl-1-propanol). Sometimes when referring to esters of butanol, the terms "butyl ester" and "butanol ester" may be used interchangeably.

본 명세서에 사용되는 바와 같이, "프로판올"은 프로판올 이성체들, 아이소프로판올 또는 1-프로판올을 말한다.As used herein, “propanol” refers to propanol isomers, isopropanol or 1-propanol.

본 명세서에 사용되는 바와 같이, "펜탄올" 은 펜탄올 이성체들, 1-펜탄올, 3-메틸-1-부탄올, 2-메틸-1-부탄올, 2,2-다이메틸-1-프로판올, 3-펜탄올, 2-펜탄올, 3-메틸-2-부탄올, 또는 2-메틸-2-부탄올을 말한다.As used herein, “pentanol” refers to pentanol isomers, 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

본 명세서에 사용되는 바와 같이, 용어 "알코올 당량"은 알코올 에스테르의 완벽한 가수분해 및 소정량의 알코올 에스테르로부터의 알코올의 후속적인 회수에 의해 수득되는 알코올의 중량을 말한다.As used herein, the term "alcohol equivalent" refers to the weight of the alcohol obtained by complete hydrolysis of the alcohol ester and subsequent recovery of the alcohol from the predetermined amount of alcohol ester.

본 명세서에 사용되는 바와 같이, 용어 "수성 상 타이터"는 발효 브로쓰 중 특정 알코올 (예를 들어, 부탄올)의 농도를 말한다.As used herein, the term "aqueous phase titer" refers to the concentration of certain alcohols (eg butanol) in fermentation broth.

본 명세서에 사용되는 바와 같이, 용어 "유효 타이터"는 발효 배지 1리터 당 알코올 에스테르화에 의해 생성된 알코올 에스테르의 알코올 당량 또는 발효에 의해 생성된 특정 알코올 (예를 들어, 부탄올)의 총량을 말한다. 예를 들어, 단위 부피의 발효물 중 부탄올의 유효 타이터는 (i) 발효 배지 중 부탄올의 양; (ii) 유기 추출제로부터 회수되는 부탄올의 양; (iii) 가스 스트리핑(gas stripping)이 사용될 경우, 가스 상으로부터 회수되는 부탄올의 양; 및 (iv) 유기 상 또는 수성 상 중 부틸 에스테르의 알코올 당량을 포함한다.As used herein, the term "effective titer" refers to the alcohol equivalent of an alcohol ester produced by alcohol esterification per liter of fermentation medium or the total amount of a specific alcohol (eg butanol) produced by fermentation. Say. For example, an effective titer of butanol in a unit volume of fermentation may include (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; (iii) the amount of butanol recovered from the gas phase when gas stripping is used; And (iv) alcohol equivalents of butyl ester in organic or aqueous phase.

본 명세서에 사용되는 바와 같이, 원위치 생성물 제거(in situ product removal; ISPR)는 발효와 같은 생물학적 과정으로부터의 특정 발효 생성물의 선택적 제거를 의미하며, 이는 상기 생성물이 생성될 때 생물학적 과정에서 생성물 농도를 제어하기 위한 것이다.As used herein, in situ product removal (ISPR) refers to the selective removal of a particular fermentation product from a biological process, such as fermentation, which determines the product concentration in the biological process when the product is produced. To control.

본 명세서에 사용되는 바와 같이, "추출제" 또는 "ISPR 추출제"는 부탄올과 같은 임의의 생성물 알코올을 추출하기 위하여 사용되는 또는 생성물 알코올 및 카르복실산 또는 지질로부터 촉매에 의해 생성되는 임의의 생성물 알코올 에스테르를 추출하기 위하여 사용되는 유기 용매를 의미한다. 가끔, 본 명세서에 사용되는 바와 같이, 용어 "용매"는 "추출제"와 동의어로 사용될 수 있다. 본 명세서에 개시된 공정들에 있어서, 추출제는 수불혼화성이다.As used herein, an "extractant" or "ISPR extractant" is used to extract any product alcohol, such as butanol or any product produced by a catalyst from product alcohol and carboxylic acid or lipids. An organic solvent used for extracting an alcohol ester. Sometimes, as used herein, the term "solvent" may be used synonymously with "extractant". In the processes disclosed herein, the extractant is water immiscible.

용어 "수불혼화성" 또는 "수불용성"은 하나의 액체 상을 형성하는 그러한 방식으로 발효 브로쓰와 같은 수성 용액과 혼합되는 것이 불가능한 화학 성분, 예를 들어 추출제 또는 용매를 말한다.The term "water immiscible" or "water insoluble" refers to a chemical component, for example extractant or solvent, which is impossible to mix with an aqueous solution such as fermentation broth in such a way to form one liquid phase.

본 명세서에 사용되는 바와 같이, 용어 "수성 상"은 발효 브로쓰를 수불혼화성 유기 추출제와 접촉시킴으로써 수득되는 2상 혼합물의 수성 상을 말한다. 발효 추출을 포함하는 본 명세서에 개시된 공정의 실시 형태에서, 용어 "발효 브로쓰"는 또한 구체적으로 2상 발효 추출에서의 수성 상을 말한다.As used herein, the term "aqueous phase" refers to the aqueous phase of a biphasic mixture obtained by contacting fermentation broth with a water immiscible organic extractant. In embodiments of the process disclosed herein including fermentation extraction, the term “fermentation broth” also specifically refers to the aqueous phase in two-phase fermentation extraction.

본 명세서에 사용되는 바와 같이, 용어 "유기 상"은 발효 브로쓰와 수불혼화성 유기 추출제를 접촉시켜 수득되는 2상 혼합물의 비수성 상을 말한다.As used herein, the term "organic phase" refers to the non-aqueous phase of the biphasic mixture obtained by contacting the fermentation broth with a water immiscible organic extractant.

본 명세서에 사용되는 바와 같이, 용어 "지방산"은 C₄ 내지 C₂₈ 탄소 원자 (가장 일반적으로는 C₁₂ 내지 C₂₄ 탄소 원자)를 갖는 카르복실산 (예를 들어, 지방족 모노카르복실산)을 말하며, 이는 포화되거나 또는 불포화된다. 또한 지방산은 분지형 또는 비분지형일 수 있다. 지방산은 동물 또는 식물 지방, 오일 또는 왁스 중 에스테르화된 형태로 함유되거나 또는 그로부터 유래될 수 있다. 지방산은 지방 또는 지방 오일 중 글리세라이드의 형태로 자연적으로 나타날 수 있거나 또는 지방의 가수분해에 의해 또는 합성에 의해 수득될 수 있다. 용어 "지방산"은 단일 화학종 또는 지방산들의 혼합물을 설명할 수 있다. 게다가, 용어 지방산은 유리 지방산을 또한 포함한다.As used herein, the term "fatty acid" refers to C ₄ to C ₂₈ carbon atoms (most commonly C ₁₂ To C ₂₄ carbon atoms), for example aliphatic monocarboxylic acids, which are saturated or unsaturated. Fatty acids can also be branched or unbranched. Fatty acids may be contained or derived from esterified forms in animal or vegetable fats, oils or waxes. Fatty acids may appear naturally in the form of glycerides in fats or fatty oils or may be obtained by hydrolysis or synthesis of fats. The term "fatty acid" may describe a single species or a mixture of fatty acids. In addition, the term fatty acid also includes free fatty acids.

본 명세서에 사용되는 바와 같이, 용어 "지방 알코올"은 C₄ 내지 C₂₂ 탄소 원자의 지방족 사슬을 갖는 알코올을 말하며, 이는 포화되거나 또는 불포화된다.As used herein, the term "fatty alcohol" refers to an alcohol having an aliphatic chain of C ₄ to C ₂₂ carbon atoms, which is saturated or unsaturated.

본 명세서에 사용되는 바와 같이 용어 "지방 알데히드"는 C₄ 내지 C₂₂ 탄소 원자의 지방족 사슬을 갖는 알데히드를 말하며, 이는 포화되거나 또는 불포화된다.As used herein, the term “fatty aldehyde” refers to an aldehyde having an aliphatic chain of C ₄ to C ₂₂ carbon atoms, which is saturated or unsaturated.

본 명세서에 사용되는 바와 같이, 용어 "카르복실산"은 일반 화학식 -COOH를 갖는 임의의 유기 화합물을 말하며, 여기서 탄소 원자는 이중 결합에 의해 산소 원자에 결합되어 카르보닐기(-C=O)를 만들고 단일 결합에 의해 하이드록실기(-OH)에 결합된다. 카르복실산은 양성자화 카르복실산의 형태로, 카르복실산의 염 (예를 들어, 암모늄, 나트륨 또는 칼륨 염)의 형태로, 또는 양성자화 카르복실산과 카르복실산 염의 혼합물로서 존재할 수 있다. 용어 카르복실산은 단일 화학종 (예를 들어, 올레산)을 설명하거나, 또는 바이오매스 유래된 지방산 에스테르 또는 트라이글리세라이드, 다이글리세라이드, 모노글리세라이드 및 인지질의 가수분해에 의해 생성될 수 있는 카르복실산들의 혼합물을 설명할 수 있다.As used herein, the term "carboxylic acid" refers to any organic compound having the general formula -COOH, wherein carbon atoms are bonded to oxygen atoms by double bonds to form carbonyl groups (-C = O) It is bonded to the hydroxyl group (-OH) by a single bond. The carboxylic acid may be present in the form of a protonated carboxylic acid, in the form of a salt of the carboxylic acid (eg, ammonium, sodium or potassium salt), or as a mixture of the protonated carboxylic acid and the carboxylic acid salt. The term carboxylic acid refers to a single species (eg oleic acid) or carboxyl which can be produced by hydrolysis of biomass derived fatty acid esters or triglycerides, diglycerides, monoglycerides and phospholipids. Describe a mixture of acids.

본 명세서에 사용되는 바와 같이, "천연 오일"은 식물 (예를 들어, 바이오매스) 또는 동물로부터 수득되는 지질을 말한다. 본 명세서에 사용되는 바와 같이, "식물 유래된 오일"은 특히 식물로부터 수득되는 지질을 말한다. 가끔, "지질"은 "오일" 및 "아실 글리세라이드"와 동의어로 사용될 수 있다. 천연 오일은 탤로우(tallow), 옥수수유, 카놀라유, 카프릭/카프릴릭 트라이글리세라이드, 피마자유, 코코넛유, 면실유, 어유, 호호바유, 라드, 아마인유, 니츠풋(neetsfoot) 오일, 오이티시카(oiticica)유, 야자유, 땅콩유, 평지씨유, 미강유, 잇꽃유, 대두유, 해바라기유, 동유, 자트로파(jatropha)유 및 식물유 블렌드를 포함하지만, 이에 한정되지 않는다.As used herein, "natural oil" refers to a lipid obtained from a plant (eg biomass) or animal. As used herein, "plant derived oil" refers especially to lipids obtained from plants. Sometimes, "lipid" can be used synonymously with "oil" and "acyl glyceride". Natural oils include tallow, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, neatsfoot oil, o It includes, but is not limited to, iticica oil, palm oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tung oil, jatropha oil and vegetable oil blends.

본 명세서에 사용되는 바와 같이, 용어 "분리"는 "회수"와 동의어이며, 초기 혼합물 중 화학적 화합물의 순도 또는 농도보다 더 큰 순도 또는 더 높은 농도의 화합물을 수득하기 위해 초기 혼합물로부터 상기 화합물을 제거하는 것을 말한다.As used herein, the term “separation” is synonymous with “recovery” and removes the compound from the initial mixture to obtain a higher purity or higher concentration of the compound than the purity or concentration of the chemical compound in the initial mixture. I say that.

본 명세서에 사용되는 바와 같이, "재조합 미생물"은 재조합 DNA 기술의 사용에 의해, 예를 들어 숙주 세포를 조작하여 부탄올과 같은 알코올을 생성하는 생합성 경로와 같은 생합성 경로를 포함하게 함으로써 변형된 미생물, 예를 들어 박테리아 또는 효모를 말한다.As used herein, a "recombinant microorganism" refers to a microorganism modified by the use of recombinant DNA technology, for example to include a biosynthetic pathway, such as a biosynthetic pathway that manipulates host cells to produce alcohols such as butanol, Eg bacteria or yeast.

본 발명은 발효 용기 내의 알코올 생성 미생물을, 발효 과정 중 단계에서 바이오매스 지질과 같은 천연 오일로부터 유래된 지방산과 접촉시키는 생성물 알코올 (예를 들어, 발효 알코올)의 생성 방법을 제공한다. 미생물의 발효 성장 동안의 이러한 지방산 보충은 미생물의 발효성 탄소 소비량, 성장 속도, 및 바이오매스 생성을, 특히 피루베이트 데카르복실라아제 활성이 감소되거나 제거된 재조합 미생물에 대하여 증가시킬 수 있다.The present invention provides a method for producing a product alcohol (eg, fermentation alcohol) by contacting an alcohol producing microorganism in a fermentation vessel with a fatty acid derived from a natural oil such as biomass lipids at a stage in the fermentation process. Such fatty acid supplementation during microbial fermentation growth can increase microbial fermentable carbon consumption, growth rate, and biomass production, particularly for recombinant microorganisms with reduced or eliminated pyruvate decarboxylase activity.

일부 실시 형태에서, 오일 중 글리세라이드는 지방산으로 화학적으로 전환될 수 있으며, 상기 지방산은 발효성 탄소 공급원으로부터 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 브로쓰와 접촉된다. 다른 실시 형태에서, 오일 중 글리세라이드는 촉매에 의해 (예를 들어, 효소에 의해) 지방산으로 가수분해될 수 있으며, 상기 지방산은 재조합 미생물을 포함하는 발효 브로쓰와 접촉된다. 일부 실시 형태에서, 지방산은 발효를 위하여 발효성 탄소 공급원을 공급하는 바이오매스에서 발견되는 지질의 가수분해에 의해 수득될 수 있다. 또한 지방산을 ISPR 추출제로 사용하여 발효 브로쓰로부터 생성물 알코올을 제거할 수 있다.In some embodiments, the glycerides in the oil may be chemically converted to fatty acids, which fatty acids are contacted with a fermentation broth comprising recombinant microorganisms that produce product alcohols from fermentable carbon sources. In another embodiment, glycerides in oil may be hydrolyzed to fatty acids by catalyst (eg, by enzymes), which fatty acids are contacted with fermentation broths comprising recombinant microorganisms. In some embodiments, fatty acids may be obtained by hydrolysis of lipids found in biomass that supplies a fermentable carbon source for fermentation. Fatty acids can also be used as ISPR extractants to remove product alcohol from fermentation broth.

지방산은 포화 지방산, 단일불포화 지방산, 다중불포화 지방산 및 그 혼합물일 수 있다. 예를 들어, 팔미트산과 올레산의 혼합물을 포함하는 자연 발생 지방산 조성물을 갖는 오일 (예를 들어, 옥수수유)은 발효 용기 내에서 발효 브로쓰와 접촉될 수 있는 유리 팔미트산과 유리 올레산의 혼합물을 생성하도록 가수분해될 수 있다. 일부 실시 형태에서, 발효 용기 내의 카르복실산 (예를 들어 지방산)의 농도는 수성 상에서의 용해 한도를 넘어서며, 유기 상 및 수성 상을 포함하는 2상 발효 혼합물이 생성되게 한다. 일부 실시 형태에서, 발효 브로쓰 중 카르복실산의 농도는 전형적으로 약 0.8 g/L 이하이며, 브로쓰 중 카르복실산의 용해도에 의해 제한된다.Fatty acids may be saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids and mixtures thereof. For example, an oil (eg corn oil) having a naturally occurring fatty acid composition comprising a mixture of palmitic acid and oleic acid may contain a mixture of free palmitic acid and free oleic acid which may be contacted with fermentation broth in a fermentation vessel. May be hydrolyzed to produce. In some embodiments, the concentration of carboxylic acid (eg fatty acid) in the fermentation vessel is beyond the solubility limit in the aqueous phase, resulting in a two-phase fermentation mixture comprising an organic phase and an aqueous phase. In some embodiments, the concentration of carboxylic acid in the fermentation broth is typically about 0.8 g / L or less and limited by the solubility of the carboxylic acid in the broth.

미생물의 성장 속도 및/또는 발효성 탄소 소비량은 지방산의 존재 하에서, 지방산의 부재 하에서의 미생물의 성장 속도 및 발효성 탄소 소비량보다 더 크다. 이에 상응하여, 본 발명의 방법에 따른 지방산 보충에 의해, 그러한 지방산 보충의 부재 하에서 성취될 수 있는 것보다 증가된 세포 농도 및 증가된 알코올 생성이 성취될 수 있다. 일부 실시 형태에서, 미생물은 부탄올 생성 미생물이거나 또는 전형적으로 생존 및 성장을 위하여 C2 기질, 예를 들어 에탄올의 보충을 필요로 하는 다른 미생물일 수 있다. 그러한 실시 형태에서, 본 발명의 방법에 따른 지방산 보충에 의해 그러한 C2 의존성 미생물이 에탄올 보충의 부재 하에 생존하고 성장하는 것이 허용된다. 일부 실시 형태에서, 미생물은 피루베이트로부터의 아세틸-CoA의 생성에 있어서 결함이 있을 수 있다. 일부 실시 형태에서, 미생물은 지방산 생합성으로의 경로가 변형되도록 하나 이상의 피루베이트 데카르복실라아제(PDC) 유전자 내의 파괴 돌연변이에 의해 대사적으로 조작된다. 일부 실시 형태에서, 미생물은 PDC1 및 PDC5 유전자와 같은 2개의 PDC 유전자 내에서의 파괴 돌연변이에 의해 대사적으로 조작되며, 이는 지방산 생합성으로의 경로가 변경되게 한다. 따라서, 본 발명의 방법은 재조합 미생물의 발효 성장에 최적인 환경을 제공함으로써 향상된 알코올 생산성을 획득할 수 있다.The growth rate and / or fermentable carbon consumption of microorganisms is greater than the growth rate and fermentable carbon consumption of microorganisms in the presence of fatty acids, in the absence of fatty acids. Correspondingly, by fatty acid replenishment according to the methods of the invention, increased cell concentration and increased alcohol production can be achieved than can be achieved in the absence of such fatty acid replenishment. In some embodiments, the microorganism may be a butanol producing microorganism or other microorganisms that typically require supplementation of a C2 substrate, such as ethanol, for survival and growth. In such embodiments, fatty acid supplementation according to the methods of the present invention allows such C2 dependent microorganisms to survive and grow in the absence of ethanol supplementation. In some embodiments, the microorganism may be defective in the production of acetyl-CoA from pyruvate. In some embodiments, the microorganism is metabolically manipulated by disruptive mutations in one or more pyruvate decarboxylase (PDC) genes so that the pathway to fatty acid biosynthesis is modified. In some embodiments, the microorganism is metabolized by disruptive mutations in two PDC genes, such as the PDC1 and PDC5 genes, which causes the pathway to fatty acid biosynthesis to be altered. Thus, the method of the present invention can obtain improved alcohol productivity by providing an environment that is optimal for fermentation growth of recombinant microorganisms.

본 발명을 도면을 참고로 하여 설명할 것이다. 도 1은 본 발명의 실시 형태에 따른 발효 알코올의 생성을 위한 예시적인 공정 흐름도를 도시한다. 나타낸 바와 같이, 공급재료(12)는 액화 용기(10) 내의 유입구에 도입되고 액화되어 공급재료 슬러리(16)를 생성할 수 있다. 공급재료(12)는 발효성 탄소 공급원 (예를 들어, 발효성 당, 예컨대 글루코스)을 공급하는 가수분해성 전분을 함유하며, 호밀, 밀, 옥수수, 사탕수수, 보리, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 또는 그 혼합물과 같은 그러나 이에 한정되지 않는 바이오매스일 수 있거나, 또는 달리 바이오매스로부터 유래될 수 있다. 일부 실시 형태에서, 공급재료(12)는 분획화된 바이오매스의 하나 이상의 성분일 수 있으며, 다른 실시 형태에서, 공급재료(12)는 제분된 비분획화 바이오매스일 수 있다. 일부 실시 형태에서, 공급재료(12)는 옥수수, 예를 들어 건식 제분된 비분획화 옥수수알일 수 있으며, 비용해 고형물은 배아, 섬유 및 글루텐을 포함할 수 있다. 비용해 고형물은 공급재료(12)의 비발효성 부분이다. 도면에 나타낸 실시 형태들을 참고로 한 본 발명에서의 논의 목적을 위하여, 공급재료(12)는 흔히 제분된 비분획화 구성 옥수수로 기재될 것이며, 여기서 비용해 고형물은 그로부터 분리되지 않았다. 그러나, 본 명세서에 개시된 예시적인 방법 및 시스템은, 당업자에게 명백한 바와 같이, 분획화되든지 그렇지 않든지 간에 상이한 공급재료들을 위하여 변경될 수 있다. 일부 실시 형태에서, 공급재료(12)는 고 올레익 옥수수일 수 있어서, 그로부터 유래된 옥수수유는 올레산 함량이 약 55 중량% 이상의 올레산인 고 올레익 옥수수유가 된다. 일부 실시 형태에서, 고 올레익 옥수수유 중 올레산 함량은 약 24 중량%인 보통 옥수수유 중 올레산 함량과 비교하여 최대 약 65 중량%일 수 있다. 고 올레익 오일은 본 발명의 방법에서 사용함에 있어서 일부 이점을 제공할 수 있으며, 그 이유는 상기 오일의 가수분해는 발효 브로쓰와의 접촉을 위한, 올레산 함량이 높은 유리 지방산을 제공하기 때문이다. 일부 실시 형태에서, 지방산 또는 그 혼합물은 불포화 지방산을 포함한다. 불포화 지방산의 존재는 융점을 감소시켜 취급에 있어서 이점을 제공한다. 불포화 지방산들 중, 단일불포화된 것들, 즉, 단일 탄소-탄소 이중 결합을 보유하는 것들은 공정 상의 고려 사항에 적합한 열 및 산화 안정성을 희생시키지 않고서 융점과 관련하여 이점을 제공할 수 있다.The present invention will be explained with reference to the drawings. 1 shows an exemplary process flow diagram for the production of fermented alcohol according to an embodiment of the invention. As shown, feedstock 12 may be introduced and liquefied into an inlet in liquefaction vessel 10 to produce feedstock slurry 16. Feedstock 12 contains hydrolyzable starch that supplies a fermentable carbon source (eg, fermentable sugars such as glucose), rye, wheat, corn, sugar cane, barley, cellulosic material, lignocellulosic Or may be derived from biomass, such as, but not limited to, based materials, or mixtures thereof. In some embodiments, feedstock 12 may be one or more components of fractionated biomass, and in other embodiments, feedstock 12 may be milled unfractionated biomass. In some embodiments, feedstock 12 may be corn, for example dry milled unfractionated corn grains, and costly solids may include embryos, fibers, and gluten. Inexpensive solids are the non-fermentable part of the feed material 12. For purposes of discussion in the present invention with reference to the embodiments shown in the figures, feedstock 12 will often be described as milled, unfractionated construct corn, where no solids have been separated therefrom. However, the exemplary methods and systems disclosed herein may be modified for different feeds, whether or not fractionated, as will be apparent to those skilled in the art. In some embodiments, feedstock 12 may be high oleic corn such that the corn oil derived therefrom is high oleic corn oil having an oleic acid content of at least about 55% by weight oleic acid. In some embodiments, the oleic acid content in high oleic corn oil may be up to about 65 weight percent compared to the oleic acid content in normal corn oil, which is about 24 weight percent. High oleic oils can provide some advantages in use in the process of the present invention, since the hydrolysis of the oils provides free fatty acids with high oleic acid content for contact with the fermentation broth. . In some embodiments, the fatty acid or mixture thereof comprises unsaturated fatty acid. The presence of unsaturated fatty acids reduces the melting point and provides an advantage in handling. Among unsaturated fatty acids, monounsaturated ones, that is, those possessing a single carbon-carbon double bond, can provide advantages in terms of melting point without sacrificing thermal and oxidative stability suitable for process considerations.

공급재료(12)의 액화 공정은 공급재료(12) 중 다당류를 예를 들어 덱스트린 및 올리고당류를 포함하는 당으로 가수분해시키는 것을 포함하며, 통상적인 공정이다. 임의의 공지된 액화 공정과, 상응하는 액화 용기 - 산업계에 의해 보통 이용됨 - 가 사용될 수 있으며, 이는 산 공정, 산-효소 공정 또는 효소 공정을 포함하지만 이에 한정되지 않는다. 그러한 공정들은 단독으로 또는 조합되어 사용될 수 있다. 일부 실시 형태에서, 효소 공정이 이용될 수 있으며, 적절한 효소(14), 예를 들어, 알파-아밀라아제가 액화 용기(10) 내의 유입구에 도입된다. 또한 물이 액화 용기(10)에 도입될 수 있다. 일부 실시 형태에서, 당화 효소, 예를 들어, 글루코아밀라아제가 액화 용기(10)에 또한 도입될 수 있다. 추가의 실시 형태에서, 리파아제를 액화 용기(10)에 또한 도입하여 오일의 하나 이상의 성분의 유리 지방산으로의 전환을 촉매할 수 있다.The liquefaction process of feedstock 12 involves the hydrolysis of polysaccharides in feedstock 12 into sugars including, for example, dextrins and oligosaccharides, and is a common process. Any known liquefaction process and the corresponding liquefaction vessel, commonly used by industry, may be used, including but not limited to acid processes, acid-enzyme processes or enzyme processes. Such processes may be used alone or in combination. In some embodiments, an enzymatic process may be used, and an appropriate enzyme 14, for example alpha-amylase, is introduced at the inlet in the liquefaction vessel 10. Water may also be introduced into the liquefaction vessel 10. In some embodiments, glycosylating enzymes, such as glucoamylases, may also be introduced into the liquefaction vessel 10. In further embodiments, lipases may also be introduced into the liquefaction vessel 10 to catalyze the conversion of one or more components of the oil to free fatty acids.

공급재료(12)의 액화에 의해 생성된 공급재료 슬러리(16)는 당, 오일(26),및 공급재료(12)를 형성하는 바이오매스로부터 유래되는 비용해 고형물을 포함한다. 일부 실시 형태에서, 오일은 발효 브로쓰 조성물의 약 0 중량% 내지 적어도 약 2 중량%의 양으로 있다. 일부 실시 형태에서, 오일은 공급재료의 0.5 중량% 이상의 양으로 있다. 공급재료 슬러리(16)는 액화 용기(10)의 유출구로부터 배출될 수 있다. 일부 실시 형태에서, 공급재료(12)는 옥수수 또는 옥수수알이며, 공급재료 슬러리(16)는 옥수수 매시 슬러리이다.Feedstock slurry 16 produced by liquefaction of feedstock 12 includes sugars, oils 26, and undissolved solids derived from the biomass forming feedstock 12. In some embodiments, the oil is in an amount from about 0% to at least about 2% by weight of the fermentation broth composition. In some embodiments, the oil is in an amount of at least 0.5% by weight of the feedstock. Feedstock slurry 16 may be discharged from the outlet of liquefaction vessel 10. In some embodiments, feedstock 12 is corn or corn grains, and feedstock slurry 16 is a corn mash slurry.

하나 이상의 물질(42)이 공급재료 슬러리(16)에 첨가될 수 있다. 물질(42)은 오일(26) 중 글리세라이드를 유리 지방산(FFA; 28)으로 가수분해시킬 수 있다. 예를 들어, 공급재료(12)가 옥수수이면, 오일(26)은 공급재료 구성 옥수수유이며, 유리 지방산(28)은 옥수수유 지방산(corn oil fatty acid; COFA)이다. 따라서, 공급재료 슬러리(16)에 물질(42)을 도입한 후, 오일(26) 중 글리세라이드의 적어도 일부분은 FFA(28)로 가수분해되어 FFA(28)을 갖는 공급재료 슬러리(18)를 생성한다.One or more materials 42 may be added to the feed slurry 16. Material 42 may hydrolyze glycerides in oil 26 into free fatty acids (FFA) 28. For example, if feedstock 12 is corn, oil 26 is feedstock constituent corn oil and free fatty acid 28 is corn oil fatty acid (COFA). Thus, after introducing the material 42 into the feedstock slurry 16, at least a portion of the glycerides in the oil 26 are hydrolyzed to the FFA 28 to feed the feedstock slurry 18 with the FFA 28. Create

일부 실시 형태에서, 하나 이상의 물질(42)은 오일(26) 중 글리세라이드를 유리 지방산(28)(FFA)으로 촉매에 의해 가수분해시킬 수 있는 하나 이상의 촉매(42)이다. 따라서, 공급재료 슬러리(16)에 촉매(42)를 도입한 후, 오일(26) 중 글리세라이드의 적어도 일부분은 FFA(28)로 가수분해되어 FFA(28) 및 촉매(42)를 갖는 공급재료 슬러리(18)를 생성한다.In some embodiments, the one or more materials 42 are one or more catalysts 42 capable of catalyzing the glycerides in the oil 26 to free fatty acids 28 (FFA). Thus, after introducing the catalyst 42 into the feedstock slurry 16, at least a portion of the glycerides in the oil 26 are hydrolyzed to the FFA 28 to feed the feedstock with the FFA 28 and the catalyst 42. Slurry 18 is produced.

오일(26)의 가수분해에 의한 생성된 산/오일 조성물은 전형적으로 약 17 중량% 이상의 FFA이다. 일부 실시 형태에서, 오일(26)의 가수분해에 의한 생성된 산/오일 조성물은 20 중량% 이상의 FFA, 약 25 중량% 이상의 FFA, 약 30 중량% 이상의 FFA, 약 35 중량% 이상의 FFA, 약 40 중량% 이상의 FFA, 약 45 중량% 이상의 FFA, 약 50 중량% 이상의 FFA, 약 55 중량% 이상의 FFA, 약 60 중량% 이상의 FFA, 약 65 중량% 이상의 FFA, 약 70 중량% 이상의 FFA, 약 75 중량% 이상의 FFA, 약 80 중량% 이상의 FFA, 약 85 중량% 이상의 FFA, 약 90 중량% 이상의 FFA, 약 95 중량% 이상의 FFA, 또는 약 99 중량% 이상의 FFA이다.The acid / oil composition produced by hydrolysis of oil 26 is typically at least about 17% by weight of FFA. In some embodiments, the resulting acid / oil composition by hydrolysis of oil 26 is at least 20 wt% FFA, at least about 25 wt% FFA, at least about 30 wt% FFA, at least about 35 wt% FFA, about 40 At least about 50% by weight, at least about 45% by weight of FFA, at least about 50% by weight of FFA, at least about 55% by weight of FFA, at least about 60% by weight of FFA, at least about 65% by weight of FFA, at least about 70% by weight of FFA At least about FFA, at least about 80 wt% FFA, at least about 85 wt% FFA, at least about 90 wt% FFA, at least about 95 wt% FFA, or at least about 99 wt% FFA.

대안적으로, 일부 실시 형태에서, 물질(들)(42)은 재조합 미생물(32)과 접촉시키기 위한 FFA(28)가 되도록 오일(26)과 화학 반응할 수 있는 하나 이상의 반응물 또는 용매를 대안적으로 구성할 수 있다. 예를 들어, 옥수수유 지방산은 물질(42)로서의 물 및 NaOH를 사용한 염기 가수분해에 의해 오일(26)로서의 옥수수유로부터 합성될 수 있으며, 이는 본 출원과 공계류 중인, 공히 소유된, 2010년 7월 28일자로 출원되고 전체적으로 본 명세서에 참고로 포함된 미국 가특허 출원 제61/368,436호에 추가로 기재된 바와 같다. 또한, 예를 들어 오일(26)로서의 옥수수유 트라이글리세라이드를 반응물(42)로서의 수성 수산화암모늄과 반응시켜 지방산 (및 지방 아미드)을 수득할 수 있으며, 이는 전체적으로 본 명세서에 참고로 포함된 문헌[Roe, et al., Am. Oil Chem. Soc. 29:18-22, 1952]에 추가로 기재된 바와 같다. 도면에 나타낸 실시 형태들을 참고로 하여 본 발명에서 논의하고자 하는 목적을 위하여, 물질(들)(42)은 흔히 재조합 미생물(32)의 발효 성장 동안 보충되는 FFA(28)로의 바이오매스 지질의 가수분해를 위한 물질(들)(42)로서 하나 이상의 촉매를 구성하는 것으로 기재될 것이다. 그러나, 본 명세서에 개시된 예시적인 방법 및 시스템은 물질(들)(42)이 바이오매스 지질을 FFA(28)로 화학적으로 전환시킬 수 있는 반응물(들) 및/또는 용매(들)가 되도록 변경될 수 있음이 이해되어야 한다.Alternatively, in some embodiments, the substance (s) 42 may alternatively comprise one or more reactants or solvents capable of chemically reacting with the oil 26 to be the FFA 28 for contacting the recombinant microorganism 32. It can be configured as. For example, corn oil fatty acid can be synthesized from corn oil as oil 26 by base hydrolysis with water as material 42 and NaOH, which was owned in 2010, co-pending with the present application. As further described in US Provisional Patent Application 61 / 368,436, filed on July 28 and incorporated herein by reference in its entirety. Also, for example, corn oil triglycerides as oil 26 may be reacted with aqueous ammonium hydroxide as reactant 42 to obtain fatty acids (and fatty amides), which are incorporated by reference in their entirety. Roe, et al., Am. Oil Chem. Soc. 29: 18-22, 1952. For the purpose of discussing the present invention with reference to the embodiments shown in the figures, the substance (s) 42 is often hydrolyzed of biomass lipids to FFA 28 which is supplemented during fermentation growth of recombinant microorganisms 32. It will be described as constituting one or more catalysts as material (s) 42 for. However, the exemplary methods and systems disclosed herein may be modified such that the substance (s) 42 are reactant (s) and / or solvent (s) capable of chemically converting biomass lipids to FFA 28. It should be understood that it can.

일부 실시 형태에서, 촉매(42)는 하나 이상의 효소, 예를 들어 하이드롤라아제 효소, 예컨대 리파아제 효소일 수 있다. 사용되는 리파아제 효소는 예를 들어 압시디아(Absidia), 아크로모박터(Achromobacter), 아에로모나스(Aeromonas), 알칼리게네스(Alcaligenes), 알테르나리아(Alternaria), 아스페르길루스(Aspergillus), 아크로모박터, 아우레오바시디움(Aureobasidium), 바실루스(Bacillus), 뷰베리아(Beauveria), 브로코트릭스(Brochothrix), 칸디다(Candida), 크로모박터(Chromobacter), 코프리누스(Coprinus), 푸사륨(Fusarium), 게오트리쿰(Geotricum), 한세눌라(Hansenula), 후미콜라(Humicola), 하이포자이마(Hyphozyma), 락토바실루스(Lactobacillus), 메타리지움(Metarhizium), 뮤코르(Mucor), 넥트리아(Nectria), 뉴로스포라(Neurospora), 파에실로마이세스(Paecilomyces), 페니실륨(Penicillium), 슈도모나스(Pseudomonas), 리족토니아(Rhizoctonia), 리조뮤코르(Rhizomucor), 리조푸스(Rhizopus), 로도스포리듐(Rhodosporidium), 로도토룰라(Rhodotorula), 사카로마이세스(Saccharomyces), 수스(Sus), 스포로볼로마이세스(Sporobolomyces), 서모마이세스(Thermomyces), 티아로스포렐라(Thiarosporella), 트리코더마(Trichoderma), 베르티실륨(Verticillium), 및/또는 야로위아(Yarrowia)의 스트레인을 포함하는 임의의 공급원으로부터 유래될 수 있다. 바람직한 태양에서, 리파아제 공급원은 압시디아 블라케슬레에나(Absidia blakesleena), 압시디아 코라임비페라(Absidia corymbifera), 아크로모박터 이오파구스(Achromobacter iophagus), 알칼리게네스 sp., 알테르나리아 브라시시올라(Alternaria brassiciola), 아스페르길루스 플라부스(Aspergillus flavus), 아스페르길루스 니게르(Aspergillus niger), 아우레오바시디움 풀룰란스(Aureobasidium pullulans), 바실루스 퓨밀루스(Bacillus pumilus), 바실루스 스테아로서모필루스(Bacillus strearothermophilus), 바실루스 섭틸리스(Bacillus subtilis), 브로코트릭스 서모소하타(Brochothrix thermosohata), 칸디다 사일린드라세아(Candida cylindracea) (칸디다 루고사(Candida rugosa)), 칸디다 파라리포라이티카(Candida paralipolytica), 칸디다 안타크티카(Candida Antarctica) 리파아제 A, 칸디다 안타르티카(Candida antartica) 리파아제 B, 칸디다 에르노비이(Candida ernobii), 칸디다 데포르만스(Candida deformans), 크로모박터 비스코숨(Chromobacter viscosum), 코프리누스 시네리우스(Coprinus cinerius), 푸사륨 옥시스포룸(Fusarium oxysporum), 푸사륨 솔라니(Fusarium solani), 푸사륨 솔라니 피시(Fusarium solani pisi), 푸사륨 로세움 쿨모룸(Fusarium roseum culmorum), 게오트리쿰 페니실라툼(Geotricum penicillatum), 한세눌라 아노말라(Hansenula anomala), 후미콜라 브레비스포라(Humicola brevispora), 후미콜라 브레비스(Humicola brevis) var. 서모이데아(thermoidea), 후미콜라 인솔렌스(Humicola insolens), 락토바실루스 쿠르바투스(Lactobacillus curvatus), 리조푸스 오리자에(Rhizopus oryzae), 페니실륨 사이클로퓸(Penicillium cyclopium), 페니실륨 크루스토숨(Penicillium crustosum), 페니실륨 엑스판숨(Penicillium expansum), 페니실륨 sp. I, 페니실륨 sp. II, 슈도모나스 아에루기노사(Pseudomonas aeruginosa), 슈도모나스 알칼리게네스(Pseudomonas alcaligenes), 슈도모나스 세파시아(Pseudomonas cepacia) (동의어: 부르크홀데리아 세파시아(Burkholderia cepacia)), 슈도모나스 플루오레센스(Pseudomonas fluorescens), 슈도모나스 프라기(Pseudomonas fragi), 슈도모나스 말토필리아(Pseudomonas maltophilia), 슈도모나스 멘도시나(Pseudomonas mendocina), 슈도모나스 메피티카 리포라이티카(Pseudomonas mephitica lipolytica), 슈도모나스 알칼리게네스, 슈도모나스 플란타리(Pseudomonas plantari), 슈도모나스 슈도알칼리게네스(Pseudomonas pseudoalcaligenes), 슈도모나스 퓨티다(Pseudomonas putida), 슈도모나스 스투체리(Pseudomonas stutzeri), 및 슈도모나스 위스콘시넨시스(Pseudomonas wisconsinensis), 리족토니아 솔라니(Rhizoctonia solani), 리조뮤코르 미에헤이(Rhizomucor miehei), 리조푸스 자포니쿠스(Rhizopus japonicus), 리조푸스 미크로스포루스(Rhizopus microsporus), 리조푸스 노도수스(Rhizopus nodosus), 로도스포리듐 토룰로이데스(Rhodosporidium toruloides), 로도토룰라 글루티니스(Rhodotorula glutinis), 사카로마이세스 세레비지애, 스포로볼로마이세스 쉬바타누스(Sporobolomyces shibatanus), 수스 스크로파(Sus scrofa), 서모마이세스 라누기노수스(Thermomyces lanuginosus; 이전에는 후미콜라 라누기노세(Humicola lanuginose)), 티아로스포렐라 파세올리나(Thiarosporella phaseolina), 트리코더마 하르지아눔(Trichoderma harzianum), 트리코더마 레에세이(Trichoderma reesei), 및 야로위아 리포라이티카(Yarrowia lipolytica)로 이루어진 군으로부터 선택된다. 추가의 바람직한 태양에서, 리파아제는 서몸사이세스 라누기노수스(Thermomcyces lanuginosus), 아스페르길루스 sp. 리파아제, 아스페르길루스 니게르 리파아제, 칸디다 안타르티카 리파아제 B, 슈도모나스 sp. 리파아제, 페니실륨 로쿠에포르티(Penicillium roqueforti) 리파아제, 페니실륨 카멤베르티이(Penicillium camembertii) 리파아제, 뮤코르 자바니쿠스(Mucor javanicus) 리파아제, 부르크홀데리아 세파시아 리파아제, 알칼리게네스 sp. 리파아제, 칸디다 루고사 리파아제, 칸디다 파라프실로시스(Candida parapsilosis) 리파아제, 칸디다 데포르만스 리파아제, 게오트리쿰 칸디둠(Geotrichum candidum) 유래의 리파아제 A 및 B, 뉴로스포라 크라사(Neurospora crassa) 리파아제, 넥트리아 헤마토코카(Nectria haematococca) 리파아제, 푸사륨 헤테로스포룸(Fusarium heterosporum) 리파아제, 리조푸스 델레마르(Rhizopus delemar) 리파아제, 리조뮤코르 미에헤이(Rhizomucor miehei) 리파아제, 리조푸스 아리주스(Rhizopus arrhizus) 리파아제, 및 리조푸스 오리자에 리파아제로 이루어진 군으로부터 선택된다. 효소 촉매(42)로 적합한, 상업적인 적합한 리파아제 제제는 리폴라아제(Lipolase)(등록상표) 100 L, 리펙스(Lipex)(등록상표) 100L, 리포클린(Lipoclean)(등록상표) 2000T, 리포자임(Lipozyme)(등록상표) CALB L, 노보자임(Novozym)(등록상표) CALA L, 및 팔라타아제(Palatase) 20000L - 노보자임즈(Novozymes)로부터 입수가능함 - , 또는 슈도모나스 플루오레센스, 슈도모나스 세파시아, 뮤코르 미에헤이, 비육돈 췌장, 칸디다 사일린드라세아, 리조푸스 니베우스(Rhizopus niveus), 칸디다 안타크티카, 리조푸스 아리주스 또는 아스페르길루스 유래의 것 - 시그마알드리치(SigmaAldrich)로부터 입수가능함 - 을 포함하지만, 이에 한정되지 않는다.In some embodiments, catalyst 42 may be one or more enzymes, such as hydrolase enzymes such as lipase enzymes. Lipase enzymes used are, for example, Absidia , Achromobacter , Aeromonas , Alcaligenes , Alternaria , Aspergillus , Achromobacter, brother Leofoo saryum Bridge Stadium (Aureobasidium), Bacillus (Bacillus), view Beria (Beauveria), Brocco matrix (Brochothrix), candidiasis (Candida), chromotherapy bakteo (Chromobacter), Coffs Linus (Coprinus), ( Fusarium , Geotricum , Hansenula , Humicola , Hyphozyma , Lactobacillus , Metarhizium , Mucor , and Nectria (Nectria), Neuro spokes La (Neurospora), par indeed My process (Paecilomyces), Penny silryum (Penicillium), Pseudomonas (Pseudomonas), Li Estonian (Rhizoctonia), Rhizopus Mu cor (Rhizomucor), Rhizopus crispus (Rhizopus), also Rhodosporidium , Also torulra (Rhodotorula), Saka in my process (Saccharomyces), Versus (Sus), as MY Seth (Sporobolomyces), Thermo Mai Seth (Thermomyces), Tia ball to Spokane Spokane Relais (Thiarosporella), Trichoderma (Trichoderma), Berti It may be derived from any source, including strains of Verticillium , and / or Yarrowia . In a preferred embodiment, the lipase source is Absidia blakesleena ), Absidia corymbifera , Achromobacter iophagus ), alkaligenes sp., Alternaria brassiciola), Aspergillus Playa booth (Aspergillus flavus ), Aspergillus niger niger ), Aureobasidium pullulans), Bacillus pyumil Ruth (Bacillus pumilus), as a brush loose Bacillus stearate (Bacillus strearothermophilus ), Bacillus subtilis subtilis), Thermo Matrix Brocco Soha other (Brochothrix thermosohata), Candida four days Lindlar Seah (Candida cylindracea) (Lu Candida Test (Candida rugosa )), Candida paralipolytica ( Candida paralipolytica), Candida hits the greater Utica (Candida Antarctica) lipase A, Candida not tar urticae (Candida antartica) lipase B, Candida El nobiyi (Candida Only ernobii), Candida to Fortis's (Candida deformans ), Chromobacter viscosum ), Coprinus cinerius ), Fusarium oxysporum ), Fusarium soni solani ), Fusarium solani pisi ), Fusarium Roseum Kummorum roseum culmorum ), Geotricum penicillatum , Hansenula annomala anomala ), Humicola brevispora), trailing Coke brevis (Humicola brevis ) var. Thermoidea , Humicola insolens ), Lactobacillus curvatus ), Rizopus orizae ( Rhizopus oryzae ), Penicillium cyclopium cyclopium), Penny silryum Crewe Stoke breath (Penicillium crustosum ), Penicillium expansum ), penicillium sp. I, penicillium sp. II, Pseudomonas Pseudomonas aeruginosa ), Pseudomonas alcaligenes ), Pseudomonas cepacia ) (synonym: Burkholderia cepacia)), Pseudomonas fluorescein sense (Pseudomonas fluorescens ), Pseudomonas fragi ), Pseudomonas maltophilia), Pseudomonas Men City or (Pseudomonas mendocina), Pseudomonas mepi urticae lipoic Lai urticae (Pseudomonas mephitica lipolytica), Pseudomonas Alcaligenes, Pseudomonas flange Tari (Pseudomonas plantari), Pseudomonas Pseudomonas Alcaligenes (Pseudomonas pseudoalcaligenes ), Pseudomonas putida ), Pseudomonas stutzeri ), and Pseudomonas wisconsinensis ), Rhizoctonia solani ), Rhizomucor Miehei , Rhizopus japonicus ), Rhizopus microsporus ), Rhizopus nodosus ), Rhodosporidium toruloides , Rhodotorula glutinis ), Saccharomyces cerevisiae, Sporobolomyces shibatanus shibatanus), wave to Sousse disk (Sus scrofa ), Thermomyces ranuginosus ( Thermomyces lanuginosus ; Formerly Humicola Ranuginose lanuginose )), Thiarosporella phaseolina ), Trichoderma harzianum ), Trichoderma reesei ), and Yarrowia lipolytica . In a further preferred embodiment, the lipase is thermomcyces lanuginosus ), Aspergillus sp. Lipase, aspergillus niger lipase, Candida antartica lipase B, Pseudomonas sp. Lipase, Penicillium, Penicillium roqueforti) lipase, Penny Vere silryum kamem Chantilly (Penicillium camembertii ) lipase, Mucor Javanicus javanicus ) lipase, burkholderia sefacia lipase, alkaligenes sp. Lipase, Candida Lugosa Lipase, Candida parapsilosis Lipase, Candida Deformans Lipase, Geotricum Candidum lipases A and B from candidum ), Neurospora crassa lipase, Nectria hematococa ( Nectria) haematococca ) lipase, Fusarium heterosporum heterosporum) lipase, Rizzo crispus Delle Mar (Rhizopus delemar) lipase, Rizzo Mu cor Mie Hay (Rhizomucor miehei ) lipase, Rhizopus arrhizus ) lipase, and Rizopus orizae lipase. Commercially suitable lipase formulations suitable as enzyme catalysts 42 include Lipolase® 100 L, Lipex® 100L, Lipoclean® 2000T, lipozyme Lipozyme® CALB L, Novozym® CALA L, and Palatase 20000L-available from Novozymes-or Pseudomonas fluorescens, Pseudomonas sefacia From Mucor Miehei, finishing pig pancreas, Candida silindrasea, Rhizopus niveus, Candida antarctica, lycopus arizus or aspergillus-available from SigmaAldrich -Including, but not limited to.

글리세라이드의 적어도 일부분을 가수분해시킨 후, 일부 실시 형태에서, 촉매(42)는 불활성화될 수 있다. 당업계에 공지된 임의의 방법을 이용하여 촉매(42)가 불활성화되게 할 수 있다. 예를 들어, 일부 실시 형태에서, 촉매(42)는 열의 인가에 의해, 및/또는 반응 매스의 pH를, 촉매(42)가 비가역적으로 불활성화되는 pH로 조정함으로써, 및/또는 촉매 활성을 선택적으로 불활성화시킬 수 있는 화학종 또는 생화학종을 첨가함으로써 불활성화될 수 있다. 예를 들어 도 1의 실시 형태에 나타낸 바와 같이, 열(q)은 공급재료 슬러리(18)에 인가되며 이에 의해 촉매(42)는 불활성화되게 된다. 열(q)의 인가는 공급재료 슬러리(18)가 발효 용기(30)에 공급되기 전에 공급재료 슬러리(18)에 적용될 수 있다. 그 후, 열처리된 공급재료 슬러리(18) (불활성 촉매(42)를 포함함)는 발효 용기(30) 내에 담기는 발효 브로쓰에 포함시킬 미생물(32)과 함께 발효 용기(30) 내로 도입된다. 대안적으로, 공급재료 슬러리(18)는 발효 용기(30)에 공급되며, 미생물(32)의 발효 용기 접종 전에 발효 용기 내에 있는 동안 열(q)이 가해질 수 있다. 예를 들어, 일부 실시 형태에서, 촉매 불활성화 처리는 공급재료 슬러리(18)를 열(q)을 이용하여 약 75℃ 이상의 온도로 약 5분 이상 동안, 약 75℃ 이상의 온도로 약 10분 이상 동안, 약 75℃ 이상의 온도로 약 15분 이상 동안, 약 80℃ 이상의 온도로 약 5분 이상 동안, 약 80℃ 이상의 온도로 약 10분 이상 동안, 약 80℃ 이상의 온도로 약 15분 이상 동안, 약 85℃ 이상의 온도로 약 5분 이상 동안, 약 85℃ 이상의 온도로 약 10분 이상 동안, 또는 약 85℃ 이상의 온도로 약 15분 이상 동안 가열함으로써 성취될 수 있다. 일부 실시 형태에서, 열(q)이 가해진 후, 공급재료 슬러리(18)는 발효 용기(30)에 도입되기 이전에 (또는 열(q)의 인가가 발효 용기에서 행해지는 경우 발효 용기 접종 이전에) 발효에 적절한 온도로 냉각된다. 예를 들어, 일부 실시 형태에서, 공급재료 슬러리(18)의 온도는 발효 브로쓰와 접촉하기 이전에는 약 30℃이다.After hydrolyzing at least a portion of the glycerides, in some embodiments, the catalyst 42 may be inactivated. Any method known in the art can be used to cause the catalyst 42 to be inactivated. For example, in some embodiments, the catalyst 42 may be modified by the application of heat and / or by adjusting the pH of the reaction mass to a pH at which the catalyst 42 is irreversibly inactivated, and / or It may be inactivated by adding chemical or biochemical species that can be selectively inactivated. For example, as shown in the embodiment of FIG. 1, heat q is applied to feedstock slurry 18, which causes catalyst 42 to be inactivated. Application of heat q may be applied to feedstock slurry 18 before feedstock slurry 18 is fed to fermentation vessel 30. Thereafter, the heat-treated feedstock slurry 18 (including the inert catalyst 42) is introduced into the fermentation vessel 30 together with the microorganisms 32 to be included in the fermentation broth contained in the fermentation vessel 30. . Alternatively, feedstock slurry 18 is supplied to fermentation vessel 30 and heat q may be applied while in fermentation vessel prior to inoculation of the fermentation vessel of microorganism 32. For example, in some embodiments, the catalyst deactivation process causes feedstock slurry 18 to utilize heat (q) for at least about 5 minutes at a temperature of at least about 75 ° C, at least about 10 minutes at a temperature of at least about 75 ° C. For at least about 15 minutes at a temperature of at least about 75 ° C., at least about 5 minutes at a temperature of at least about 80 ° C., at least about 10 minutes at a temperature of at least about 80 ° C., for at least about 15 minutes at a temperature of at least about 80 ° C., By heating at a temperature of at least about 85 ° C. for at least about 5 minutes, at a temperature of at least about 85 ° C. for at least about 10 minutes, or at a temperature of at least about 85 ° C. for at least about 15 minutes. In some embodiments, after the heat q is applied, the feedstock slurry 18 is not introduced into the fermentation vessel 30 (or before fermentation vessel inoculation if the application of heat q is done in the fermentation vessel). ) Cooled to a temperature suitable for fermentation. For example, in some embodiments, the temperature of feedstock slurry 18 is about 30 ° C. before contacting the fermentation broth.

발효 용기 내에서 촉매(42)가 지방산(28)에 의한 알코올의 에스테르화를 하지 못하게 하는 것이 바람직할 때 촉매(42)의 불활성화가 바람직하다. 일부 실시 형태에서, 유기 산 (예를 들어, 지방산) 및 촉매 (예를 들어, 리파아제)에 의한 발효 배지에서의 생성물 알코올의 에스테르화에 의한 알코올 에스테르의 생성이 바람직하며, 이는 2010년 7월 28일자로 출원된, 본 출원과 공계류 중인, 공히 소유된 미국 특허 출원 제61/368,429호; 2010년 9월 2일자로 출원된 미국 가특허 출원 제61/379,546호; 및 2011년 2월 7일자로 출원된 미국 가특허 출원 제61/440,034호에 추가로 기재된 바와 같은데, 이들 전부는 전체적으로 본 명세서에 참고로 포함된다. 예를 들어, 부탄올 생성에 있어서, 발효 용기 내의 활성 촉매(42)(슬러리(18)를 통하여 도입됨)는 원위치에서 지방산 부틸 에스테르(fatty acid butyl ester; FABE)가 형성되도록 지방산(28) (슬러리(18)를 통하여 도입됨)에 의한 부탄올의 에스테르화를 촉매할 수 있다. 지방산의 알코올 에스테르가 바람직한 그러한 실시 형태에서, 본 명세서에 개시된 방법은 생성물 알코올을 포함하는 발효 브로쓰와의 접촉 이전에 촉매(42)를 불활성화시키는 것이 제외되도록 변경될 수 있다. 따라서, 도 1 내지 도 5의 예시적인 공정 흐름도를 참조하면, 이들 대안적인 실시 형태들은 촉매(42)가 발효 용기(30) 내에서 지방산(28)을 이용하여 생성물 알코올을 에스테르화하도록 촉매(42)를 함유하는 공정 스트림에의 열(q)의 인가를 제외함으로써 성취될 수 있다. 게다가, 일부 실시 형태에서, 도 1 내지 도 5의 예시적인 공정 흐름도는 촉매(42) 대신 물질(들)(42)로서 하나 이상의 반응물 또는 용매를 사용하여 오일(26)을 FFA(28)로 화학적으로 전환시키는 데 있어서 필요하지 않을 경우 열(q)이 제외되도록 변경될 수 있다.Deactivation of the catalyst 42 is preferred when it is desirable to prevent the catalyst 42 from esterifying the alcohol by the fatty acids 28 in the fermentation vessel. In some embodiments, the production of alcohol esters by esterification of product alcohols in fermentation medium with organic acids (eg fatty acids) and catalysts (eg lipases) is preferred, which is July 28, 2010. Commonly owned US patent application Ser. No. 61 / 368,429, filed co-pending with the present application; United States Provisional Patent Application 61 / 379,546, filed September 2, 2010; And US Provisional Patent Application 61 / 440,034, filed February 7, 2011, all of which are incorporated herein by reference in their entirety. For example, in the production of butanol, the active catalyst 42 (introduced through slurry 18) in the fermentation vessel is such that fatty acid 28 (slurry) is formed so that fatty acid butyl ester (FABE) is formed in situ. Esterification of butanol) (introduced via (18)). In those embodiments in which alcohol esters of fatty acids are preferred, the method disclosed herein can be modified to exclude inactivation of catalyst 42 prior to contact with fermentation broth comprising product alcohol. Thus, referring to the exemplary process flow diagrams of FIGS. 1-5, these alternative embodiments provide a catalyst 42 for the catalyst 42 to esterify the product alcohol with fatty acids 28 in the fermentation vessel 30. Can be achieved by excluding the application of heat q to the process stream containing c). In addition, in some embodiments, the exemplary process flow diagrams of FIGS. 1-5 are used to convert oil 26 to FFA 28 using one or more reactants or solvents as material (s) 42 instead of catalyst 42. The column q can be changed to exclude if it is not necessary to switch to.

발효 용기(30)는 슬러리(18)를 발효시켜 부탄올과 같은 생성물 알코올을 생성하도록 구성된다. 특히, 미생물(32)은 슬러리(18) 중 발효성 당을 대사하며, 생성물 알코올을 배출한다. 미생물(32)은 박테리아, 시아노박테리아, 사상균(filamentous fungi) 및 효모의 군으로부터 선택된다. 일부 실시 형태에서, 미생물(32)은 박테리아, 예를 들어 이. 콜라이(E. coli)일 수 있다. 일부 실시 형태에서, 미생물(32)은 발효 재조합 미생물일 수 있다. 슬러리는 예를 들어 올리고당류 형태의 당 및 물을 포함할 수 있으며, 약 20 g/L 미만의 단량체성 글루코스, 더 바람직하게는 약 10 g/L 미만 또는 약 5 g/L 미만의 단량체성 글루코스를 포함할 수 있다. 단량체성 글루코스의 양을 측정하기에 적합한 방법은 당업계에 잘 알려져 있다. 당업계에 공지된 그러한 적합한 방법은 HPLC를 포함한다.Fermentation vessel 30 is configured to ferment slurry 18 to produce product alcohols such as butanol. In particular, microorganism 32 metabolizes fermentable sugars in slurry 18 and releases product alcohol. The microorganism 32 is selected from the group of bacteria, cyanobacteria, filamentous fungi and yeast. In some embodiments, microorganism 32 is bacteria, such as E. coli. E. coli. In some embodiments, microorganism 32 may be a fermentative recombinant microorganism. The slurry may comprise, for example, sugars and water in the form of oligosaccharides, less than about 20 g / L monomeric glucose, more preferably less than about 10 g / L or less than about 5 g / L monomeric glucose. It may include. Suitable methods for measuring the amount of monomeric glucose are well known in the art. Such suitable methods known in the art include HPLC.

일부 실시 형태에서, 슬러리(18)는 슬러리(18) 중 복합 당류 (예를 들어, 올리고당류)를 단당류로 분해시키기 위하여 당화 공정에 처해지는데, 상기 단당류는 미생물(32)에 의해 쉽게 대사될 수 있다. 당해 산업계가 일상적으로 이용하는 임의의 공지된 당화 공정이 이용될 수 있으며, 이는 산 공정, 산-효소 공정 또는 효소 공정을 포함하지만 이에 한정되지 않는다. 일부 실시 형태에서, 동시적 당화 및 발효(simultaneous saccharification and fermentation; SSF)가 발효 용기(30) 내부에서 일어날 수 있으며, 이는 도 1에 나타낸 바와 같다. 일부 실시 형태에서, 효소(38), 예를 들어 글루코아밀라아제는 전분 또는 올리고당류를 미생물(32)에 의해 대사될 수 있는 글루코스로 분해시키기 위하여 발효 용기(30) 내의 유입구에 도입될 수 있다.In some embodiments, slurry 18 is subjected to a saccharification process to break down complex sugars (eg, oligosaccharides) in slurry 18 into monosaccharides, which can be readily metabolized by microorganism 32. have. Any known saccharification process that is commonly used in the industry may be used, including but not limited to acid processes, acid-enzyme processes or enzyme processes. In some embodiments, simultaneous saccharification and fermentation (SSF) can occur within fermentation vessel 30, as shown in FIG. 1. In some embodiments, enzyme 38, such as glucoamylase, may be introduced at the inlet in fermentation vessel 30 to break down starch or oligosaccharides into glucose that may be metabolized by microorganism 32.

선택적으로, 에탄올(33)을 발효 용기(30)에 공급하여 발효 브로쓰에 포함시킬 수 있다. 일부 실시 형태에서, 부탄올 생합성 경로를 갖는 재조합 미생물이 부탄올 생성용 미생물(32)로 사용될 때, 미생물(32)은 생존 및 성장을 위하여 C2 기질 (예를 들어, 에탄올)의 보충을 필요로 할 수 있다. 따라서, 일부 실시 형태에서, 에탄올(33)이 발효 용기(30)에 공급될 수 있다.Optionally, ethanol 33 may be supplied to fermentation vessel 30 to include in the fermentation broth. In some embodiments, when a recombinant microorganism having a butanol biosynthetic pathway is used as the butanol producing microorganism 32, the microorganism 32 may require supplementation of a C2 substrate (eg, ethanol) for survival and growth. have. Thus, in some embodiments, ethanol 33 may be supplied to fermentation vessel 30.

그러나, 놀랍게도, 유리 지방산 (예를 들어, FFA(28))이 발효 용기에 존재하는 본 발명의 방법은 주어진 재조합 미생물의 생명력을 해치지 않고서 상기 재조합 미생물을 위하여 전형적으로 공급되는 에탄올(33)의 양을 감소시킬 수 있음이 밝혀졌다. 또한, 일부 실시 형태에서, 본 발명의 방법은, 에탄올(33)이 보충될 때 실현될 수 있는 알코올 (예를 들어, 부탄올) 생성 속도와 비견되는 에탄올 보충을 하지 않을 때의 생성 속도를 제공한다. 하기 예 1 내지 예 14에 제시된 비교예로 추가로 입증되는 바와 같이, 발효 용기에 지방산은 있지만 에탄올은 없는 때의 부탄올 생성 속도는 발효 용기에 지방산과 에탄올 모두 없는 때의 부탄올 생성 속도보다 더 클 수 있다. 따라서, 일부 실시 형태에서, 에탄올(33) 보충량은 통상적인 공정에 비하여 감소된다. 예를 들어, C2 기질의 보충을 필요로 하는 미생물에 있어서 발효 용기에 첨가되는 에탄올의 전형적인 양은 약 5 g/L의 무수 에탄올(즉, 발효 배지 1리터 당 5 g의 무수 에탄올)이다. 일부 실시 형태에서, 발효에 어떠한 에탄올(33)도 보충되지 않는다. 후자의 경우, 에탄올(33) 스트림은 발효 용기로부터 전적으로 제외된다. 따라서, 본 발명의 일부 실시 형태에서, 보충 에탄올(33)과 관련된 비용과, 에탄올(33)의 저장조와 관련된 그리고 부탄올 발효 동안 상기 에탄올을 발효 용기에 공급하는 것과 관련된 불편함을 감소시키거나 또는 제거하는 것이 가능하다. 게다가, 에탄올 보충과는 관계없이, 일부 실시 형태에서, 본 발명의 방법은 발효 동안 지방산의 존재에 의해 미생물(32)에 의한 더욱 높은 글루코스 흡수율을 제공할 수 있다. 본 발명에 개시된 방법에 따르면, 지방산은 발효 과정 중 단계에서 FFA(28)로서 발효 용기(30) 내로 도입되거나, 슬러리(16)의 공급재료 오일(26)로부터 가수분해되거나, 또는 달리 바이오매스 지질과 같은 천연 오일로부터 가수분해될 수 있다. 또한 지방산은 ISPR 추출제(29)로서 발효 용기 내로 도입될 수 있다.Surprisingly, however, the method of the present invention in which free fatty acids (eg, FFA 28) are present in a fermentation vessel provides an amount of ethanol 33 that is typically supplied for the recombinant microorganism without harming the viability of a given recombinant microorganism. It has been found that can be reduced. In addition, in some embodiments, the method of the present invention provides a rate of production when no ethanol replenishment is comparable to the rate of alcohol (eg, butanol) production that can be realized when ethanol 33 is replenished. . As further demonstrated by the comparative examples set forth in Examples 1-14 below, the butanol production rate when fatty acids in the fermentation vessel but no ethanol can be greater than the butanol production rate when both fatty acids and ethanol are absent in the fermentation vessel. have. Thus, in some embodiments, the ethanol 33 replenishment amount is reduced compared to conventional processes. For example, for microorganisms that require replenishment of C2 substrate, the typical amount of ethanol added to the fermentation vessel is about 5 g / L of absolute ethanol (ie, 5 g of absolute ethanol per liter of fermentation medium). In some embodiments, the fermentation is not supplemented with any ethanol 33. In the latter case, the ethanol 33 stream is wholly excluded from the fermentation vessel. Thus, in some embodiments of the invention, it reduces or eliminates the cost associated with supplemental ethanol 33 and the inconvenience associated with the reservoir of ethanol 33 and associated with feeding the ethanol to the fermentation vessel during butanol fermentation. It is possible to. In addition, regardless of ethanol replenishment, in some embodiments, the methods of the present invention may provide higher glucose uptake rates by microorganisms 32 by the presence of fatty acids during fermentation. According to the process disclosed herein, the fatty acid is introduced into the fermentation vessel 30 as FFA 28 in the stages of the fermentation process, hydrolyzed from the feedstock oil 26 of the slurry 16, or otherwise biomass lipids. It can be hydrolyzed from natural oils such as. Fatty acids may also be introduced into the fermentation vessel as ISPR extractant 29.

발효 용기(30)에서, 알코올은 미생물(32)에 의해 생성된다. 원위치 생성물 제거(ISPR)를 이용하여 생성물 알코올을 발효 브로쓰로부터 제거할 수 있다. 일부 실시 형태에서, ISPR은 액체-액체 추출을 포함한다. 액체-액체 추출은 미국 특허 공개 제2009/0305370호에 기재되어 있는 방법에 따라 실시될 수 있으며, 상기 미국 특허 공개의 개시 내용은 전체적으로 본 명세서에 포함된다. 미국 특허 공개 제2009/0305370호에는 액체-액체 추출을 이용하여 발효 브로쓰로부터 부탄올을 생성 및 회수하는 방법이 기재되어 있으며, 상기 방법은 발효 브로쓰를 수불혼화성 추출제와 접촉시켜 수성 상 및 유기 상을 포함하는 2상 혼합물을 형성하는 단계를 포함한다. 전형적으로, 추출제는 포화된, 단일불포화된, 다중불포화된 (및 그 혼합물의) C₁₂ 내지 C₂₂ 지방 알코올, C₁₂ 내지 C₂₂ 지방산, C₁₂ 내지 C₂₂ 지방산 에스테르, C₁₂ 내지 C₂₂ 지방 알데히드 및 그 혼합물로 이루어진 군으로부터 선택되는 유기 추출제일 수 있으며, 이는 발효 브로쓰와 접촉하여 수성 상 및 유기 상을 포함하는 2상 혼합물을 형성한다. 또한, 추출제는 포화된, 단일불포화된, 다중불포화된 (및 그 혼합물의) C₄ 내지 C₂₂ 지방 알코올, C₄ 내지 C₂₈ 지방산, C₄ 내지 C₂₈ 지방산 에스테르, C₄ 내지 C₂₂ 지방 알데히드 및 그 혼합물로 이루어진 군으로부터 선택되는 유기 추출제일 수 있으며, 이는 발효 브로쓰와 접촉하여 수성 상 및 유기 상을 포함하는 2상 혼합물을 형성한다. 또한, 슬러리(18)로부터의 유리 지방산(28)은 ISPR 추출제(28)로서의 역할을 할 수 있다. 예를 들어, 유리 지방산(28)이 옥수수유 지방산(COFA)일 때, ISPR 추출제(28)는 COFA이다. ISPR 추출제(FFA)(28)는 발효 브로쓰와 접촉하여 수성 상(34) 및 유기 상을 포함하는 2상 혼합물을 형성한다. 발효 브로쓰에 존재하는 생성물 알코올은 유기 상 내로 우선적으로 분배되어 알코올 함유 유기 상(36)을 형성한다. 일부 실시 형태에서, 발효 용기(30)는 수성 상 및 유기 상을 포함하는 2상 혼합물을 형성하는 하나 이상의 추가의 ISPR 추출제(29)를 수용하기 위한 하나 이상의 유입구를 가지며, 이때 생성물 알코올은 유기 상 내로 분배된다.In the fermentation vessel 30, alcohol is produced by the microorganism 32. In situ product removal (ISPR) can be used to remove the product alcohol from the fermentation broth. In some embodiments, the ISPR comprises liquid-liquid extraction. Liquid-liquid extraction can be performed according to the method described in US Patent Publication No. 2009/0305370, the disclosure of which is incorporated herein in its entirety. US Patent Publication No. 2009/0305370 describes a method for producing and recovering butanol from fermentation broth using liquid-liquid extraction, which method comprises contacting the fermentation broth with a water immiscible extractant to provide an aqueous phase and Forming a biphasic mixture comprising an organic phase. Typically, extractants are saturated, monounsaturated, polyunsaturated (and mixtures thereof) C ₁₂ to C ₂₂ fatty alcohols, C ₁₂ to C ₂₂ fatty acids, C ₁₂ to C ₂₂ fatty acid esters, C ₁₂ to C ₂₂ Organic extractant selected from the group consisting of fatty aldehydes and mixtures thereof, which are contacted with fermentation broth to form a biphasic mixture comprising an aqueous phase and an organic phase. The extractant may also be saturated, monounsaturated, polyunsaturated (and mixtures thereof) C ₄ to C ₂₂ fatty alcohols, C ₄ to C ₂₈ fatty acids, C ₄ to C ₂₈ fatty acid esters, C ₄ to C ₂₂ fats. It may be an organic extractant selected from the group consisting of aldehydes and mixtures thereof, which contact with fermentation broth to form a biphasic mixture comprising an aqueous phase and an organic phase. In addition, free fatty acid 28 from slurry 18 may serve as ISPR extractant 28. For example, when the free fatty acid 28 is corn oil fatty acid (COFA), the ISPR extractant 28 is COFA. ISPR extractant (FFA) 28 is contacted with the fermentation broth to form a biphasic mixture comprising an aqueous phase 34 and an organic phase. The product alcohol present in the fermentation broth is preferentially distributed into the organic phase to form the alcohol containing organic phase 36. In some embodiments, fermentation vessel 30 has one or more inlets for receiving one or more additional ISPR extractants 29 forming a two-phase mixture comprising an aqueous phase and an organic phase, wherein the product alcohol is organic Is distributed into the phase.

2상 혼합물은 발효 용기(30)로부터 스트림(39)로서 제거되고 용기(35) 내로 도입될 수 있으며, 여기서 알코올 함유 유기 상(36)이 수성 상(34)으로부터 분리된다. 알코올 함유 유기 상(36)은 사이펀 작용(siphoning), 경사분리(decantation), 흡인(aspiration), 원심분리, 중력 방식 침강조의 이용, 막 보조 상 분할 등을 포함하지만 이에 한정되지 않는 당업계에 공지된 방법을 이용하여 2상 스트림(39)의 수성 상(34)으로부터 분리된다. 전부의 또는 일부의 수성 상(34)은 발효 배지 (예시된 바와 같음)로서 발효 용기(30) 내로 재순환되거나, 또는 달리 버려지고 신선 배지로 대체되거나, 또는 임의의 남아있는 생성물 알코올의 제거를 위하여 처리되고 그 후 발효 용기(30)로 재순환될 수 있다. 그 후, 알코올 함유 유기 상(36)을 분리기(50)에서 처리하여 생성물 알코올(54)을 회수하고, 그 후, 생성된 알코올 린 추출제(27)를, 생성물 알코올의 추가의 추출을 위하여 슬러리(18)로부터의 신선 FFA(28)와 조합하여 및/또는 신선 추출제(29)와 함께 발효 용기(30) 내로 다시 재순환시킬 수 있다. 대안적으로, (슬러리(18)로부터의) 신선 FFA(28) 및/또는 추출제(29)를 발효 용기에 계속적으로 첨가하여 2상 혼합물 스트림(39) 중 제거되는 ISPR 추출제(들)를 대체할 수 있다.The biphasic mixture can be removed as a stream 39 from the fermentation vessel 30 and introduced into the vessel 35 where the alcohol containing organic phase 36 is separated from the aqueous phase 34. Alcohol-containing organic phase 36 is known in the art, including but not limited to siphoning, decantation, aspiration, centrifugation, use of gravity settling baths, membrane assisted phase separation, and the like. It is separated from the aqueous phase 34 of the two-phase stream 39 using known methods. All or part of the aqueous phase 34 is recycled into fermentation vessel 30 as fermentation medium (as illustrated), or otherwise discarded and replaced with fresh medium, or for removal of any remaining product alcohol. It may be processed and then recycled to fermentation vessel 30. The alcohol containing organic phase 36 is then treated in separator 50 to recover product alcohol 54, after which the resulting alcohol lean extractant 27 is slurry for further extraction of product alcohol. It may be recycled back into fermentation vessel 30 in combination with fresh FFA 28 from 18 and / or with fresh extractant 29. Alternatively, fresh FFA 28 and / or extractant 29 (from slurry 18) may be continuously added to the fermentation vessel to remove ISPR extractant (s) removed in the biphasic mixture stream 39. Can be replaced.

일부 실시 형태에서, 임의의 추가의 ISPR 추출제(29)는 외인성 유기 추출제, 예를 들어 올레일 알코올, 베헤닐 알코올, 세틸 알코올, 라우릴 알코올, 미리스틸 알코올, 스테아릴 알코올, 1-운데칸올, 올레산, 라우르산, 미리스트산, 스테아르산, 메틸 미리스테이트, 메틸 올레에이트, 운데카날, 라우릭 알데히드, 20-메틸운데카날 및 그 혼합물일 수 있다. 일부 실시 형태에서, ISPR 추출제(29)는 카르복실산 또는 유리 지방산일 수 있으며, 일부 실시 형태에서, 카르복실산 또는 유리 지방산은 4 내지 28개의 탄소, 다른 실시 형태에서 4 내지 22개의 탄소, 다른 실시 형태에서 8 내지 22개의 탄소, 다른 실시 형태에서 10 내지 28개의 탄소, 다른 실시 형태에서 7 내지 22개의 탄소, 다른 실시 형태에서 12 내지 22개의 탄소, 다른 실시 형태에서 4 내지 18개의 탄소, 다른 실시 형태에서 12 내지 22개의 탄소, 그리고 또 다른 실시 형태에서 12 내지 18개의 탄소의 사슬을 갖는다. 일부 실시 형태에서, ISPR 추출제(29)는 하기 지방산들 중 하나 이상이다: 아잘레익(azaleic)산, 카프르산, 카프릴산, 피마자유, 코코넛유(즉, 라우르산, 미리스트산, 팔미트산, 카프릴산, 카프르산, 스테아르산, 카프로익산, 아라키드산, 올레산 및 리놀레산을 포함하는 지방산들의 자연 발생 조합물로서), 다이머(dimer), 아이소스테아르산, 라우르산, 아마인유, 미리스트산, 올레산, 올리브유, 야자유, 팔미트산, 야자핵유, 땅콩유, 펠라르곤산, 리시놀레산, 세바식산, 대두유, 스테아르산, 톨유, 탤로우, #12 하이드록시 스테아르산 또는 임의의 종자유. 일부 실시 형태에서, ISPR 추출제(29)는 이산(diacid), 예를 들어, 아젤라산 및 세바식산 중 하나 이상이다. 따라서, 일부 실시 형태에서, ISPR 추출제(29)는 2가지 이상의 상이한 지방산의 혼합물일 수 있다. 일부 실시 형태에서, ISPR 추출제(29)는 천연 오일로부터 유래되는 글리세라이드의 화학적 또는 효소적 가수분해에 의해 유도되는 유리 지방산일 수 있다. 예를 들어, 일부 실시 형태에서, ISPR 추출제(29)는 도 4의 실시 형태를 참고로 하여 이후에 설명되는 바와 같이 바이오매스 지질과 같은 천연 오일의 효소적 가수분해에 의해 수득되는 유리 지방산(28')일 수 있다. 일부 실시 형태에서, ISPR 추출제(29)는 지방산, 지방 알코올, 지방 아미드, 지방산 메틸 에스테르, 지방산의 저급 알코올 에스테르, 지방산 글리콜 에스테르, 하이드록실화 트라이글리세라이드 및 그 혼합물로 이루어진 군으로부터 선택되는 지방산 추출제일 수 있으며, 이는 예를 들어 2010년 7월 28일자로 출원된, 본 출원과 공계류 중인, 공히 소유된 미국 가특허 출원 제61/368,436호에 기재된 바와 같이 바이오매스 지질과 같은 천연 오일의 화학적 전환에 의해 수득된다. 그러한 실시 형태에서, 추출제(29) 생성용 바이오매스 지질은 동일하거나 또는 상이한 바이오매스 공급원으로부터의 것일 수 있으며, 상기 공급원으로부터 공급재료(12)가 수득된다. 예를 들어, 일부 실시 형태에서, 추출제(29) 생성용 바이오매스 지질은 대두로부터 유래될 수 있으며, 반면에 공급재료(12)의 바이오매스 공급원은 옥수수이다. 당업자에게 명백함에 틀림없는 바와 같이, 공급재료(12)에 대하여 추출제(29)를 위한 다양한 바이오매스 공급원들의 임의의 가능한 조합이 사용될 수 있다. 일부 실시 형태에서, 추가의 ISPR 추출제(29)는 COFA를 포함한다.In some embodiments, any additional ISPR extractant 29 may be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undone Canol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal and mixtures thereof. In some embodiments, ISPR extractant 29 may be a carboxylic acid or free fatty acid, and in some embodiments, the carboxylic acid or free fatty acid may be 4 to 28 carbons, in other embodiments 4 to 22 carbons, 8 to 22 carbons in other embodiments, 10 to 28 carbons in other embodiments, 7 to 22 carbons in other embodiments, 12 to 22 carbons in other embodiments, 4 to 18 carbons in other embodiments, In another embodiment, 12 to 22 carbons, and in still other embodiments, a chain of 12 to 18 carbons. In some embodiments, the ISPR extractant 29 is one or more of the following fatty acids: azaleic acid, capric acid, caprylic acid, castor oil, coconut oil (ie, lauric acid, myristic acid) , As a naturally occurring combination of fatty acids including palmitic acid, caprylic acid, capric acid, stearic acid, caproic acid, arachidic acid, oleic acid and linoleic acid), dimer, isostearic acid, lauric acid Linseed oil, myristic acid, oleic acid, olive oil, palm oil, palmitic acid, palm kernel oil, peanut oil, pelagonate, ricinoleic acid, sebacic acid, soybean oil, stearic acid, tall oil, tallow, # 12 hydroxy stearic Acid or any seed oil. In some embodiments, ISPR extractant 29 is one or more of diacids such as azelaic acid and sebacic acid. Thus, in some embodiments, ISPR extractant 29 may be a mixture of two or more different fatty acids. In some embodiments, ISPR extractant 29 may be a free fatty acid derived by chemical or enzymatic hydrolysis of glycerides derived from natural oils. For example, in some embodiments, ISPR extractant 29 may be a free fatty acid obtained by enzymatic hydrolysis of natural oils, such as biomass lipids, as described below with reference to the embodiment of FIG. 4. 28 '). In some embodiments, ISPR extractant 29 is a fatty acid selected from the group consisting of fatty acids, fatty alcohols, fatty amides, fatty acid methyl esters, lower alcohol esters of fatty acids, fatty acid glycol esters, hydroxylated triglycerides, and mixtures thereof. Extractant, which is, for example, a natural oil such as biomass lipid, as described in commonly owned US Provisional Patent Application No. 61 / 368,436, filed July 28, 2010. Obtained by chemical conversion. In such embodiments, the biomass lipids for producing the extractant 29 may be from the same or different biomass sources, from which the feedstock 12 is obtained. For example, in some embodiments, the biomass lipids for producing extractant 29 may be derived from soybeans, while the biomass source of feedstock 12 is corn. As will be apparent to those skilled in the art, any possible combination of various biomass sources for extractant 29 may be used for feedstock 12. In some embodiments, additional ISPR extractant 29 comprises COFA.

원위치 추출 발효(extractive fermentation)가 발효 용기(30)에서 배치식 모드 또는 연속식 모드로 수행될 수 있다. 원위치 추출 발효에 있어서, 유기 추출제는 발효 시작시에 발효 배지와 접촉되어 2상 발효 배지를 형성할 수 있다. 대안적으로, 유기 추출제는 미생물이 배양물의 광학 밀도를 측정함으로써 결정될 수 있는 원하는 양의 성장을 이룬 후에 발효 배지와 접촉할 수 있다. 또한, 유기 추출제는 발효 배지 중 생성물 알코올 수준이 소정의 수준에 도달하는 시점에 발효 배지와 접촉할 수 있다. 부탄올 생성의 경우, 예를 들어 ISPR 추출제는 부탄올 농도가 미생물에게 유독해지는 수준에 도달하기 전에 한 번에 발효 배지와 접촉시킬 수 있다. 발효 배지를 ISPR 추출제와 접촉시킨 후에, 부탄올 생성물은 추출제 내로 분배되어, 미생물을 함유하는 수성 상 중의 농도가 감소되고, 이럼으로써 저해성 부탄올 생성물에 대한 생산 미생물의 노출이 제한된다.In-situ fermentation (extractive fermentation) may be performed in the fermentation vessel 30 in batch mode or continuous mode. In in situ extraction fermentation, the organic extractant may be contacted with the fermentation medium at the start of fermentation to form a two phase fermentation medium. Alternatively, the organic extractant can be contacted with the fermentation medium after the microorganism has achieved the desired amount of growth that can be determined by measuring the optical density of the culture. In addition, the organic extractant may be contacted with the fermentation broth when the product alcohol level in the fermentation broth reaches a predetermined level. In the case of butanol production, for example, the ISPR extractant may be contacted with the fermentation medium at one time before the butanol concentration reaches a level that is toxic to microorganisms. After contacting the fermentation medium with the ISPR extractant, the butanol product is partitioned into the extractant to reduce the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the producing microorganisms to the inhibitory butanol product.

사용되는 ISPR 추출제의 부피는 후술되는 바와 같이 발효 배지의 부피, 발효 용기의 크기, 부탄올 생성물에 대한 추출제의 분배 계수 및 선택되는 발효 모드를 비롯한 많은 요인에 따라 달라진다. 추출제의 부피는 발효 용기의 사용 부피(working volume)의 약 3% 내지 약 60%일 수 있다. 추출 효율에 따라, 발효 배지 중 부탄올의 수성 상 타이터는 예를 들어 약 1 g/L 내지 약 85 g/L, 약 10 g/L 내지 약 40 g/L, 약 10 g/L 내지 약 20 g/L, 약 15 g/L 내지 약 50 g/L 또는 약 20 g/L 내지 약 60 g/L일 수 있다. 일부 실시 형태에서, 알코올 에스테르화 후 생성된 발효 브로쓰는 유리(즉, 비에스테르화) 알코올을 포함할 수 있으며, 일부 실시 형태에서, 알코올 에스테르화 후 발효 브로쓰 중 유리 알코올의 농도는 생성물 알코올이 부탄올일 때 1, 3, 6, 10, 15, 20, 25, 30 25, 40, 45, 50, 55, 또는 60 g/L 이하이거나, 또는 생성물 알코올이 에탄올일 때, 알코올 에스테르화 후 발효 브로쓰 중 유리 알코올의 농도는 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 또는 100 g/L 이하이다. 이론에 구애되지 않고서, 더욱 높은 부탄올 타이터는, 부분적으로는 발효 배지로부터 독성 부탄올 생성물을 제거하고 이럼으로써 그 수준을 미생물에게 유독한 수준 미만으로 유지함에 의해 추출 발효법으로 수득될 수 있다고 여겨진다.The volume of ISPR extractant used depends on many factors, including the volume of the fermentation medium, the size of the fermentation vessel, the partition coefficient of the extractant to the butanol product, and the fermentation mode selected, as described below. The volume of extractant may be from about 3% to about 60% of the working volume of the fermentation vessel. Depending on the extraction efficiency, the aqueous phase titer of butanol in the fermentation medium is for example about 1 g / L to about 85 g / L, about 10 g / L to about 40 g / L, about 10 g / L to about 20 g / L, about 15 g / L to about 50 g / L or about 20 g / L to about 60 g / L. In some embodiments, the fermentation broth produced after alcohol esterification may comprise free (ie, non-esterified) alcohol, and in some embodiments, the concentration of free alcohol in the fermentation broth after alcohol esterification is determined by the product alcohol. Fermentation bro after alcohol esterification when 1, 3, 6, 10, 15, 20, 25, 30 25, 40, 45, 50, 55, or 60 g / L when butanol or the product alcohol is ethanol The concentration of free alcohol in the bath is 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / L or less. Without being bound by theory, it is believed that higher butanol titers can be obtained by extractive fermentation, in part by removing toxic butanol products from the fermentation medium, thereby maintaining their levels below those toxic to microorganisms.

원위치 추출 발효의 배치식 모드에서, 소정 부피의 유기 추출제가 발효 용기에 첨가되며, 추출제는 당해 공정 동안 제거되지 않는다. 이 모드는 발효 배지 중 저해성 부탄올 생성물의 농도를 최소화하기 위하여 더 큰 부피의 유기 추출제를 필요로 한다. 결과적으로, 발효 배지의 부피는 더 작고, 생성된 생성물의 양은 연속식 모드를 사용하여 수득되는 것보다 더 적다. 예를 들어, 배치식 모드에서의 추출제의 부피는 일 실시 형태에서는 발효 용기 사용 부피의 20% 내지 약 60%, 그리고 다른 실시 형태에서는 약 30% 내지 약 60%일 수 있다.In the batch mode of in situ extraction fermentation, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. This mode requires a larger volume of organic extractant to minimize the concentration of inhibitory butanol product in the fermentation medium. As a result, the volume of the fermentation medium is smaller and the amount of product produced is less than that obtained using the continuous mode. For example, the volume of extractant in batch mode may be from 20% to about 60% of the fermentation vessel use volume in one embodiment, and from about 30% to about 60% in another embodiment.

가스 스트리핑 (예시되어 있지 않음)을 ISPR 추출제와 함께 사용하여 생성물 알코올을 발효 배지로부터 제거할 수 있다.Gas stripping (not illustrated) may be used with the ISPR extractant to remove the product alcohol from the fermentation medium.

도 1의 실시 형태에서, 생성물 알코올은 원위치에서 발효 브로쓰로부터 추출되며, 이때 2상 혼합물(39)의 분리는 별도의 용기(35)에서 일어난다. 일부 실시 형태에서, 2상 혼합물(39)의 분리는 이후에 설명되는 도 2 및 도 3의 실시 형태들에 나타낸 바와 같이 발효 용기에서 일어날 수 있으며, 여기서 알코올 함유 유기 상 스트림(36)은 발효 용기(30)로부터 직접적으로 배출된다. 또한 수성 상 스트림(34)은 발효 용기(30)로부터 직접적으로 배출되고, 임의의 남아있는 생성물 알코올의 제거를 위하여 처리되고, 재순환되거나, 또는 버려지고 신선 발효 배지로 대체될 수 있다. 유기 추출제(들)에 의한 생성물 알코올의 추출은 발효 브로쓰로부터 미생물을 제거하거나 또는 제거하지 않고서 행해질 수 있다. 미생물은 여과 또는 원심분리를 포함하지만 이에 한정되지 않는 당업계에 공지된 수단에 의해 발효 브로쓰로부터 제거될 수 있다. 예를 들어, 수성 상 스트림(34)은 미생물(32), 예를 들어 효모를 포함할 수 있다. 미생물(32)은 예를 들어 원심분리기 (예시되어 있지 않음)에서 수성 상 스트림으로부터 용이하게 분리될 수 있다. 그 후 미생물(32)은 발효 용기(30)로 재순환될 수 있으며, 이는 시간이 지남에 따라 알코올 생성의 생성 속도를 증가시키고 이럼으로써 알코올 생성 효율을 증가시킬 수 있다.In the embodiment of FIG. 1, the product alcohol is extracted from the fermentation broth in situ, with the separation of the biphasic mixture 39 taking place in a separate vessel 35. In some embodiments, separation of the biphasic mixture 39 can occur in a fermentation vessel, as shown in the embodiments of FIGS. 2 and 3 described below, wherein the alcohol containing organic phase stream 36 is a fermentation vessel. It is discharged directly from 30. The aqueous phase stream 34 may also be discharged directly from the fermentation vessel 30 and treated, recycled, or discarded and replaced with fresh fermentation medium for removal of any remaining product alcohol. Extraction of the product alcohol by the organic extractant (s) can be done with or without the removal of microorganisms from the fermentation broth. The microorganism may be removed from the fermentation broth by means known in the art, including but not limited to filtration or centrifugation. For example, aqueous phase stream 34 may comprise microorganism 32, for example yeast. The microorganism 32 can be easily separated from the aqueous phase stream, for example in a centrifuge (not illustrated). The microorganism 32 may then be recycled to the fermentation vessel 30, which may increase the rate of production of alcohol production over time, thereby increasing the alcohol production efficiency.

연속식 모드의 원위치 추출 발효에서, 추출제의 부피는 일 실시 형태에서는 발효 용기 사용 부피의 약 3% 내지 약 50%, 다른 실시 형태에서는 약 3% 내지 약 30%, 다른 실시 형태에서는 3% 내지 약 20%, 그리고 다른 실시 형태에서는 3% 내지 약 10%일 수 있다. 생성물이 반응기로부터 계속적으로 제거되기 때문에, 더 적은 부피의 추출제가 필요하며, 이는 더 큰 부피의 발효 배지가 사용될 수 있게 한다.In the in situ extraction fermentation in continuous mode, the volume of extractant in one embodiment is from about 3% to about 50% of the volume used in the fermentation vessel, in another embodiment from about 3% to about 30%, in other embodiments from 3% to About 20%, and in other embodiments from 3% to about 10%. As the product is continuously removed from the reactor, less volume of extractant is needed, which allows larger volumes of fermentation medium to be used.

원위치 추출 발효에 대한 대안으로서, 생성물 알코올은 발효 용기(30)의 하류의 발효 브로쓰로부터 추출될 수 있다. 그러한 예에서, 발효 브로쓰는 발효 용기(30)로부터 제거되고, 용기(35)에서 2상 혼합물(39) - 이는 그 후 유기 상(36) 및 수성 상(34)으로 분리됨 - 이 수득되도록 ISPR 추출제와 접촉시키기 위하여 용기(35) 내로 도입될 수 있다. 대안적으로, ISPR 추출제는 용기(35)에 도입되기 이전에 별도의 용기 (예시되어 있지 않음)에서 발효 브로쓰에 첨가될 수 있다.As an alternative to in situ extraction fermentation, the product alcohol may be extracted from the fermentation broth downstream of the fermentation vessel 30. In such an example, the fermentation broth is removed from the fermentation vessel 30 and in the vessel 35 an ISPR extraction such that a biphasic mixture 39, which is then separated into an organic phase 36 and an aqueous phase 34, is obtained. It may be introduced into the container 35 to make contact with the agent. Alternatively, the ISPR extractant may be added to the fermentation broth in a separate vessel (not illustrated) prior to introduction into vessel 35.

비제한적 가공 실시예(prophetic example)로서, 도 1의 실시 형태를 참고로 하면, 명목상 약 4 중량%의 옥수수유를 함유할 수 있는 (공급재료(12)로서) 분쇄된 통옥수수의 수성 현탁물을 약 85℃ 내지 120℃에서 30분 내지 2시간 동안 (액화 효소(14)로서) 아밀라아제로 처리하고, 생성된 액화 매시(16)를 65℃ 내지 30℃로 냉각시키고, 적어도 30%로부터 적어도 99%만큼 높은, 지질 중 이용가능한 지방산 내용물의 유리 지방산으로의 전환율을 생성하기에 충분한 시간 동안 pH 4.5 내지 7.5 (일부 실시 형태에서, pH 5.5 내지 6.5)에서 0.1 ppm 내지 10 ppm (일부 실시 형태에서, 0.5 ppm 내지 1.0 ppm)의 리파아제 (촉매(42)로서)로 처리할 수 있다. 선택적으로, 액화되고 리파아제 처리된 매시(18)는 발효 이전에 리파아제(42)를 불활성화시키기 위하여 가열될 수 있다. 매시(18)는 (예를 들어, 열교환기를 사용하여) 약 30℃로 냉각되고 약 25% 내지 30 중량%의 건조 옥수수 고형물로 발효 용기(30)에 로딩될 수 있다. (당화 효소(38)로서) 글루코아밀라아제의 첨가에 의한 발효 동안의 액화 매시(18)의 당화에 의해 글루코스가 생성될 수 있다. 생성된 발효 브로쓰는 리파아제(42)로 처리되지 않은 액화 매시를 사용하면 발효 브로쓰에 존재할 수 있는 옥수수유 (예를 들어, 약 1.2 중량%의 옥수수유)의 양보다 유의하게 더 적게 함유할 수 있다. 특히, 리파아제(42) 처리에 의해 옥수수유 지질(26) (트라이글리세라이드(TG))이 FFA(28)로서의 COFA (그리고 약간의 다이글리세라이드(DG) 또는 모노글리세라이드(MG))로 전환될 수 있으며, 이는 발효 브로쓰와 접촉된다. 발효 브로쓰 중 미생물의 성장 속도 및/또는 글루코스 소비량은 COFA의 존재 하에서, 지방산의 부재 하에서의 미생물의 성장 속도 및 발효성 탄소 소비량보다 더 클 수 있다. 예를 들어, 실시예에서 이후에 하기에 기재되어 있는 바와 같이, 도 6 내지 도 8은 보충된 지방산의 존재 하에서의 부탄올 생성 미생물에 의한 증가된 글루코스 소비량을 도시한다.As a non-limiting processing example, referring to the embodiment of FIG. 1, an aqueous suspension of ground corn products (as feedstock 12) that may contain nominally about 4% by weight of corn oil. Was treated with amylase (as liquefied enzyme 14) at about 85 ° C. to 120 ° C. for 30 minutes to 2 hours, the resulting liquefied mash 16 was cooled to 65 ° C. to 30 ° C. and at least 99% from at least 30%. 0.1 ppm to 10 ppm (in some embodiments, at pH 4.5 to 7.5 (in some embodiments, pH 5.5 to 6.5) for a time sufficient to produce a conversion of the available fatty acid content in the lipid to free fatty acids, as high as% 0.5 ppm to 1.0 ppm) of lipase (as catalyst 42). Optionally, the liquefied and lipase treated mash 18 may be heated to inactivate the lipase 42 prior to fermentation. Mash 18 may be cooled to about 30 ° C. (eg, using a heat exchanger) and loaded into fermentation vessel 30 with about 25% to 30% by weight of dry corn solids. Glucose may be produced by saccharification of the liquefied mash 18 during fermentation (as glycosylation enzyme 38) by addition of glucoamylase. The resulting fermentation broth may contain significantly less than the amount of corn oil (eg, about 1.2 wt% corn oil) that may be present in the fermentation broth using liquefied mash that has not been treated with lipase 42. have. In particular, lipase 42 treatment converts corn oil lipid 26 (triglyceride (TG)) to COFA (and some diglyceride (DG) or monoglyceride (MG)) as FFA 28 It may be in contact with the fermentation broth. The growth rate and / or glucose consumption of the microorganisms in the fermentation broth may be greater than the growth rate and fermentable carbon consumption of the microorganisms in the presence of COFA, in the absence of fatty acids. For example, as described below in the Examples below, FIGS. 6-8 show increased glucose consumption by butanol producing microorganisms in the presence of supplemented fatty acids.

일부 실시 형태에서, 도 1의 시스템 및 방법은 공급재료 슬러리(16) (오일(26)을 가짐) 및 촉매(42)가 슬러리(18) (FFA(28)를 가짐)을 생성하도록 발효 용기(30)에 도입되어 접촉되도록 변경될 수 있다. 그 후 발효 용기 온도를 상승시켜 촉매(42)를 열 불활성화시킬 수 있다. 그 후 발효 용기 온도를 감소시킬 수 있으며, 발효 용기에 미생물(32)을 접종할 수 있고, 이에 의해 슬러리(18)의 당류는 생성물 알코올이 생성되도록 발효될 수 있다.In some embodiments, the system and method of FIG. 1 includes a fermentation vessel 16 such that feedstock slurry 16 (with oil 26) and catalyst 42 produce slurry 18 (with FFA 28). 30) may be introduced and contacted. The fermentation vessel temperature may then be raised to thermally inactivate the catalyst 42. The fermentation vessel temperature can then be reduced and the microorganism 32 can be inoculated into the fermentation vessel, whereby the saccharides of the slurry 18 can be fermented to produce product alcohol.

일부 실시 형태에서, 도 1의 시스템 및 방법은, 당업자에게 명백함에 틀림없는 바와 같이, 발효 용기(30) 이전에 발효 용기(30)에서의 동시적 당화 및 발효(SSF)가 별도의 당화 용기(60) (도 2 참조)로 대체되도록 변경될 수 있다. 따라서, 슬러리(18)는 SSF 공정에서 발효 전에 또는 발효 동안 당화될 수 있다. 또한, 공급재료 오일(26)의 가수분해를 위한 촉매(42)가 당화 효소(38) 전에, 상기 효소 후에, 또는 상기 효소와 동시에 도입될 수 있음이 명백함에 틀림없다. 따라서, 일부 실시 형태에서, 효소(38) 및 촉매(42)의 첨가는 단계적(예를 들어, 촉매(42), 이어서 효소(38), 또는 그 반대)일 수 있거나, 또는 실질적으로 동시적(즉, 사람 또는 기계가 상기 첨가를 한꺼번에 수행하는 데 걸리는 시간에서와 정확하게 동일한 시간에, 또는 사람 또는 기계가 상기 첨가를 두 번으로 수행하는 데 걸리는 시간에서와 같이 하나의 효소/촉매 직후에 다른 하나의 촉매 효소)일 수 있다.In some embodiments, the system and method of FIG. 1 may be characterized in that simultaneous saccharification and fermentation (SSF) in fermentation vessel 30 prior to fermentation vessel 30 may be a separate saccharification vessel ( 60) (see FIG. 2). Thus, the slurry 18 may be saccharified before or during the fermentation in the SSF process. It should also be clear that the catalyst 42 for hydrolysis of the feedstock oil 26 can be introduced before the saccharification enzyme 38, after the enzyme or simultaneously with the enzyme. Thus, in some embodiments, the addition of enzyme 38 and catalyst 42 may be stepwise (eg, catalyst 42, then enzyme 38, or vice versa), or substantially concurrent ( That is, at the exact same time as the time it takes for a person or machine to carry out the addition all at once, or another one immediately after one enzyme / catalyst, such as at the time it takes for the person or machine to perform the addition twice. Catalytic enzyme).

예를 들어, 도 2의 실시 형태에 나타낸 바와 같이, 도 1의 시스템 및 방법은, 발효 용기(30) 이전에 발효 용기(30)에서의 동시적 당화 및 발효(SSF)가 별도의 당화 용기(60)로 대체되도록 변경될 수 있다. 도 2는 효소(38)를 수용하는 별도의 당화 용기(60)의 포함을 제외하고는 도 1과 실질적으로 동일하며, 이때 촉매(42)는 액화, 당화 공급재료 스트림(62)에 도입된다. 공급재료 슬러리(16)는 효소(38), 예를 들어 글루코아밀라아제와 함께 당화 용기(60) 내로 도입되며 이에 의해 슬러리(16) 중 올리고당류의 형태의 당류는 단당류로 분해될 수 있다. 액화, 당화 공급재료 스트림(62)은 촉매(42)가 도입되는 당화 용기(60)에서 배출된다. 공급재료 스트림(62)은 단당류, 오일(26), 및 비용해 고형물 - 공급재료로부터 유래됨 - 을 포함한다. 오일(26)은 촉매(42)의 도입에 의해 가수분해되어 유리 지방산(28) 및 촉매(42)를 갖는 액화, 당화 공급재료 슬러리(64)를 생성한다.For example, as shown in the embodiment of FIG. 2, the system and method of FIG. 1 allows simultaneous saccharification and fermentation (SSF) in the fermentation vessel 30 prior to the fermentation vessel 30 to a separate saccharification vessel ( 60). FIG. 2 is substantially the same as FIG. 1 except for the inclusion of a separate saccharification vessel 60 containing the enzyme 38, wherein the catalyst 42 is introduced into the liquefaction, saccharification feedstock stream 62. Feedstock slurry 16 is introduced into saccharification vessel 60 with enzyme 38, for example glucoamylase, whereby sugars in the form of oligosaccharides in slurry 16 can be broken down into monosaccharides. Liquefaction, saccharification feedstock stream 62 exits saccharification vessel 60 into which catalyst 42 is introduced. Feedstock stream 62 comprises monosaccharides, oil 26, and insoluble solids, derived from the feedstock. Oil 26 is hydrolyzed by introduction of catalyst 42 to produce a liquefied, saccharified feedstock slurry 64 having free fatty acid 28 and catalyst 42.

대안적으로, 일부 실시 형태에서, 촉매(42)를 당화 효소(38)와 함께 첨가하여 글루코스 생성 및 오일 지질(26)의 유리 지방산(28)으로의 가수분해를 동시에 할 수 있다. 효소(38) 및 촉매(42)는 단계적이거나 (예를 들어, 촉매(42), 그 후 효소(38), 또는 그 반대) 또는 동시적일 수 있다. 대안적으로, 일부 실시 형태에서, 슬러리(62)는 발효 용기에 도입될 수 있으며, 이때 촉매(42)는 발효 용기(30)에 직접적으로 첨가된다.Alternatively, in some embodiments, catalyst 42 may be added along with glycosylating enzyme 38 to simultaneously produce glucose and hydrolyze oil lipid 26 to free fatty acid 28. Enzyme 38 and catalyst 42 may be staged (eg, catalyst 42, then enzyme 38, or vice versa) or concurrent. Alternatively, in some embodiments, slurry 62 may be introduced into the fermentation vessel, with catalyst 42 added directly to fermentation vessel 30.

도 2의 실시 형태에서, 열(q)은 공급재료 슬러리(64)에 인가되고 이에 의해 촉매(42)는 불활성화되며, 그 후, 열처리 슬러리(64)는 단당류를 대사하여 생성물 알코올 (예를 들어, 부탄올)을 생성하는 알코올 생성 미생물(32)과 함께 발효 용기(30)에 도입된다. 대안적으로, 슬러리(64)는 발효 용기(30)에 공급되고, 미생물(32)의 접종 전에 발효 용기에 있는 동안 열(q)이 가해질 수 있다.In the embodiment of FIG. 2, heat q is applied to feedstock slurry 64, whereby catalyst 42 is inactivated, and heat treatment slurry 64 then metabolizes monosaccharides to produce product alcohol (eg, For example, butanol) is introduced into the fermentation vessel 30 together with the alcohol producing microorganism 32. Alternatively, slurry 64 may be supplied to fermentation vessel 30 and heat q may be applied while in fermentation vessel before inoculation of microorganism 32.

도 1을 참고로 하여 상기에 기재된 바와 같이, 유리 지방산(28)은 또한 수성 상으로부터의 생성물 알코올을 우선적으로 분배하기 위한 ISPR 추출제로서의 역할을 할 수 있다. 일부 실시 형태에서, 하나 이상의 추가의 ISPR 추출제(29)가 또한 발효 용기(30) 내로 도입될 수 있다. 2상 혼합물의 분리는 발효 용기(30) 내에서 일어나고, 이에 의해 알코올 함유 유기 상 스트림(36) 및 수성 상 스트림(34)이 발효 용기(30)로부터 직접적으로 배출된다. 대안적으로, 2상 혼합물의 분리는 도 1의 실시 형태에 제공된 바와 같이 별도의 용기(35)에서 행해질 수 있다. 도 2의 실시 형태의 남아있는 공정 작업은 도 1과 동일하며, 따라서 이는 다시 상세하게 설명되지 않을 것이다.As described above with reference to FIG. 1, the free fatty acids 28 may also serve as ISPR extractants for preferentially dispensing the product alcohol from the aqueous phase. In some embodiments, one or more additional ISPR extractants 29 may also be introduced into fermentation vessel 30. Separation of the biphasic mixture occurs in the fermentation vessel 30, whereby the alcohol containing organic phase stream 36 and the aqueous phase stream 34 are withdrawn directly from the fermentation vessel 30. Alternatively, the separation of the biphasic mixture can be done in a separate vessel 35 as provided in the embodiment of FIG. 1. The remaining process operations of the embodiment of FIG. 2 are the same as in FIG. 1, and thus will not be described in detail again.

또 다른 실시 형태에서, 공급재료(12)로부터 유래된 오일(26)은, FFA(28)를 갖는 공급재료 슬러리(18)가 액화 용기(10)로부터 직접적으로 배출되고 발효 용기(30)에 공급될 수 있도록, 액화 이전에 또는 액화 동안 FFA(28)로 촉매에 의해 가수분해될 수 있다. 예를 들어, 오일(26)을 갖는 공급재료(12)는 오일(26) 중 글리세라이드의 적어도 일부분의 FFA(28)로의 가수분해를 위하여 촉매(42)와 함께 액화 용기(10)에 공급될 수 있다. 공급재료(12) 중 전분을 가수분해시키는 효소(14) (예를 들어, 알파-아밀라아제)를 또한 용기(10)에 도입하여 액화 공급재료를 생성할 수 있다. 효소(14) 및 촉매(42)의 첨가는 단계적이거나 또는 동시적일 수 있다. 예를 들어, 촉매(42)가 도입될 수 있고, 그 후 오일(26)의 적어도 일부분이 가수분해된 후 효소(14)가 도입될 수 있다. 대안적으로, 효소(14)가 도입될 수 있으며, 그 후 촉매(42)가 도입될 수 있다. 액화 공정은 열(q)의 인가를 포함할 수 있다. 그러한 실시 형태에서, 촉매(42)는, 오일(26)이 가수분해될 수 있도록 촉매(42)를 불활성화시키는 온도보다 공정 온도가 낮을 때 액화 이전에 또는 액화 동안 도입될 수 있다. 그 후, 열(q)의 인가는 2배 목적의 액화와, 불활성화가 요구될 경우 촉매(42)의 불활성화를 제공할 수 있다.In another embodiment, the oil 26 derived from the feedstock 12 is fed with the feedstock slurry 18 having the FFA 28 discharged directly from the liquefaction vessel 10 and fed to the fermentation vessel 30. May be hydrolyzed by catalyst to FFA 28 prior to or during liquefaction. For example, feedstock 12 with oil 26 may be supplied to liquefaction vessel 10 with catalyst 42 for hydrolysis of at least a portion of the glycerides in oil 26 to FFA 28. Can be. Enzyme 14 (eg, alpha-amylase) that hydrolyzes starch in feed 12 may also be introduced into vessel 10 to produce a liquefied feedstock. The addition of enzyme 14 and catalyst 42 may be stepwise or simultaneous. For example, catalyst 42 may be introduced, and then enzyme 14 may be introduced after at least a portion of oil 26 is hydrolyzed. Alternatively, enzyme 14 may be introduced, followed by catalyst 42. The liquefaction process may include the application of heat q. In such embodiments, the catalyst 42 may be introduced before or during liquefaction when the process temperature is lower than the temperature at which the catalyst 26 is inactivated so that the oil 26 can be hydrolyzed. The application of heat q can then provide liquefaction for double purpose and inactivation of catalyst 42 if inactivation is desired.

도 1 및 도 2와 관련된 이전에 기재된 실시 형태들 중 임의의 것을 포함하는 일부 실시 형태에서, 비용해 고형물은 발효 용기(30) 내로의 도입 이전에 공급재료 슬러리로부터 제거될 수 있다. 예를 들어, 도 3의 실시 형태에 나타낸 바와 같이, 공급재료 슬러리(16)는 비용해 고형물을 고체 상 또는 습윤 케이크(24)로 배출하도록 구성된 분리기(20)의 유입구 내로 도입된다. 예를 들어, 일부 실시 형태에서, 분리기(20)는 필터 프레스(filter press), 진공 여과기, 또는 비용해 고형물을 공급재료 슬러리(16)로부터 분리하기 위한 원심분리기를 포함할 수 있다. 선택적으로, 일부 실시 형태에서, 분리기(20)는 또한 공급재료 슬러리(16)에 존재하는 오일(26)의 일부 또는 실질적으로 전부를 제거하도록 구성될 수 있다. 그러한 실시 형태에서, 분리기(20)는 사이펀 작용, 흡인, 경사분리, 원심분리, 중력 방식 침강조의 이용, 막 보조 상 분할 등을 포함하지만 이에 한정되지 않는, 수성 공급스트림으로부터 오일을 제거하기 위한 당업계에 공지된 임의의 적합한 분리기일 수 있다. 당 및 물을 포함하는 남아있는 공급재료는 수성 스트림(22)으로서 발효 용기(30)에 배출된다.In some embodiments, including any of the previously described embodiments associated with FIGS. 1 and 2, cost-effective solids may be removed from the feedstock slurry prior to introduction into fermentation vessel 30. For example, as shown in the embodiment of FIG. 3, feedstock slurry 16 is introduced into the inlet of separator 20, which is configured to costly discharge solids into solid phase or wet cake 24. For example, in some embodiments, separator 20 may include a filter press, a vacuum filter, or a centrifuge for separating inexpensive solids from feedstock slurry 16. Optionally, in some embodiments, separator 20 may also be configured to remove some or substantially all of oil 26 present in feedstock slurry 16. In such embodiments, separator 20 is intended for removing oil from an aqueous feedstream, including but not limited to siphoning, suction, decantation, centrifugation, use of gravity settling baths, membrane assisted phase separation, and the like. It may be any suitable separator known in the art. The remaining feed comprising sugar and water is discharged to fermentation vessel 30 as an aqueous stream 22.

일부 실시 형태에서, 분리기(20)는 오일(26)을 제거하지만, 비용해 고형물은 제거하지 못한다. 따라서, 발효 용기(30)에 공급되는 수성 스트림(22)은 비용해 고형물을 포함한다. 일부 실시 형태에서, 분리기(20)는 당 및 물을 함유하는 수성 층(즉, 스트림(22)), 비용해 고형물을 함유하는 고형물 층(즉, 습윤 케이크(24)), 및 오일 층(즉, 오일 스트림(26))을 포함하는 원심분리 생성물을 생성하기 위하여 공급재료 슬러리(16)를 교반시키거나 회전시키는 트라이캔터 원심분리기(tricanter centrifuge; 20)를 포함한다. 원심분리를 통하여 비용해 고형물을 공급재료 슬러리(16)로부터 제거하기 위한 방법 및 시스템은, 2010년 6월 18일자로 출원된, 본 출원과 공계류 중인, 공히 소유된 미국 가특허 출원 제61/356,290호에 상세하게 설명되어 있으며, 상기 미국 가특허 출원은 전체적으로 본 명세서에 참고로 포함된다.In some embodiments, separator 20 removes oil 26 but does not costly remove solids. Thus, the aqueous stream 22 fed to the fermentation vessel 30 inexpensively contains solids. In some embodiments, separator 20 includes an aqueous layer containing sugar and water (i.e. stream 22), a solids layer containing insoluble solids (i.e. wet cake 24), and an oil layer (i.e. And a tricanter centrifuge 20 that agitates or rotates the feed slurry 16 to produce a centrifuge product comprising oil stream 26). A method and system for costly removal of solids from feedstock slurry 16 via centrifugation is a commonly owned U.S. Provisional Patent Application No. 61 /, filed June 18, 2010. 356,290, which is incorporated herein by reference in its entirety.

어떤 경우든, 촉매(42)는 제거되는 오일(26)과 접촉하여 촉매(42)를 포함하는 FFA(28)의 스트림을 생성할 수 있으며, 이는 도 3에 나타낸 바와 같다. 그 후 열(q)을 FFA(28)의 스트림에 인가할 수 있으며, 이에 의해 촉매(42)는 불활성화되게 된다. 그 후, 불활성 촉매(42) 및 FFA(28)의 스트림은 발효 용기(30) 내로 스트림(22) 및 미생물(32)과 함께 도입될 수 있다. 대안적으로, FFA(28) 및 활성 촉매(42)는 용기(40)로부터 발효 용기(30)로 공급될 수 있으며, 활성 촉매(42)는 그 후 미생물(32)의 접종 전에 발효 용기 내에 있는 동안 열(q)이 가해져서 불활성화될 수 있다.In either case, catalyst 42 may be contacted with oil 26 to be removed to produce a stream of FFA 28 comprising catalyst 42, as shown in FIG. 3. Heat q can then be applied to the stream of FFA 28, which causes catalyst 42 to be inactivated. Thereafter, the inert catalyst 42 and the stream of FFA 28 can be introduced together with the stream 22 and the microorganism 32 into the fermentation vessel 30. Alternatively, the FFA 28 and the active catalyst 42 may be supplied from the vessel 40 to the fermentation vessel 30, which is then placed in the fermentation vessel before inoculation of the microorganism 32. Heat may be applied to deactivate it.

FFA(28)는 ISPR 추출제(28)로서의 역할을 할 수 있으며, 발효 용기(30)에서 2상 혼합물을 형성한다. SSF에 의해 생성된 생성물 알코올은 FFA(28)로 구성된 유기 상(36) 내로 분배된다. 일부 실시 형태에서, 하나 이상의 추가의 ISPR 추출제(29)가 또한 발효 용기(30) 내로 도입될 수 있다. 따라서, (예를 들어, 공급재료로부터의) 오일(26)은 촉매에 의해 FFA(28)로 가수분해되고, 이럼으로써 ISPR 추출제를 또한 생성하면서 ISPR 추출제 중 지질의 증가 속도를 감소시킬 수 있다. 유기 상(36)은 용기(35)에서 2상 혼합물(39)의 수성 상(34)으로부터 분리될 수 있다. 일부 실시 형태에서, 2상 혼합물(39)의 분리는 도 2 및 도 3에서 설명되는 실시 형태들에 나타낸 바와 같이 발효 용기에서 일어날 수 있으며, 여기서 알코올 함유 유기 상 스트림(36)은 발효 용기(30)로부터 직접적으로 배출된다. 유기 상(36)은 생성물 알코올(54)의 회수 및 회수된 추출제(27)의 선택적 재순환을 위하여 분리기(50)에 도입될 수 있으며, 이는 도 1에 나타낸 바와 같다. 도 3의 실시 형태의 남아있는 공정 작업은 도 1과 동일하며, 따라서 이는 다시 상세하게 설명되지 않을 것이다.FFA 28 may serve as ISPR extractant 28 and form a biphasic mixture in fermentation vessel 30. The product alcohol produced by the SSF is partitioned into an organic phase 36 composed of FFA 28. In some embodiments, one or more additional ISPR extractants 29 may also be introduced into fermentation vessel 30. Thus, the oil 26 (eg from the feedstock) is hydrolyzed to the FFA 28 by the catalyst, thereby reducing the rate of increase of lipids in the ISPR extractant while also producing the ISPR extractant. have. Organic phase 36 may be separated from aqueous phase 34 of biphasic mixture 39 in vessel 35. In some embodiments, separation of the biphasic mixture 39 can occur in a fermentation vessel as shown in the embodiments described in FIGS. 2 and 3, where the alcohol containing organic phase stream 36 is a fermentation vessel 30. Discharged directly). Organic phase 36 may be introduced to separator 50 for recovery of product alcohol 54 and for selective recycling of recovered extractant 27, as shown in FIG. 1. The remaining process operations of the embodiment of FIG. 3 are the same as in FIG. 1, and therefore will not be described in detail again.

습윤 케이크(24)가 원심분리기(20)를 통하여 제거될 때, 일부 실시 형태에서, 공급재료(12)로부터의 오일, 예를 들어 공급재료가 옥수수일 때 옥수수유의 일부분은 습윤 케이크(24) 내에 남아있다. 습윤 케이크(24)는, 일단 수성 용액(22)이 원심분리기(20)로부터 배출되었으면, 원심분리기에서 추가의 물로 세척될 수 있다. 습윤 케이크(24)의 세척에 의해 습윤 케이크에 존재하는 당 (예를 들어, 올리고당류)이 회수될 것이며, 회수된 당 및 물은 액화 용기(10)로 재순환될 수 있다. 세척 후, 습윤 케이크(20)는 임의의 적합한 공지된 방법을 통하여 주정잔액박(Dried Distillers' Grains with Soluble; DDGS)을 형성하도록 건조될 수 있다. 분리기(20) 내에 형성된 습윤 케이크(24)로부터의 DDGS의 형성은 몇몇 효과를 갖는다. 비용해 고형물은 발효 용기에 가지 않기 때문에, DDGS는 포획된 추출제 및/또는 생성물 알코올, 예를 들어 부탄올을 갖지 않으며, 이것은 발효 용기의 조건에 처해지지 않고, 이것은 발효 용기에 존재하는 미생물과 접촉하지 않는다. 모든 이들 효과는 DDGS를 예를 들어 동물 사료로서 가공하여 판매하는 것을 더욱 용이하게 한다. 일부 실시 형태에서, 오일(26)은 습윤 케이크(24)로부터 별도로 배출되지 않으며, 오히려 오일(26)은 습윤 케이크(24)의 일부로 포함되고 궁극적으로는 DDGS에 존재한다. 그러한 경우에, 오일은 DDGS로부터 분리되고, 동일하거나 상이한 알코올 발효 과정에서의 후속적인 사용을 위하여 ISPR 추출제(29)로 전환될 수 있다. 원심분리를 통하여 비용해 고형물을 공급재료(16)로부터 제거하기 위한 방법 및 시스템은, 2010년 6월 18일자로 출원된, 본 출원과 공계류 중인, 공히 소유된 미국 가특허 출원 제61/356,290호에 상세하게 설명되어 있으며, 상기 미국 가특허 출원은 전체적으로 본 명세서에 참고로 포함된다.When wet cake 24 is removed through centrifuge 20, in some embodiments, a portion of the corn oil from feedstock 12, such as corn oil when feedstock is corn, is in wet cake 24. Remains. The wet cake 24 may be washed with additional water in the centrifuge once the aqueous solution 22 has been withdrawn from the centrifuge 20. The washing of the wet cake 24 will recover the sugars (eg, oligosaccharides) present in the wet cake, and the recovered sugars and water can be recycled to the liquefaction vessel 10. After washing, the wet cake 20 can be dried to form Dried Distillers' Grains with Soluble (DDGS) via any suitable known method. Formation of DDGS from wet cake 24 formed in separator 20 has several effects. Since inexpensive solids do not go to the fermentation vessel, the DDGS has no entrained extractant and / or product alcohol, for example butanol, which is not subject to the conditions of the fermentation vessel, which is in contact with the microorganisms present in the fermentation vessel. I never do that. All these effects make it easier to process DDGS for example as animal feed. In some embodiments, the oil 26 is not drained separately from the wet cake 24, but rather the oil 26 is included as part of the wet cake 24 and ultimately present in the DDGS. In such a case, the oil can be separated from the DDGS and converted to ISPR extractant 29 for subsequent use in the same or different alcoholic fermentation process. A method and system for costly removal of solids from feedstock 16 via centrifugation is a commonly owned U.S. Provisional Patent Application 61 / 356,290, filed June 18, 2010. The U.S. Provisional Patent Application is described in detail in its entirety, and is incorporated herein by reference in its entirety.

또 다른 실시 형태 (예시되어 있지 않음)에서, 당업자에게 명백함에 틀림없는 바와 같이, 당화는 분리기(20)와 액화 용기(10) 사이에 위치하는 별도의 당화 용기(60)에서 일어날 수 있다 (도 2 참조).In another embodiment (not illustrated), as must be apparent to those skilled in the art, saccharification may occur in a separate saccharification vessel 60 located between separator 20 and liquefaction vessel 10 (FIG. 2).

예를 들어 도 4의 실시 형태에 나타낸 또 다른 실시 형태에서, 천연 오일(26')은 촉매(42)가 또한 공급되고 이에 의해 오일(26') 중 글리세라이드의 적어도 일부분이 가수분해되어 FFA(28')를 형성하는 용기(40)에 공급된다. 촉매(42)는 열(q)의 인가에 의한 것과 같이 후속적으로 불활성화될 수 있다. 그 후, 불활성 촉매(42) 및 FFA(28')를 함유하는 용기(40)로부터의 생성물 스트림을, 공급재료 오일(26), 그리고 일부 실시 형태에서는 비용해 고형물이 분리기(20)에 의해 이전에 제거된 수성 공급재료 스트림(22)과 함께 발효 용기(30) 내로 도입한다 (예를 들어, 도 3의 실시 형태 참조). 당화 효소(38) 및 미생물(32)이 또한 발효 용기(30) 내로 도입되며, 이에 의해 생성물 알코올이 SSF에 의해 생성된다.For example, in another embodiment shown in the embodiment of FIG. 4, natural oil 26 ′ is also supplied with catalyst 42 whereby at least a portion of the glycerides in oil 26 ′ are hydrolyzed to produce FFA ( 28 ') to a container 40 forming a container. Catalyst 42 may subsequently be deactivated, such as by application of heat q. Thereafter, the product stream from the vessel 40 containing the inert catalyst 42 and the FFA 28 ′ is transferred to the feedstock oil 26, and in some embodiments, to the solids by the separator 20. Into the fermentation vessel 30 together with the aqueous feed stream 22 removed (see, eg, the embodiment of FIG. 3). Saccharification enzyme 38 and microorganism 32 are also introduced into fermentation vessel 30 whereby product alcohol is produced by SSF.

대안적으로, 오일(26') 및 촉매(42)는 용기(40)를 사용하기보다는 오히려, 오일(26')이 FFA(28')로 가수분해되는 발효 용기(30)에 직접적으로 공급될 수 있다. 그 후, 활성 촉매(42)는 미생물(32)의 접종 전에 발효 용기 내에 있는 동안 열(q)이 가해져서 불활성화될 수 있다. 대안적으로, FFA(28') 및 활성 촉매(42)는 용기(40)로부터 발효 용기(30)로 공급될 수 있으며, 활성 촉매(42)는 그 후 미생물(32)의 접종 전에 발효 용기 내에 있는 동안 열(q)이 가해져서 불활성화될 수 있다. 그러한 실시 형태에서, 오일(26)이 제거된 스트림(22)이라기보다는 오히려 오일(26)을 포함하는 공급재료 슬러리(16)가 발효 용기(30)에 공급되고 활성 촉매(42)와 접촉될 수 있다. 따라서, 활성 촉매(42)를 사용하여 오일(26)을 FFA(28)로 가수분해시키고, 이럼으로써 발효 용기 중 오일의 존재에 기인하는, 시간이 지남에 따른 추출제의 분배 계수의 열화 및/또는 손실을 감소시킬 수 있다.Alternatively, oil 26 'and catalyst 42 may be supplied directly to fermentation vessel 30 where oil 26' is hydrolyzed to FFA 28 'rather than using vessel 40. Can be. Thereafter, the active catalyst 42 may be deactivated by applying heat q while in the fermentation vessel before inoculation of the microorganism 32. Alternatively, the FFA 28 ′ and the active catalyst 42 may be supplied from the vessel 40 to the fermentation vessel 30, where the active catalyst 42 is then in the fermentation vessel before inoculation of the microorganism 32. During this time, heat q can be applied to inactivate it. In such embodiments, rather than the stream 22 from which the oil 26 has been removed, a feedstock slurry 16 comprising the oil 26 may be supplied to the fermentation vessel 30 and contacted with the active catalyst 42. have. Thus, the active catalyst 42 is used to hydrolyze the oil 26 to the FFA 28, thereby degrading the partition coefficient of the extractant over time, due to the presence of oil in the fermentation vessel, and / or Or loss can be reduced.

일부 실시 형태에서, 도 4의 시스템 및 방법은, 당업자에게 명백함에 틀림없는 바와 같이, 발효 용기(30) 이전에 발효 용기(30)에서의 동시적 당화 및 발효가 별도의 당화 용기(60)로 대체되도록 변경될 수 있다.In some embodiments, the system and method of FIG. 4 may be used to allow simultaneous saccharification and fermentation in fermentation vessel 30 to a separate saccharification vessel 60 prior to fermentation vessel 30, as will be apparent to those skilled in the art. Can be changed to replace.

일부 실시 형태에서, 천연 오일(26')은 탤로우, 옥수수유, 카놀라유, 카프릭/카프릴릭 트라이글리세라이드, 피마자유, 코코넛유, 면실유, 어유, 호호바유, 라드, 아마인유, 니츠풋 오일, 오이티시카(oiticica)유, 야자유, 땅콩유, 평지씨유, 미강유, 잇꽃유, 대두유, 해바라기유, 동유, 자트로파유, 식물유 블렌드 및 그 혼합물일 수 있다. 일부 실시 형태에서, 천연 오일(26')은 2가지 이상의 천연 오일의 혼합물, 예를 들어 야자유와 대두유의 혼합물이다. 일부 실시 형태에서, 천연 오일(26')은 식물 유래된 오일이다. 일부 실시 형태에서, 식물 유래된 오일은 발효 과정에서 사용될 수 있는 바이오매스로부터 유래되지만 반드시 그러할 필요는 없는 것일 수 있다. 바이오매스는 (도 4에 나타낸 바와 같이 스트림(22)으로서) 공급재료(12)가 수득되는 동일하거나 상이한 공급원일 수 있다. 따라서, 예를 들어, 일부 실시 형태에서, 오일(26')은 옥수수로부터 유래될 수 있으며, 반면에 공급재료(12)는 사탕수수일 수 있다. 예를 들어, 일부 실시 형태에서, 오일(26')은 옥수수로부터 유래될 수 있으며, 공급재료(12)의 바이오매스 공급원도 옥수수이다. 당업자에게 명백함에 틀림없는 바와 같이, 공급재료(12)에 대하여 오일(26')의 상이한 바이오매스 공급원들의 임의의 가능한 조합이 사용될 수 있다. 도 4의 실시 형태의 남아있는 공정 작업은 도 1과 동일하며, 따라서 이는 다시 상세하게 설명되지 않을 것이다.In some embodiments, natural oil 26 'is tallow, corn oil, canola oil, capric / caprylic triglycerides, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard, linseed oil, nitzfoot Oils, oiticica oils, palm oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tung oil, jatropha oil, vegetable oil blends and mixtures thereof. In some embodiments, natural oil 26 'is a mixture of two or more natural oils, such as a mixture of palm oil and soybean oil. In some embodiments, natural oil 26 'is a plant derived oil. In some embodiments, the plant derived oil may be from, but need not be, a biomass that may be used in the fermentation process. The biomass can be the same or different source from which feedstock 12 is obtained (as stream 22 as shown in FIG. 4). Thus, for example, in some embodiments, oil 26 'may be derived from corn while feedstock 12 may be sugar cane. For example, in some embodiments, oil 26 'may be derived from corn, and the biomass source of feedstock 12 is also corn. As will be apparent to those skilled in the art, any possible combination of different biomass sources of oil 26 ′ may be used for feedstock 12. The remaining process operations of the embodiment of FIG. 4 are the same as in FIG. 1, and thus will not be described in detail again.

본 발명의 일부 실시 형태에서, 공급재료(12)에 존재하는 바이오매스 오일은 알코올 발효에 따른 단계에서 FFA(28)로 전환될 수 있다. 그 후 FFA(28)는 발효 용기 내에 도입되고 발효 브로쓰와 접촉될 수 있는데, 이는 알코올 생성 미생물의 개선된 성장 속도 및/또는 발효성 탄소 소비를 성취하기 위한 것이다. 또한 FFA(28)는 ISPR 추출제(28)로서의 역할을 할 수 있다. 예를 들어, 도 5의 실시 형태에서, 공급재료(12)를 액화시켜 공급재료로부터 유래되는 오일(26)을 포함하는 공급재료 슬러리(16)를 생성한다. 또한 공급재료 슬러리(16)는 공급재료로부터의 비용해 고형물을 포함할 수 있다. 대안적으로, 비용해 고형물은 분리기, 예를 들어 원심분리기 (예시되어 있지 않음)를 통하여 슬러리(16)로부터 분리될 수 있다. 오일(26)을 함유하는 공급재료 슬러리(16)는 당화 효소(38) 및 미생물(32)을 포함하는 발효 브로쓰를 함유하는 발효 용기(30)에 직접적으로 도입된다. 생성물 알코올은 발효 용기(30) 내에서 SSF에 의해 생성된다. 대안적으로, 일부 실시 형태에서, 당해 공정은 도 2와 관련하여 논의된 바와 같이 별도의 당화 용기를 포함하도록 변경될 수 있다.In some embodiments of the invention, the biomass oil present in feedstock 12 may be converted to FFA 28 in a step following alcohol fermentation. FFA 28 may then be introduced into the fermentation vessel and contacted with the fermentation broth, to achieve improved growth rates and / or fermentable carbon consumption of the alcohol producing microorganisms. FFA 28 may also serve as ISPR extractant 28. For example, in the embodiment of FIG. 5, the feedstock 12 is liquefied to produce a feedstock slurry 16 comprising oil 26 derived from the feedstock. Feedstock slurry 16 may also include insoluble solids from the feedstock. Alternatively, the insoluble solids may be separated from the slurry 16 via a separator, for example a centrifuge (not illustrated). Feedstock slurry 16 containing oil 26 is introduced directly into fermentation vessel 30 containing fermentation broth comprising saccharifying enzyme 38 and microorganism 32. Product alcohol is produced by SSF in fermentation vessel 30. Alternatively, in some embodiments, the process can be modified to include a separate saccharification container as discussed in connection with FIG. 2.

ISPR 추출제(29)를 발효 용기(30)에 도입하여 2상 혼합물을 형성하며, 생성물 알코올은 ISPR 추출제(29)의 유기 상 내로의 분배에 의해 제거된다. 또한 오일(26)은 유기 상 내로 분배된다. 2상 혼합물의 분리는 발효 용기(30) 내에서 일어나고, 이에 의해 알코올 함유 유기 상 스트림(36) 및 수성 상 스트림(34)이 발효 용기(30)로부터 직접적으로 배출된다. 대안적으로, 2상 혼합물의 분리는 도 1의 실시 형태에 제공된 바와 같이 별도의 용기(35)에서 행해질 수 있다. 오일(26)을 포함하는 유기 상 스트림(36)을 분리기(50) 내로 도입하여 생성물 알코올(54)을 추출제(29)로부터 회수한다. 생성된 알코올 린 추출제(27)는 회수된 추출제(29) 및 오일(26)을 포함한다. 추출제(27)는 촉매(42)와 접촉되며, 이에 의해 오일(26) 중 글리세라이드의 적어도 일부분이 가수분해되어 FFA(28)를 형성한다. 그 후, FFA(28)를 포함하는 추출제(27)에 열(q)을 가하여 촉매(42)를 불활성화시킨 후 이를 다시 발효 용기(30) 내로 재순환시킬 수 있다. 그러한 재순환된 추출제 스트림(27)은 별도의 스트림이거나 또는 신선한 메이크업(make-up) 추출제 스트림(29)과 조합된 스트림일 수 있다. 그러면, 알코올 함유 유기 상(36)을 발효 용기(30)로부터 후속적으로 꺼내는 것은 새롭게 도입된 공급재료 슬러리(16)로부터의 추가의 오일(26) 및 생성물 알코올에 더하여 (신선한 추출제(29) 및 재순환된 추출제(27)로서) FFA(28) 및 ISPR 추출제(29)를 포함할 수 있다. 그 후, 유기 상(36)은 생성물 알코올이 회수되도록 처리되고, 방금 기재된 것과 동일한 방식으로 추가의 오일(26)의 가수분해를 위하여 촉매(42)와 접촉된 후 발효 용기(30) 내로 다시 재순환될 수 있다. 일부 실시 형태에서, 메이크업 ISPR 추출제(29)의 사용은 발효 과정이 시간이 지남에 따라 작동될 때 단계적으로 폐지될 수 있으며, 그 이유는 상기 과정 그 자체가 FFA(28)를 생성물 알코올 추출을 위한 메이크업 ISPR 추출제로서 생성할 수 있기 때문이다. 따라서, ISPR 추출제는 FFA(28)를 포함하는 재순환된 추출제(27)의 스트림일 수 있다.ISPR extractant 29 is introduced into fermentation vessel 30 to form a biphasic mixture, with the product alcohol being removed by distribution of ISPR extractant 29 into the organic phase. Oil 26 is also dispensed into the organic phase. Separation of the biphasic mixture occurs in the fermentation vessel 30, whereby the alcohol containing organic phase stream 36 and the aqueous phase stream 34 are withdrawn directly from the fermentation vessel 30. Alternatively, the separation of the biphasic mixture can be done in a separate vessel 35 as provided in the embodiment of FIG. 1. An organic phase stream 36 comprising oil 26 is introduced into separator 50 to recover product alcohol 54 from extractant 29. The resulting alcohol lean extractant 27 includes the recovered extractant 29 and oil 26. Extractant 27 is contacted with catalyst 42, whereby at least a portion of the glycerides in oil 26 are hydrolyzed to form FFA 28. Thereafter, heat q may be applied to the extractant 27 including the FFA 28 to inactivate the catalyst 42 and then recycle it back into the fermentation vessel 30. Such recycled extractant stream 27 may be a separate stream or a stream combined with a fresh make-up extractant stream 29. Subsequently, the alcohol-containing organic phase 36 is subsequently removed from the fermentation vessel 30 in addition to the additional oil 26 and product alcohol from the freshly introduced feedstock slurry 16 (fresh extractor 29). And as recycled extractant 27) FFA 28 and ISPR extractant 29. The organic phase 36 is then treated to recover the product alcohol, contacted with the catalyst 42 for hydrolysis of the additional oil 26 in the same manner as just described and then recycled back into the fermentation vessel 30. Can be. In some embodiments, the use of make-up ISPR extractant 29 may be phased out when the fermentation process is operated over time, because the process itself may cause FFA 28 to produce product alcohol extraction. This is because it can be produced as a make-up ISPR extractant. Thus, the ISPR extractant may be a stream of recycled extractant 27 comprising FFA 28.

따라서, 도 1 내지 도 5는 발효 성장 동안 미생물과 접촉하고 이에 의해 미생물의 성장 속도 및/또는 발효성 탄소 소비량이 유리 지방산의 존재 하에서 더욱 커지게 하여 알코올 생산성의 개선을 가능하게 하는 데 사용될 수 있는, 식물 유래된 오일과 같은 천연 오일(26')의 촉매적 가수분해에 의해 생성된 FFA(28'), 및 바이오매스 유래된 오일(26)의 가수분해에 의해 생성된 FFA(28)와, 발효 공정들을 포함하는 방법 및 시스템의 다양한 비제한적 실시 형태들을 제공한다.Thus, FIGS. 1-5 can be used to contact microorganisms during fermentation growth and thereby allow the growth rate and / or fermentable carbon consumption of the microorganisms to be greater in the presence of free fatty acids to enable improvement of alcohol productivity. FFA 28 'produced by catalytic hydrolysis of natural oil 26', such as plant derived oil, and FFA 28 produced by hydrolysis of biomass derived oil 26, Various non-limiting embodiments of methods and systems including fermentation processes are provided.

상기 논의 및 실시예들로부터 당업자는 본 발명의 필수적인 특징을 확인할 수 있고, 본 발명으로부터 벗어나지 않고서도 다양한 용도 및 조건에 맞도록 본 발명을 다양하게 변화시키고 변경시킬 수 있다. 예를 들어, 일부 실시 형태에서, 본 발명에 따른 지방산 보충은 발효 전에, 즉 발효 용기(30) 내에서의 발효 이전에 미생물(32)의 시드(seed) 배양 동안 이용될 수 있다. 전형적으로, 효모와 같은 미생물(32)은 시드 배양에 의해 원하는 세포 농도로 성장시킨 후 수확하고, 발효 용기(30) 내에 접종할 수 있으며, 이는 당업계에 공지된 바와 같다. 따라서, 일부 실시 형태에 따르면, 시드 배양 배지를 FFA(28)와 접촉시킬 수 있고 이에 의해 향상된 성장 속도 및 미생물 바이오매스 생성이 성취될 수 있는데, 이는 알코올 발효 과정의 시드 배양기와 관련된 발효 전 시간을 감소시킬 수 있다. 따라서, 본 발명에 따른 지방산 보충은, 본 발명으로부터 벗어나지 않고서 전체 공정 효율을 향상시키기 위하여 알코올 발효 과정 중 다양한 단계에, 예를 들어 발효 전 배양 및 발효 동안 이용될 수 있음이 명백함에 틀림없다.Those skilled in the art can identify essential features of the present invention from the above discussion and examples, and various changes and modifications can be made to the present invention to suit various applications and conditions without departing from the present invention. For example, in some embodiments, fatty acid supplements according to the present invention may be used during seed culture of microorganisms 32 prior to fermentation, ie, prior to fermentation in fermentation vessel 30. Typically, microorganisms 32, such as yeast, can be grown to seed cell concentration by seed culture, harvested, and seeded in fermentation vessel 30, as is known in the art. Thus, according to some embodiments, the seed culture medium may be contacted with the FFA 28 whereby improved growth rates and microbial biomass production may be achieved, which may reduce the time before fermentation associated with the seed incubator of the alcohol fermentation process. Can be reduced. Thus, it must be apparent that fatty acid supplements according to the invention can be used at various stages of the alcoholic fermentation process, for example during pre-fermentation and during fermentation, in order to improve the overall process efficiency without departing from the invention.

도 1 내지 도 5를 참고로 하여 설명되는 전술한 실시 형태들 중 임의의 것을 포함하는 일부 실시 형태에서, 발효 용기(30) 내의 발효 브로쓰는 적어도 하나의 발효성 탄소 공급원으로부터의 부탄올로의 생합성 경로를 통하여 부탄올을 생성하도록 유전자 변형된(즉, 유전자 조작된) 적어도 하나의 재조합 미생물(32)을 포함한다. 특히, 재조합 미생물을 적합한 탄소 기질을 함유하는 발효 배지에서 성장시킬 수 있다. 추가의 탄소 기질은 단당류, 예를 들어 프룩토스; 올리고당류, 예를 들어 락토스, 말토스 또는 수크로스; 다당류, 예를 들어 전분 또는 셀룰로오스; 또는 그 혼합물과, 재생가능한 공급재료로부터의 정제되지 않은 혼합물, 예를 들어 유장(cheese whey) 투과물, 옥수수 침지액, 사탕무 당밀 및 보리 맥아를 포함할 수 있지만, 이에 한정되지 않는다. 다른 탄소 기질은 에탄올, 락테이트, 석시네이트 또는 글리세롤을 포함할 수 있다.In some embodiments, including any of the foregoing embodiments described with reference to FIGS. 1-5, the fermentation broth in fermentation vessel 30 is a biosynthetic pathway from but at least one fermentable carbon source to butanol. At least one recombinant microorganism 32 genetically modified (ie, genetically engineered) to produce butanol through. In particular, recombinant microorganisms can be grown in fermentation medium containing a suitable carbon substrate. Additional carbon substrates include monosaccharides such as fructose; Oligosaccharides such as lactose, maltose or sucrose; Polysaccharides such as starch or cellulose; Or mixtures thereof, and crude mixtures from renewable feedstocks, such as, but not limited to, whey permeate, corn steep liquor, beet molasses and barley malt. Other carbon substrates can include ethanol, lactate, succinate or glycerol.

또한, 탄소 기질은 C1 기질, 예를 들어, 이산화탄소 또는 메탄올 - 이에 대해서는 중요 생화학적 중간체로의 대사적 전환이 입증됨 - 일 수도 있다. 메틸요구성 유기체(methylotrophic organism)는 또한, C1 및 C2 기질에 더하여 다수의 다른 탄소 함유 화합물, 예를 들어 메틸아민, 글루코사민 및 다양한 아미노산을 대사 활성에 이용하는 것으로 공지되어 있다. 예를 들어, 메틸요구성 효모는 메틸아민 유래의 탄소를 이용하여 트레할로스 또는 글리세롤을 형성하는 것으로 공지되어 있다 (문헌[Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK]). 이와 유사하게, 다양한 종의 칸디다는 알라닌 또는 올레산을 대사할 것이다 (문헌[Sulter et al., Arch. Microbiol. 153:485-489, 1990]). 그러므로, 본 발명에 이용되는 탄소 공급원은 광범위한 탄소 함유 기질을 포함할 수 있으며 유기체의 선택에 의해서만 한정되는 것으로 생각된다.The carbon substrate may also be a C1 substrate, such as carbon dioxide or methanol, in which metabolic conversion to important biochemical intermediates is demonstrated. Methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and various amino acids in addition to the C1 and C2 substrates for metabolic activity. For example, methylyoconstitutive yeast is known to form trehalose or glycerol using carbon derived from methylamine (Bellion et al., Microb. Growth C1 Compd., Int. Symp., 7th). (1993), 415-32, Editor (s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK]. Similarly, Candida of various species will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153: 485-489, 1990). Therefore, the carbon source used in the present invention may include a wide range of carbon containing substrates and is believed to be limited only by the choice of organism.

상술된 탄소 기질 및 그 혼합물 모두가 적합한 것으로 고려되지만, 일부 실시형태에서, 탄소 기질은 글루코스, 프룩토스 및 수크로스, 또는 C5 당을 사용하도록 변형된 효모를 위한 자일로스 및/또는 아라비노스와 같은 C5 당과의 이들 기질의 혼합물이다. 수크로스는 재생산가능한 당 공급원, 예를 들어 사탕수수, 사탕무, 카사바(cassava), 단 수수(sweet sorghum) 및 그 혼합물로부터 유래될 수 있다. 글루코스 및 덱스트로스는 옥수수, 밀, 호밀, 보리, 귀리 및 그 혼합물과 같은 곡물을 비롯한 전분 계의 공급재료의 당화를 통하여 재생산가능한 곡물 공급원으로부터 유래될 수 있다. 게다가, 발효성 당은 예를 들어, 본 명세서에 참고로 포함된 미국 특허 공개 제2007/0031918 A1호에 기재된 바와 같이, 전처리 및 당화 과정을 통하여 재생가능한 셀룰로오스계 또는 리그노셀룰로오스계 바이오매스로부터 유래될 수 있다. (수성 스트림(22)로부터의) 적절한 탄소 공급원 외에, 발효 브로쓰는 적합한 미네랄, 염, 보조인자, 완충제 및 다른 성분들 - 당업자에게 공지되어 있으며 다이하이드록시산 데하이드라타아제(DHAD)를 포함하는 효소 경로의 촉진 및 배양물의 성장에 적합함 - 을 함유해야 한다.Although all of the above-described carbon substrates and mixtures thereof are considered suitable, in some embodiments, the carbon substrate is such as xylose and / or arabinose for yeast modified to use glucose, fructose and sucrose, or C5 sugars. Mixtures of these substrates with C5 sugars. Sucrose can be derived from a reproducible sugar source, for example sugar cane, sugar beet, cassava, sweet sorghum and mixtures thereof. Glucose and dextrose may be derived from reproducible grain sources through the saccharification of feedstocks of starch systems, including grains such as corn, wheat, rye, barley, oats and mixtures thereof. In addition, the fermentable sugars are derived from cellulosic or lignocellulosic biomass that is renewable through pretreatment and saccharification, as described, for example, in US Patent Publication No. 2007/0031918 A1, which is incorporated herein by reference. Can be. In addition to a suitable carbon source (from aqueous stream 22), the fermentation broth is suitable minerals, salts, cofactors, buffers and other ingredients-known to those skilled in the art and include dihydroxy acid dehydratase (DHAD) Suitable for the promotion of enzymatic pathways and for the growth of cultures.

생합성 경로를 통하여 부탄올을 생성하는 재조합 미생물은 클로스트리듐(Clostridium), 자이모모나스(Zymomonas), 에스케리키아(Escherichia), 살모넬라(Salmonella), 세라티아(Serratia), 에르위니아(Erwinia), 클렙시엘라(Klebsiella), 시겔라(Shigella), 로도코쿠스(Rhodococcus), 슈도모나스, 바실루스, 락토바실루스, 엔테로코쿠스, 알칼리게네스, 클렙시엘라, 파에니바실루스(Paenibacillus), 아르트로박터(Arthrobacter), 코리네박테륨(Corynebacterium), 브레비박테륨(Brevibacterium), 쉬조사카로마이세스(Schizosaccharomyces), 클루이베로마이세스(Kluyveromyces), 야로위아, 피키아(Pichia), 칸디다, 한세눌라 또는 사카로마이세스 속의 구성원을 포함할 수 있다. 일 실시 형태에서, 재조합 미생물은 에스케리키아 콜라이, 락토바실러스 플란타룸(Lactobacillus plantarum) 및 사카로마이세스 세레비지에로 이루어진 군으로부터 선택될 수 있다. 일 실시 형태에서, 재조합 미생물은 사카로마이세스, 자이고사카로마이세스(Zygosaccharomyces), 쉬조사카로마이세스(Schizosaccharomyces), 데케라(Dekkera), 토룰롭시스(Torulopsis), 브레타노마이세스(Brettanomyces) 및 칸디다의 일부 종으로부터 선택되는 크랩트리 양성 효모이다. 크랩트리 양성 효모의 종은 사카로마이세스 세레비지애, 사카로마이세스 클루이베리(Saccharomyces kluyveri), 쉬조사카로마이세스 폼베(Schizosaccharomyces pombe), 사카로마이세스 바야누스(Saccharomyces bayanus), 사카로마이세스 미키타에(Saccharomyces mikitae), 사카로마이세스 파라독수스(Saccharomyces paradoxus), 자이고사카로마이세스 로욱시이(Zygosaccharomyces rouxii), 및 칸디다 글라브라타(Candida glabrata)를 포함하지만, 이에 한정되지 않는다. 예를 들어, 어떠한 미생물이 부탄올을 생성하는지 뿐만 아니라 미생물에 의한 발효를 이용한 부탄올의 생성도 공지되어 있으며, 이는 본 명세서에 참고로 포함된 미국 특허 공개 제2009/0305370호에 개시되어 있다. 일부 실시 형태에서, 미생물은 부탄올 생합성 경로를 포함한다. 적합한 아이소부탄올 생합성 경로는 당업계에 공지되어 있다 (예를 들어, 본 명세서에 참고로 포함된 미국 특허 공개 제2007/0092957호 참조). 일부 실시 형태에서, 경로의 기질의 생성물로의 전환을 촉매하는 적어도 하나, 적어도 2가지, 적어도 3가지 또는 적어도 4가지의 폴리펩티드는 미생물 내의 비상동성 폴리뉴클레오티드에 의해 코딩된다. 일부 실시 형태에서, 경로의 기질의 생성물로의 전환을 촉매하는 모든 폴리펩티드는 미생물 내의 비상동성 폴리뉴클레오티드에 의해 코딩된다. 일부 실시 형태에서, 미생물은 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함한다. 피루베이트 데카르복실라아제 활성이 실질적으로 없는 미생물이 본 명세서에 참고로 포함된 미국 특허 공개 제2009/0305363호에 기재되어 있다.Recombinant microorganism generating butanol via a biosynthetic pathway is Clostridium (Clostridium), Eisai thigh eggplant (Zymomonas), Escherichia (Escherichia), Salmonella (Salmonella), Serratia marcescens (Serratia), El Winiah (Erwinia), keulrep Ciel La (Klebsiella), Shigella (Shigella), also co kusu (Rhodococcus), Pseudomonas, Bacillus, Lactobacillus, Enterobacter nose Syracuse, Alcaligenes, keulrep when Ella, wave Enigma Bacillus (Paenibacillus), are Trojan bakteo (Arthrobacter ), Corynebacterium , Brevibacterium , Schizosaccharomyces , Kluyveromyces , Yarrowia, Pichia , Candida, Hansenula or Saccharomyces It may include members of the Seth. In one embodiment, the recombinant microorganism is Escherichia coli, Lactobacillus Planta Room (Lactobacillus plantarum ) and Saccharomyces cerevisiae. In one embodiment, the recombinant microorganism is Saccharomyces to my process, my process (Zygosaccharomyces), Shh irradiated with Xi Kosaka Caro My process (Schizosaccharomyces), having Mosquera (Dekkera), sat rulrop sheath (Torulopsis), Breda Gaetano My process (Brettanomyces ) And Crabtree positive yeast selected from some species of Candida. Species of Crabtree positive yeast are Saccharomyces and Saccharomyces. kluyveri , Schizosaccharomyces pombe), saccharose in my process bar Janus (Saccharomyces bayanus), my process Miki to the other saccharide (Saccharomyces mikitae ), Saccharomyces paradoxus ), Zygosaccharomyces rouxii ), and Candida glabrata . For example, not only which microorganism produces butanol, but also the production of butanol using fermentation by the microorganism is disclosed in US Patent Publication No. 2009/0305370, which is incorporated herein by reference. In some embodiments, the microorganism comprises a butanol biosynthetic pathway. Suitable isobutanol biosynthesis pathways are known in the art (see, eg, US Patent Publication No. 2007/0092957, incorporated herein by reference). In some embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze the conversion of a substrate to a product of a pathway are encoded by nonhomologous polynucleotides in a microorganism. In some embodiments, all polypeptides that catalyze the conversion of pathway substrates to products are encoded by nonhomologous polynucleotides in a microorganism. In some embodiments, the microorganism comprises reducing or eliminating pyruvate decarboxylase activity. Microorganisms substantially free of pyruvate decarboxylase activity are described in US Patent Publication No. 2009/0305363, which is incorporated herein by reference.

실시예에서 사용되는 것들을 포함하는 소정의 스트레인의 제작이 본 명세서에 제공된다.Provided herein are fabrication of certain strains, including those used in the Examples.

사카로마이세스 세레비지애 스트레인 BP1083 ("NGCI-070")의 제작Production of Saccharomyces cerevisiae strain BP1083 ("NGCI-070")

스트레인 BP1064는 CEN.PK 113-7D (CBS 8340; CBS (Centraalbureau voor Schimmelcultures) 진균류 생물다양성 센터, 네덜란드)로부터 유래하며, 하기의 유전자의 결실을 함유한다: URA3, HIS3, PDC1, PDC5, PDC6 및 GPD2. BP1064는 플라스미드 pYZ090 (서열 번호 1, 미국 가특허 출원 제61/246,844호에 개시됨) 및 pLH468 (서열 번호 2)로 형질전환되어 스트레인 NGCI-070 (BP1083, PNY1504)을 생성하였다.Strain BP1064 is derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6 and GPD2. . BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1, disclosed in US Provisional Patent Application 61 / 246,844) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083, PNY1504).

전체 코딩 서열이 완전히 제거된 결실을 형질전환체의 선발을 위한 G418 내성 마커 또는 URA3 유전자 중 어느 하나, 및 표적 유전자의 상류 및 하류의 상동성 영역을 함유하는 PCR 단편과의 상동성 재조합에 의해 생성하였다. loxP 부위가 측면에 있는 G418 내성 마커를 Cre 리콤비나아제(recombinase)를 사용하여 제거하였다. URA3 유전자를 상동성 재조합에 의해 제거하여, 스카리스(scarless) 결실을 생성하거나, loxP 부위가 측면에 있다면, Cre 리콤비나아제를 사용하여 제거하였다.Deletion in which the entire coding sequence has been completely removed is generated by homologous recombination with a PCR fragment containing either the G418 resistance marker or the URA3 gene for selection of the transformant and the homologous regions upstream and downstream of the target gene. It was. G418 resistance markers flanked by loxP sites were removed using Cre recombinase. The URA3 gene was removed by homologous recombination to produce a scarless deletion or, if the loxP site was flanked, using Cre recombinase.

스카리스 결실 절차를 문헌[Akada et al., Yeast, 23:399-405 (2006)]으로부터 수정하였다. 일반적으로, 각각의 스카리스 결실을 위한 PCR 카세트를 중첩 PCR에 의하여 4개의 단편, 즉, A-B-U-C를 조합함으로써 만들었다. 프로모터(URA3 유전자의 상류의 250 bp) 및 종결자 (URA3 유전자의 하류의 150 bp)와 함께 천연 CEN.PK 113-7D URA3 유전자로 이루어진 PCR 카세트는 선발가능한/역선발가능한 마커, URA3 (단편 U)을 함유하였다. 각각 500 bp 길이의 단편 A 및 C는 표적 유전자의 바로 상류의 500 bp(단편 A) 및 표적 유전자의 3' 500 bp(단편 C)에 해당한다. 단편 A 및 C를 상동성 재조합에 의한 염색체 내로의 카세트의 통합을 위하여 사용하였다. 단편 B(500 bp 길이)는 표적 유전자의 바로 하류의 500 bp에 해당하며, 이를 상동성 재조합에 의한 염색체로부터의 URA3 마커 및 단편 C의 절제(excision)를 위해 사용하였는데, 그 이유는 단편 B에 해당하는 서열의 직접 반복이 염색체 내로의 카세트의 통합 시에 생성되었기 때문이다. PCR 생성물 ABUC 카세트를 사용하여, URA3 마커를 먼저 상동성 재조합에 의하여 염색체 내로 통합시킨 다음, 염색체로부터 절제하였다. 초기의 통합에 의하여 유전자를 결실시켜 3' 500 bp가 제외되게 하였다. 절제 시에, 유전자의 3' 500 bp 영역도 또한 결실되었다. 이러한 방법을 사용한 유전자의 통합을 위하여, 통합될 유전자는 단편 A와 단편 B 사이의 PCR 카세트에 포함시켰다.The Scarris deletion procedure was modified from Akada et al., Yeast, 23: 399-405 (2006). In general, PCR cassettes for each scarris deletion were made by combining four fragments, namely A-B-U-C, by overlapping PCR. The PCR cassette consisting of the native CEN.PK 113-7D URA3 gene with a promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene) was selected as a selectable / reversible marker, URA3 (fragment U). ). Fragments A and C, each 500 bp in length, correspond to 500 bp directly upstream of the target gene (fragment A) and 3 ′ 500 bp (fragment C) of the target gene. Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp in length) corresponds to 500 bp immediately downstream of the target gene and was used for excision of URA3 marker and fragment C from the chromosome by homologous recombination, because This is because direct repetition of the corresponding sequences was generated upon incorporation of the cassette into the chromosome. Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome by homologous recombination and then excised from the chromosome. Initial integration caused the gene to be deleted, excluding 3 '500 bp. Upon ablation, the 3 '500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in a PCR cassette between fragment A and fragment B.

URA3 결실URA3 deletion

내인성 URA3 코딩 영역을 결실시키기 위하여, ura3::loxP-kanMX-loxP 카세트를 pLA54 주형 DNA (서열 번호 3)로부터 PCR 증폭시켰다. pLA54는 케이. 락티스 TEF1 프로모터 및 kanMX 마커를 함유하며, loxP 부위가 측면에 있어서, Cre 리콤비나아제를 사용한 재조합 및 마커의 제거를 가능하게 하였다. PCR은 퓨전(Phusion)(등록상표) DNA 폴리머라아제 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.(New England BioLabs Inc.)) 및 프라이머 BK505 및 BK506 (서열 번호 4 및 5)을 사용하여 행하였다. 각 프라이머의 URA3 부분은 URA3 프로모터의 상류의 5' 영역 및 코딩 영역의 하류의 3' 영역으로부터 유래되어, loxP-kanMX-loxP 마커의 통합이 URA3 코딩 영역의 대체로 이어지게 하였다. 표준 유전자 기술을 사용하여 PCR 생성물로 CEN.PK 113-7D를 형질전환시켰고(문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]), G418 (100 ㎍/mL)을 함유하는 YPD 상에서 30℃에서 형질전환체를 선발하였다. 형질전환체를 프라이머 LA468 및 LA492 (서열 번호 6 및 7)를 사용하여 PCR에 의해 스크리닝하여 올바른 통합을 확인하고, CEN.PK 113-7D Δura3::kanMX로 표기하였다.To delete the endogenous URA3 coding region, the ura3 :: loxP-kanMX-loxP cassette was PCR amplified from pLA54 template DNA (SEQ ID NO: 3). pLA54, It contains the Lactis TEF1 promoter and kanMX marker, and the loxP site flanked to allow recombination and removal of the marker using Cre recombinase. PCR uses Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) And primers BK505 and BK506 (SEQ ID NOs: 4 and 5) Was carried out. The URA3 portion of each primer was derived from the 5 'region upstream of the URA3 promoter and the 3' region downstream of the coding region, leading to the integration of the loxP-kanMX-loxP marker leading to replacement of the URA3 coding region. CEN.PK 113-7D was transformed with PCR products using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), G418 Transformants were selected at 30 ° C. on YPD containing (100 μg / mL). Transformants were screened by PCR using primers LA468 and LA492 (SEQ ID NOs: 6 and 7) to confirm correct integration and designated CEN.PK 113-7D Δura3 :: kanMX.

HIS3 결실HIS3 fruit

스카리스 HIS3 결실을 위한 PCR 카세트를 위한 4개의 단편을 퓨젼(등록상표) 고충실도 PCR 마스터 믹스 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스) 및 젠트라(Gentra)(등록상표) 퓨어진(Puregene)(등록상표) 이스트(Yeast)/백트(Bact) 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠(Qiagen))에 의해 제조된 주형으로서의 CEN.PK 113-7D 게놈 DNA를 사용하여 증폭시켰다. HIS3 단편 A를 프라이머 oBP452 (서열 번호 14) 및 프라이머 oBP453 (서열 번호 15) - HIS3 단편 B의 5' 말단에 대하여 상동성을 갖는 5' 테일(tail)을 함유함 - 을 이용하여 증폭시켰다. HIS3 단편 B는 HIS3 단편 A의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP454 (서열 번호 16) 및 HIS3 단편 U의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP455 (서열 번호 17)를 이용하여 증폭시켰다. HIS3 단편 U는 HIS3 단편 B의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP456 (서열 번호 18) 및 HIS3 단편 C의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP457 (서열 번호 19)을 이용하여 증폭시켰다. HIS3 단편 C는 HIS3 단편 U의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP458 (서열 번호 20) 및 프라이머 oBP459 (서열 번호 21)를 이용하여 증폭시켰다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다. HIS3 단편 AB를 HIS3 단편 A 및 HIS3 단편 B를 혼합하고, 프라이머 oBP452 (서열 번호 14) 및 oBP455 (서열 번호 17)를 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. HIS3 단편 UC를 HIS3 단편 U 및 HIS3 단편 C를 혼합하고, 프라이머 oBP456 (서열 번호 18) 및 oBP459 (서열 번호 21)를 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. 생성된 PCR 생성물을 아가로스 겔, 이어서 겔 추출 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 정제하였다. HIS3 ABUC 카세트를 HIS3 단편 AB 및 HIS3 단편 UC를 혼합하고 프라이머 oBP452 (서열 번호 14) 및 oBP459 (서열 번호 21)를 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다.Four fragments for PCR cassettes for Scarris HIS3 deletion were fused with Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, Mass.) And Gentra® Pure Amplified using CEN.PK 113-7D genomic DNA as a template prepared by the Yeast / Bact kit (Qiagen, Valencia, Calif.). HIS3 fragment A was amplified using primers oBP452 (SEQ ID NO: 14) and primers oBP453 (SEQ ID NO: 15), which contained a 5 'tail homologous to the 5' end of HIS3 fragment B. HIS3 fragment B contains primer oBP454 (SEQ ID NO: 16) containing a 5 'tail homologous to the 3' end of HIS3 fragment A and a 5 'tail homologous to the 5' end of HIS3 fragment U Amplification using primers oBP455 (SEQ ID NO: 17). HIS3 fragment U contains primer oBP456 (SEQ ID NO: 18) containing a 5 'tail homologous to the 3' end of HIS3 fragment B and a 5 'tail homologous to the 5' end of HIS3 fragment C Amplification using primers oBP457 (SEQ ID NO: 19). HIS3 fragment C was amplified using primers oBP458 (SEQ ID NO: 20) and primer oBP459 (SEQ ID NO: 21) containing a 5 'tail homologous to the 3' end of HIS3 fragment U. PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA). HIS3 fragment AB was generated by overlapping PCR by mixing HIS3 fragment A and HIS3 fragment B and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO: 17). HIS3 fragment UC was generated by overlapping PCR by mixing HIS3 fragment U and HIS3 fragment C and amplifying with primers oBP456 (SEQ ID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR product was purified with an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). HIS3 ABUC cassettes were generated by overlapping PCR by mixing HIS3 fragment AB and HIS3 fragment UC and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA).

CEN.PK 113-7D Δura3::kanMX의 적격(competent) 세포를 만들고, 프로즌-이지 이스트 트랜스포메이션(Frozen-EZ Yeast Transformation) II™ 키트 (미국 캘리포니아주 어바인 소재의 자이모 리서치 코포레이션(Zymo Research Corporation))를 사용하여 HIS3 ABUC PCR 카세트로 형질전환시켰다. 형질전환 혼합물을 30℃에서 2% 글루코스가 보충된, 우라실이 결여된 합성 완전 배지 상에 도말하였다. his3 넉아웃이 있는 형질전환체를 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 제조한 게놈 DNA를 사용하여 프라이머 oBP460 (서열 번호 22) 및 oBP461 (서열 번호 23)을 사용하여 PCR에 의해 스크리닝하였다. 올바른 형질전환체를 스트레인 CEN.PK 113-7D Δura3::kanMX Δhis3::URA3으로서 선발하였다.Create competent cells of CEN.PK 113-7D Δura3 :: kanMX and use the Frozen-EZ Yeast Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA, USA )) Was used to transform the HIS3 ABUC PCR cassette. Transformation mixtures were plated on synthetic complete medium lacking uracil at 30 ° C. supplemented with 2% glucose. Transformants with his3 knockout were prepared using primers oBP460 (SEQ ID NO: 22) using genomic DNA prepared with Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA); Screened by PCR using oBP461 (SEQ ID NO: 23). The correct transformants were selected as strain CEN.PK 113-7D Δura3 :: kanMX Δhis3 :: URA3.

Δura3 부위로부터의 KanMX 마커의 제거 및 Δhis3 부위로부터의 URA3 마커의 제거Removal of KanMX Marker from Δura3 Site and Removal of URA3 Marker from Δhis3 Site

KanMX 마커는 프로즌-이지 이스트 트랜스포메이션 II™ 키트 (미국 캘리포니아주 어바인 소재의 자이모 리서치 코포레이션)를 사용하여 CEN.PK 113-7D Δura3::kanMX Δhis3::URA3을 pRS423::PGAL1-cre (서열 번호 66, 미국 가특허 출원 제61/290,639호에 개시됨)로 형질전환시키고, 30℃에서 2% 글루코스가 보충된, 히스티딘 및 우라실이 결여된 합성 완전 배지 상에 도말함으로써 제거하였다. 형질전환체는 30℃에서 1% 갈락토스가 보충된 YP에서 대략 6시간 동안 성장시켜 Cre 리콤비나아제 및 KanMX 마커 절제를 유도하고, 회수를 위하여 30℃에서 YPD (2% 글루코스) 플레이트 상에 도말하였다. 단리체를 YPD에서 하룻밤 성장시키고, 30℃에서 5-플루오로-오로트산 (5-FOA, 0.1%)을 함유하는 합성 완전 배지 상에 도말하여, URA3 마커가 손실된 단리체를 선발하였다. 5-FOA 내성 단리체를 pRS423::PGAL1-cre 플라스미드의 제거를 위하여 YPD에서 성장시켜 YPD 상에 도말하였다. YPD+G418 플레이트, 우라실 결여 합성 완전 배지 플레이트 및 히스티딘 결여 합성 완전 배지 플레이트에서 성장을 분석함으로써 단리체를 KanMX 마커, URA3 마커, 및 pRS423::PGAL1-cre 플라스미드의 손실에 대하여 체크하였다. G418에 대하여 민감하며, 우라실 및 히스티딘에 대하여 영양요구성인 올바른 단리체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3으로서 선발하고, BP857로 표기하였다. 상기 결실들 및 마커 제거는 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 사용하여 제조된 게놈 DNA를 이용하여 Δura3의 경우 프라이머 oBP450 (서열 번호 24) 및 oBP451 (서열 번호 25), 그리고 Δhis3의 경우 프라이머 oBP460 (서열 번호 22) 및 oBP461 (서열 번호 23)을 이용하여 PCR 및 서열결정에 의해 확인하였다.KanMX markers were replaced with CEN.PK 113-7D Δura3 :: kanMX Δhis3 :: URA3 using the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). No. 66, disclosed in US Provisional Patent Application 61 / 290,639) and removed by plating onto synthetic complete medium lacking histidine and uracil, supplemented with 2% glucose at 30 ° C. Transformants were grown for approximately 6 hours in YP supplemented with 1% galactose at 30 ° C. to induce Cre recombinase and KanMX marker ablation, and plated on YPD (2% glucose) plates at 30 ° C. for recovery. It was. The isolate was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orroic acid (5-FOA, 0.1%) at 30 ° C. to isolate isolates with missing URA3 markers. 5-FOA resistant isolates were grown on YPD and plated on YPD for removal of the pRS423 :: PGAL1-cre plasmid. Isolates were checked for loss of KanMX markers, URA3 markers, and pRS423 :: PGAL1-cre plasmids by analyzing growth in YPD + G418 plates, uracil lacking synthetic complete medium plates and histidine lacking synthetic complete medium plates. The correct isolates sensitive to G418 and nutritional to uracil and histidine were selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 and designated BP857. The deletions and marker removal were carried out using primers oBP450 (SEQ ID NO.) For Δura3 using genomic DNA prepared using the Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA). 24) and oBP451 (SEQ ID NO: 25), and for Δhis3 were confirmed by PCR and sequencing using primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23).

PDC6 결실PDC6 deletion

스카리스 PDC6 결실을 위한 PCR 카세트를 위한 4개의 단편을 퓨젼(등록상표) 고충실도 PCR 마스터 믹스 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스) 및 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)에 의해 제조된 주형으로서의 CEN.PK 113-7D 게놈 DNA를 사용하여 증폭시켰다. PDC6 단편 A를 프라이머 oBP440 (서열 번호 26) 및 프라이머 oBP441 (서열 번호 27) - PDC6 단편 B의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유함 - 을 이용하여 증폭시켰다. PDC6 단편 B는 PDC6 단편 A의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP442 (서열 번호 28), 및 PDC6 단편 U의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP443 (서열 번호 29)를 이용하여 증폭시켰다. PDC6 단편 U는 PDC6 단편 B의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP444 (서열 번호 30) 및 PDC6 단편 C의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP445 (서열 번호 31)를 이용하여 증폭시켰다. PDC6 단편 C는 PDC6 단편 U의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP446 (서열 번호 32) 및 프라이머 oBP447 (서열 번호 33)을 이용하여 증폭시켰다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다. PDC6 단편 AB를 PDC6 단편 A 및 PDC6 단편 B를 혼합하고, 프라이머 oBP440 (서열 번호 26) 및 oBP443 (서열 번호 29)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. PDC6 단편 UC를 PDC6 단편 U 및 PDC6 단편 C를 혼합하고, 프라이머 oBP444 (서열 번호 30) 및 oBP447 (서열 번호 33)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. 생성된 PCR 생성물을 아가로스 겔, 이어서 겔 추출 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 정제하였다. PDC6 ABUC 카세트를 PDC6 단편 AB 및 PDC6 단편 UC를 혼합하고 프라이머 oBP440 (서열 번호 26) 및 oBP447 (서열 번호 33)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다.Four fragments for PCR cassettes for Scarris PDC6 deletion were incorporated into a Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, Mass.) And Gentra® Fused® Yeast. Amplification was performed using CEN.PK 113-7D genomic DNA as template prepared by Bait Kit (Qiagen, Valencia, CA). PDC6 fragment A was amplified using primers oBP440 (SEQ ID NO: 26) and primer oBP441 (SEQ ID NO: 27), which contained a 5 'tail homologous to the 5' end of PDC6 fragment B. PDC6 fragment B contains primer oBP442 (SEQ ID NO: 28) containing a 5 'tail homologous to the 3' end of PDC6 fragment A, and a 5 'tail homologous to the 5' end of PDC6 fragment U Amplified using primers oBP443 (SEQ ID NO: 29). PDC6 fragment U contains primer oBP444 (SEQ ID NO: 30) containing a 5 'tail homologous to the 3' end of PDC6 fragment B and a 5 'tail homologous to the 5' end of PDC6 fragment C Amplification using primers oBP445 (SEQ ID NO: 31). PDC6 fragment C was amplified using primers oBP446 (SEQ ID NO: 32) and primer oBP447 (SEQ ID NO: 33) containing a 5 'tail homologous to the 3' end of PDC6 fragment U. PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA). PDC6 fragment AB was generated by overlapping PCR by mixing PDC6 fragment A and PDC6 fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ ID NO: 29). PDC6 fragment UC was generated by overlapping PCR by mixing PDC6 fragment U and PDC6 fragment C and amplifying with primers oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR product was purified with an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). PDC6 ABUC cassettes were generated by overlapping PCR by mixing PDC6 fragment AB and PDC6 fragment UC and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA).

CEN.PK 113-7D Δura3::loxP Δhis3의 적격 세포를 만들고, 프로즌-이지 이스트 트랜스포메이션 II™ 키트 (미국 캘리포니아주 어바인 소재의 자이모 리서치 코포레이션)를 사용하여 PDC6 ABUC PCR 카세트로 형질전환시켰다. 형질전환 혼합물을 30℃에서 2% 글루코스가 보충된 우라실이 결여된 합성 완전 배지 상에 도말하였다. pdc6 넉아웃이 있는 형질전환체를 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 제조한 게놈 DNA를 사용하여 프라이머 oBP448 (서열 번호 34) 및 oBP449 (서열 번호 35)를 사용하여 PCR에 의해 스크리닝하였다. 올바른 형질전환체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3으로서 선발하였다.Eligible cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 were made and transformed with a PDC6 ABUC PCR cassette using the Frozen-Easy East Transformation II ™ kit (Zymo Research Corporation, Irvine, CA). Transformation mixtures were plated at 30 ° C. on synthetic complete medium lacking uracil supplemented with 2% glucose. Transformants with pdc6 knockout were prepared using primers oBP448 (SEQ ID NO: 34) using genomic DNA prepared with Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA); Screened by PCR using oBP449 (SEQ ID NO: 35). The correct transformants were selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3을 YPD에서 하룻밤 성장시키고, 30℃에서 5-플루오로-오로트산 (0.1%)을 함유하는 합성 완전 배지 상에 도말하여, URA3 마커가 손실된 단리체를 선발하였다. 상기 결실 및 마커 제거는 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 사용하여 제조된 게놈 DNA를 이용하여 프라이머 oBP448 (서열 번호 34) 및 oBP449 (서열 번호 35)를 이용하여 PCR 및 서열결정에 의해 확인하였다. 단리체로부터의 PDC6 유전자의 부재는 PDC6의 코딩 서열에 대하여 특이적인 프라이머, oBP554 36) 및 oBP555 (서열 번호 37)를 사용하여 음성 PCR 결과에 의해 입증하였다. 올바른 단리체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6으로서 선발하고, BP891로 표기하였다.CEN.PK 113-7D [Delta] ura3 :: loxP [Delta] his3 [Delta] pdc6 :: URA3 were grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 [deg.] C. Isolated isolates were selected. The deletion and marker removal were carried out using primers oBP448 (SEQ ID NO: 34) and oBP449 using genomic DNA prepared using the Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA). (SEQ ID NO: 35) to confirm by PCR and sequencing. The absence of the PDC6 gene from the isolate was demonstrated by negative PCR results using primers specific for the coding sequence of PDC6, oBP554 36) and oBP555 (SEQ ID NO: 37). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 and designated BP891.

PDC1 결실 ilvDSm 통합PDC1 deletion ilvDSm integration

PDC1 유전자를 결실시키고, 스트렙토코커스 뮤탄스 ATCC 번호 700610으로부터의 ilvD 코딩 영역으로 대체하였다. PDC1 결실-ilvDSm 통합을 위한 PCR 카세트를 위한, 스트렙토코쿠스 뮤탄스 유래의 ilvD 코딩 영역이 뒤따르는 단편을 퓨젼(등록상표) 고충실도 PCR 마스터 믹스 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스) 및 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)에 의해 제조된 주형으로서의 NYLA83 (본 명세서에 그리고 미국 가특허 출원 제61/246,709호에 기재되어 있음) 게놈 DNA를 사용하여 증폭시켰다. PDC1 단편 A-ilvDSm (서열 번호 141)을 프라이머 oBP513 (서열 번호 38) 및 프라이머 oBP515 (서열 번호 39) - PDC1 단편 B의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유함 - 를 이용하여 증폭시켰다. PDC1 결실-ilvDSm 통합을 위한 PCR 카세트를 위한 B, U 및 C 단편을 퓨젼(등록상표) 고충실도 PCR 마스터 믹스 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스) 및 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)에 의해 제조된 주형으로서의 CEN.PK 113-7D 게놈 DNA를 사용하여 증폭시켰다. PDC1 단편 B를 PDC1 단편 A-ilvDSm의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP516 (서열 번호 40) 및 PDC1 단편 U의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP517 (서열 번호 41)을 이용하여 증폭시켰다. PDC1 단편 U는 PDC1 단편 B의 3' 말단에 대하여 상동성을 갖는 5' 말단 테일을 함유하는 프라이머 oBP518 (서열 번호 42) 및 PDC1 단편 C의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP519 (서열 번호 43)를 이용하여 증폭시켰다. PDC1 단편 C는 PDC1 단편 U의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP520 (서열 번호 44), 및 프라이머 oBP521 (서열 번호 45)을 이용하여 증폭시켰다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 정제하였다. PDC1 단편 A-ilvDSm-B를 PDC1 단편 A-ilvDSm 및 PDC1 단편 B를 혼합하고 프라이머 oBP513 (서열 번호 38) 및 oBP517 (서열 번호 41)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. PDC1 단편 UC를 PDC1 단편 U 및 PDC1 단편 C를 PDC1 혼합하고, 프라이머 oBP518 (서열 번호 42) 및 oBP521 (서열 번호 45)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. 생성된 PCR 생성물을 아가로스 겔, 이어서 겔 추출 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 정제하였다. PDC1 A-ilvDSm-BUC 카세트 (서열 번호 142)를 PDC1 단편 A A-ilvDSm-B 및 PDC1 단편 UC를 혼합하고, 프라이머 oBP513 (서열 번호 38) 및 oBP521 (서열 번호 45)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다.The PDC1 gene was deleted and replaced with the ilvD coding region from Streptococcus mutans ATCC No. 700610. A fragment followed by an ilvD coding region from Streptococcus mutans, for a PCR cassette for PDC1 deletion-ilvDSm integration, was a Fusion® high fidelity PCR master mix (New England Biolabs, Ipswich, Mass.) And NYLA83 (herein and in U.S. Provisional Patent Application No. 61 / 246,709) as a template made by Gentra® Purified East / Back Kit (Qiagen, Valencia, Calif.) A) using genomic DNA. PDC1 fragment A-ilvDSm (SEQ ID NO: 141) was prepared using primers oBP513 (SEQ ID NO: 38) and primer oBP515 (SEQ ID NO: 39), which contained a 5 'tail homologous to the 5' end of PDC1 fragment B. Amplified. B, U and C fragments for PCR cassettes for PDC1 deletion-ilvDSm integration were fused with Fusion® High Fidelity PCR Master Mix (New England Biolabs, Ipswich, Mass.) And Gentra® Fused ( Amplification was performed using CEN.PK 113-7D genomic DNA as a template prepared by the East / Bact kit (Qiagen, Valencia, Calif.). PDC1 fragment B was prepared by primer oBP516 (SEQ ID NO: 40) containing a 5 'tail homologous to the 3' end of PDC1 fragment A-ilvDSm and a 5 'tail homologous to the 5' end of PDC1 fragment U. Amplification was carried out using primers oBP517 (SEQ ID NO: 41). PDC1 fragment U contains primer oBP518 (SEQ ID NO: 42) containing a 5 'terminal tail homologous to the 3' end of PDC1 fragment B and a 5 'tail homologous to the 5' end of PDC1 fragment C Amplified using primers oBP519 (SEQ ID NO: 43). PDC1 fragment C was amplified using primer oBP520 (SEQ ID NO: 44), and primer oBP521 (SEQ ID NO: 45) containing a 5 'tail homologous to the 3' end of PDC1 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). PDC1 fragment A-ilvDSm-B was generated by overlapping PCR by mixing PDC1 fragment A-ilvDSm and PDC1 fragment B and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41). PDC1 fragment UC was generated by overlapping PCR by PDC1 mixing PDC1 fragment U and PDC1 fragment C and amplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45). The resulting PCR product was purified with an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). Nested PCR by mixing the PDC1 A-ilvDSm-BUC cassette (SEQ ID NO: 142) with PDC1 fragment A A-ilvDSm-B and PDC1 fragment UC and amplifying with primers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45) Produced by PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA).

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6의 적격 세포를 만들고, 프로즌-이지 이스트 트랜스포메이션 II™ 키트 (미국 캘리포니아주 어바인 소재의 자이모 리서치 코포레이션)를 사용하여 PDC1 A-ilvDSm-BUC PCR 카세트로 형질전환시켰다. 형질전환 혼합물을 30℃에서 2% 글루코스가 보충된 우라실이 결여된 합성 완전 배지 상에 도말하였다. pdc1 넉아웃 ilvDSm 통합이 있는 형질전환체를 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 제조한 게놈 DNA를 사용하여 프라이머 oBP511 (서열 번호 46) 및 oBP512 (서열 번호 47)를 사용하여 PCR에 의해 스크리닝하였다. 단리체로부터의 PDC1 유전자의 부재를 PDC1의 코딩 서열에 대하여 특이적인 프라이머, oBP550 48) 및 oBP551 (서열 번호 49)을 사용하여 음성 PCR 결과에 의해 입증하였다. 올바른 형질전환체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3으로서 선발하였다.Qualified cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 were made and PDC1 A-ilvDSm-BUC PCR cassettes using the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). And transformed. Transformation mixtures were plated at 30 ° C. on synthetic complete medium lacking uracil supplemented with 2% glucose. Transformants with pdc1 knockout ilvDSm integration were prepared using primers oBP511 (SEQ ID NO: 46) using genomic DNA prepared with the Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA). ) And oBP512 (SEQ ID NO: 47) by screening by PCR. The absence of the PDC1 gene from the isolate was demonstrated by negative PCR results using primers specific for the coding sequence of PDC1, oBP550 48) and oBP551 (SEQ ID NO: 49). The correct transformants were selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3을 YPD에서 하룻밤 성장시키고, 30℃에서 5-플루오로-오로트산 (0.1%)을 함유하는 합성 완전 배지 상에 도말하여, URA3 마커가 손실된 단리체를 선발하였다. PDC1의 결실, ilvDSm의 통합, 및 마커 제거는 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 사용하여 제조된 게놈 DNA를 이용하여 프라이머 oBP511 (서열 번호 46) 및 oBP512 (서열 번호 47)를 사용하여 PCR에 의해 확인하고 서열결정하였다. 올바른 단리체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm으로서 선발하고, BP907로 표기하였다.CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3 were grown overnight in YPD and plated on a synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. , Isolates with missing URA3 markers were selected. Deletion of PDC1, integration of ilvDSm, and marker removal were carried out using primers oBP511 (genome) using genomic DNA prepared using the Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA). SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) were confirmed and sequenced by PCR. The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm and designated BP907.

PDC5 결실 sadB 통합PDC5 elimination sadB integration

PDC5 유전자를 결실시키고, 아크로모박터 자일로족시단스로부터의 sadB 코딩 영역으로 대체하였다. 먼저, PDC5 결실-sadB 통합을 위한 PCR 카세트의 세그먼트를 플라스미드 pUC19-URA3MCS 내로 클로닝하였다.The PDC5 gene was deleted and replaced with the sadB coding region from Acromobacter xyloxidans. First, a segment of the PCR cassette for PDC5 deletion-sadB integration was cloned into the plasmid pUC19-URA3MCS.

pUC19-URA3MCS는 pUC19 기반의 것이며, 다중 클로닝 부위(MCS) 내에 위치한 사카로마이세스 세레비지애로부터의 URA3 유전자의 서열을 함유한다. pUC19는 에스케리키아 콜라이에서의 복제 및 선발을 위한 베타-락타마아제를 코딩하는 유전자 및 pMB1 레플리콘을 함유한다. URA3의 코딩 서열에 더하여, 이러한 유전자의 상류 및 하류로부터의 서열을 효모에서의 URA3 유전자의 발현을 위해 포함시켰다. 벡터는 클로닝 목적을 위해 사용될 수 있으며, 효모 통합 벡터로서 사용될 수 있다.pUC19-URA3MCS is based on pUC19 and contains the sequence of the URA3 gene from Saccharomyces cerevisiae located within the multiple cloning site (MCS). pUC19 contains pMB1 replicon and a gene encoding beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequences of URA3, sequences upstream and downstream of these genes were included for expression of the URA3 gene in yeast. The vector may be used for cloning purposes and may be used as a yeast integration vector.

사카로마이세스 세레비지애 CEN.PK 113-7D 게놈 DNA 유래의 URA3 코딩 영역의 상류의 250 bp 및 상기 코딩 영역의 하류의 150 bp와 함께 URA3 코딩 영역을 포함하는 DNA를, 퓨전(등록상표) 고충실도 PCR 마스터 믹스 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.)를 사용하여, BamHI, AscI, PmeI, 및 FseI 제한 부위를 함유하는 프라이머 oBP438 (서열 번호 12) 및 XbaI, PacI, 및 NotI 제한 부위를 함유하는 프라이머 oBP439 (서열 번호 13)를 이용하여 증폭시켰다. 게놈 DNA를 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 사용하여 제조하였다. PCR 생성물 및 pUC19 (서열 번호 144)를, BamHI 및 XbaI을 이용한 절단 후 T4 DNA 리가아제로 라이게이션시켜 벡터 pUC19-URA3MCS를 생성하였다. 벡터를 프라이머 oBP264 (서열 번호 10) 및 oBP265 (서열 번호 11)를 이용하여 PCR 및 서열결정에 의해 확인하였다.DNA comprising the URA3 coding region together with 250 bp upstream of the URA3 coding region derived from Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA and 150 bp downstream of the coding region is fused (registered trademark). Primers oBP438 (SEQ ID NO: 12) and XbaI, PacI, and containing BamHI, AscI, PmeI, and FseI restriction sites, using a high fidelity PCR master mix (New England Biolabs Inc., Ipswich, Mass.). Amplification was performed using primers oBP439 (SEQ ID NO: 13) containing a NotI restriction site. Genomic DNA was prepared using Gentra® Purified Yeast / Bact kit (Qiagen, Valencia, CA). The PCR product and pUC19 (SEQ ID NO: 144) were ligated with T4 DNA ligase after digestion with BamHI and XbaI to generate vector pUC19-URA3MCS. Vectors were identified by PCR and sequencing using primers oBP264 (SEQ ID NO: 10) and oBP265 (SEQ ID NO: 11).

PDC5 단편 B 및 sadB의 코딩 서열을 pUC19-URA3MCS 내로 클로닝하여, PDC5 A-sadB-BUC PCR 카세트의 sadB-BU 부분을 생성하였다. sadB의 코딩 서열을 주형으로서 pLH468-sadB (서열 번호 67)를 사용하여, AscI 제한 부위를 함유하는 프라이머 oBP530 (서열 번호 50), 및 PDC5 단편 B의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP531 (서열 번호 51)을 이용하여 증폭시켰다. PDC5 단편 B는 sadB의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP532 (서열 번호 52), 및 PmeI 제한 부위를 함유하는 프라이머 oBP533 (서열 번호 53)을 이용하여 증폭시켰다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 정제하였다. sadB-PDC5 단편 B를, sadB 및 PDC5 단편 B PCR 생성물을 혼합하고 프라이머 oBP530 (서열 번호 50) 및 oBP533 (서열 번호 53)을 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. 생성된 PCR 생성물을 AscI 및 PmeI으로 절단하고, T4 DNA 리가아제를 사용하여 적절한 효소를 이용한 절단 후에 pUC19-URA3MCS의 상응하는 부위 내로 라이게이션시켰다. 생성된 플라스미드를 프라이머 oBP536 (서열 번호 54) 및 oBP546 (서열 번호 55) - PDC5 단편 C의 5' 말단에 대하여 상동성을 갖는 5' 테일을 함유함 - 를 사용한 sadB-단편 B-단편 U의 증폭을 위한 주형으로 사용하였다. PDC5 단편 C는 PDC5 sadB-단편 B-단편 U의 3' 말단에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP547 (서열 번호 56) 및 프라이머 oBP539 (서열 번호 57)를 이용하여 증폭시켰다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다. PDC5 sadB-단편 B-단편 U-단편 C를 PDC5 sadB-단편 B-단편 U 및 PDC5 단편 C를 혼합하고, 프라이머 oBP536 (서열 번호 54) 및 oBP539 (서열 번호 57)를 이용하여 증폭시킴으로써 중첩 PCR에 의해 생성하였다. 생성된 PCR 생성물을 아가로스 겔, 이어서 겔 추출 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)에서 정제하였다. PDC5 A-sadB-BUC 카세트 (서열 번호 143)는 PDC5 sadB-단편 B-단편 U-단편 C를 천연 PDC5 코딩 서열의 바로 상류의 50개 뉴클레오티드에 대하여 상동성을 갖는 5' 테일을 함유하는 프라이머 oBP542 (서열 번호 58), 및 프라이머 oBP539 (서열 번호 57)를 이용하여 증폭시킴으로써 생성하였다. PCR 생성물을 PCR 정제 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 이용하여 정제하였다.The coding sequences of PDC5 fragment B and sadB were cloned into pUC19-URA3MCS to generate the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. Using the coding sequence of sadB as a template, pLH468-sadB (SEQ ID NO: 67), primer oBP530 containing the AscI restriction site (SEQ ID NO: 50), and a 5 'tail having homology to the 5' end of PDC5 fragment B Amplification was performed using a primer containing oBP531 (SEQ ID NO: 51). PDC5 fragment B was amplified using primer oBP532 (SEQ ID NO: 52) containing a 5 'tail with homology to the 3' end of sadB, and primer oBP533 (SEQ ID NO: 53) containing a PmeI restriction site. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). sadB-PDC5 fragment B was generated by overlapping PCR by mixing sadB and PDC5 fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was digested with AscI and PmeI and ligated into the corresponding sites of pUC19-URA3MCS after digestion with appropriate enzymes using T4 DNA ligase. The resulting plasmid was amplified by sadB-fragment B-fragment U with primers oBP536 (SEQ ID NO: 54) and oBP546 (SEQ ID NO: 55)-containing a 5 'tail homologous to the 5' end of PDC5 fragment C. Used as a template for. PDC5 fragment C was amplified using primers oBP547 (SEQ ID NO: 56) and primer oBP539 (SEQ ID NO: 57) containing a 5 'tail homologous to the 3' end of PDC5 sadB-fragment B-fragment U. PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA). PDC5 sadB-fragment B-fragment U-fragment C was subjected to overlapping PCR by mixing PDC5 sadB-fragment B-fragment U and PDC5 fragment C and amplifying with primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57). Generated by The resulting PCR product was purified on an agarose gel followed by a gel extraction kit (Qiagen, Valencia, CA). The PDC5 A-sadB-BUC cassette (SEQ ID NO: 143) is a primer oBP542 containing a 5 'tail of PDC5 sadB-fragment B-fragment U-fragment C with homology to 50 nucleotides immediately upstream of the native PDC5 coding sequence. (SEQ ID NO: 58), and amplified using primers oBP539 (SEQ ID NO: 57). PCR products were purified using a PCR Purification Kit (Qiagen, Valencia, CA).

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm의 적격 세포를 만들고, 프로즌-이지 이스트 트랜스포메이션 II™ 키트 (미국 캘리포니아주 어바인 소재의 자이모 리서치 코포레이션)를 사용하여 PDC5 A-sadB-BUC PCR 카세트로 형질전환시켰다. 형질전환 혼합물을 30℃에서 1% 에탄올을 보충한 (글루코스 없음) 우라실이 결여된 합성 완전 배지 상에 도말하였다. pdc5 넉아웃 sadB 통합이 있는 형질전환체를 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)로 제조한 게놈 DNA를 사용하여 프라이머 oBP540 (서열 번호 59) 및 oBP541 (서열 번호 60)을 사용하여 PCR에 의해 스크리닝하였다. 단리체로부터의 PDC5 유전자의 부재를 PDC5의 코딩 서열에 대하여 특이적인 프라이머, oBP552 61) 및 oBP553 (서열 번호 62)을 사용하여 음성 PCR 결과에 의해 입증하였다. 올바른 형질전환체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3으로서 선발하였다.Create qualified cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm and use the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA) to PDC5 A-sadB Transformed with -BUC PCR cassette. Transformation mixtures were plated on synthetic complete medium lacking uracil (without glucose) supplemented with 1% ethanol at 30 ° C. Transformants with pdc5 knockout sadB integration were prepared using primers oBP540 (SEQ ID NO: 59) using genomic DNA prepared with the Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA). ) And oBP541 (SEQ ID NO: 60) by screening by PCR. The absence of the PDC5 gene from the isolate was demonstrated by negative PCR results using primers specific for the coding sequence of PDC5, oBP552 61) and oBP553 (SEQ ID NO: 62). The correct transformants were selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3을 YPE (1% 에탄올)에서 하룻밤 성장시키고, 에탄올이 보충되고 (글루코스 없음) 5-플루오로-오로트산 (0.1%)을 함유하는 완전 합성 배지 상에 30℃에서 도말하여, URA3 마커가 손실된 단리주를 선발하였다. PDC5의 결실, sadB의 통합, 및 마커 제거는 젠트라(등록상표) 퓨어진(등록상표) 이스트/백트 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 사용하여 제조된 게놈 DNA를 이용하여 프라이머 oBP540 (서열 번호 59) 및 oBP541 (서열 번호 60)을 사용하여 PCR에 의해 확인하였다. 올바른 단리체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB로서 선발하고, BP913으로 표기하였다.CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3 are grown overnight in YPE (1% ethanol), ethanol supplemented (without glucose) and 5-fluoro-orroic acid Plated at 30 ° C. on complete synthetic medium containing (0.1%), isolated isolates with missing URA3 markers were selected. Deletion of PDC5, incorporation of sadB, and marker removal were carried out using primers oBP540 (genome) using genomic DNA prepared using the Gentra® Purified Yeast / Back Kit (Qiagen, Valencia, CA). SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60) to confirm by PCR. The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB and designated BP913.

GPD2 결실GPD2 Fruit

내인성 GPD2 코딩 영역을 결실시키기 위하여, gpd2::loxP-URA3-loxP 카세트 (서열 번호 145)를 주형 DNA로서 loxP-URA3-loxP (서열 번호 68)를 사용하여 PCR 증폭시켰다. loxP-URA3-loxP는 loxP 리콤비나아제 부위가 측면에 있는 URA3 마커를 함유한다 (ATCC 번호: 77107). PCR은 퓨전(등록상표) DNA 폴리머라아제 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.) 및 프라이머 LA512 및 LA513 (서열 번호 8 및 9)을 사용하여 행하였다. 각 프라이머의 GPD2 부분은 GPD2 코딩 영역의 상류의 5' 영역 및 상기 코딩 영역의 하류의 3' 영역으로부터 유래되어, loxP-URA3-loxP 마커의 통합이 GPD2 코딩 영역의 대체로 이어지게 하였다. PCR 생성물로 BP913을 형질전환시키고, 형질전환체를 1% 에탄올이 보충되고 (글루코스 없음) 우라실이 결여된 합성 완전 배지 상에서 선발하였다. 형질전환체를 프라이머 oBP582 및 AA270 (각각 서열 번호 63 및 64)을 사용하여 PCR에 의해 올바른 통합이 확인되도록 스크리닝하였다.To delete the endogenous GPD2 coding region, the gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 145) was PCR amplified using loxP-URA3-loxP (SEQ ID NO: 68) as template DNA. loxP-URA3-loxP contains a URA3 marker flanked by loxP recombinase sites (ATCC No. 77107). PCR was performed using Fusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) And primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each primer was derived from the 5 'region upstream of the GPD2 coding region and the 3' region downstream of the coding region, resulting in the integration of loxP-URA3-loxP markers leading to replacement of the GPD2 coding region. BP913 was transformed with the PCR product and the transformant was selected on a synthetic complete medium supplemented with 1% ethanol (without glucose) and lacking uracil. Transformants were screened using primers oBP582 and AA270 (SEQ ID NOs 63 and 64, respectively) to confirm correct integration by PCR.

URA3 마커는 pRS423::PGAL1-cre (서열 번호 66)에 의한 형질전환 및 30℃에서 1% 에탄올로 보충된 히스티딘 결여 합성 완전 배지 상에의 도말에 의해 재순환시켰다. 형질전환체를 1% 에탄올이 보충되고, 5-플루오로-오로트산 (0.1%)을 함유하는 합성 완전 배지 상에 스트리킹하고, 30℃에서 인큐베이션시켜, URA3 마커가 손실된 단리체를 선발하였다. 5-FOA 내성 단리체를 pRS423::PGAL1-cre 플라스미드의 제거를 위하여 YPE (1% 에탄올)에서 성장시켰다. 상기 결실 및 마커 제거를 젠트라 퓨어진 프라이머 oBP582 (서열 번호 63) 및 oBP591 (서열 번호 65)을 이용하여 PCR에 의해 확인하였다. 올바른 단리체를 스트레인 CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP로서 선발하고, PNY1503 (BP1064)으로 표기하였다.URA3 markers were recycled by transformation with pRS423 :: PGAL1-cre (SEQ ID NO: 66) and smear on histidine deficient synthetic complete medium supplemented with 1% ethanol at 30 ° C. Transformants were streaked on synthetic complete medium supplemented with 1% ethanol, containing 5-fluoro-orotic acid (0.1%) and incubated at 30 ° C. to isolate isolates missing URA3 markers. . 5-FOA resistant isolates were grown in YPE (1% ethanol) for the removal of pRS423 :: PGAL1-cre plasmid. The deletion and marker removal was confirmed by PCR using the Gentra fused primers oBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP and designated PNY1503 (BP1064).

BP1064를 플라스미드 pYZ090 (서열 번호 1) 및 pLH468 (서열 번호 2)로 형질전환시켜 스트레인 NGCI-070 (BP1083; PNY1504)을 생성하였다.BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083; PNY1504).

스트레인 NYLA74, NYLA83, 및 NYLA84의 제작Fabrication of strains NYLA74, NYLA83, and NYLA84

에스. 세레비지애의 내인성 PDC1 및 PDC6 유전자의 삽입-불활성화. PDC1, PDC5, 및 PDC6 유전자는 하기에 기재된 바와 같이 피루베이트 데카르복실라아제의 주요한 3가지 아이소자임을 코딩한다.s. Insertion-inactivation of endogenous PDC1 and PDC6 genes in cerevisiae. The PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase as described below.

pRS425::GPM-sadB의 제작Construction of the pRS425 :: GPM-sadB

아크로모박터 자일로속시단스 유래의 부탄올 데하이드로게나아제 (서열 번호 70)를 코딩하는 DNA 단편 (미국 특허 공개 제2009/0269823호에 개시됨)을 클로닝하였다. 이차 알코올 데하이드로게나아제 (서열 번호 69)에 있어서의 sadB로 불리우는 이 유전자의 코딩 영역을, 그람(gram) 음성 유기체에 있어서 권고되는 프로토콜에 따라 젠트라(등록상표) 퓨어진(등록상표) 키트 (미국 캘리포니아주 발렌시아 소재의 퀴아젠)를 사용하여 제조한 에이. 자일로속시단스 게놈 DNA로부터, 각각 정방향 및 역방향 프라이머 N473 및 N469 (서열 번호 74 및 75)를 이용하여 표준 조건을 이용하여 증폭시켰다. PCR 생성물을 pCR(등록상표)4 블런트(BLUNT) (미국 캘리포니아주 칼스바드 소재의 인비트로겐(Invitrogen™)) 내로 토포(TOPO)(등록상표)-블런트 클로닝하여 pCR4Blunt::sadB를 생성하였으며, 이것으로 이. 콜라이 마흐(Mach)-1 세포를 형질전환시켰다. 후속적으로, 플라스미드를 4개의 클론으로부터 단리하였고, 서열을 확인하였다.A DNA fragment (disclosed in US Patent Publication No. 2009/0269823) encoding butanol dehydrogenase (SEQ ID NO: 70) from Acromobacter xyloxyxidans was cloned. The coding region of this gene, called sadB in secondary alcohol dehydrogenase (SEQ ID NO: 69), was subjected to the Gentra® Purified Kit according to the recommended protocol for gram negative organisms. Qiagen, Valencia, California, USA. From xyloxyoxydans genomic DNA, amplified using standard conditions using forward and reverse primers N473 and N469 (SEQ ID NOs: 74 and 75), respectively. PCR products were cloned into TOPO®-blunt into pCR®4 Blunt (BLUNT) (Invitrogen ™, Carlsbad, Calif.) To generate pCR4Blunt :: sadB. By this. E. coli Mach-1 cells were transformed. Subsequently, the plasmid was isolated from four clones and the sequence confirmed.

sadB 코딩 영역을 pCR4Blunt::sadB로부터 PCR 증폭시켰다. PCR 프라이머들은 효모 GPM1 프로모터 및 ADH1 종결자 (N583 및 N584, 서열 번호 76 및 77로 제공됨)와 중첩되는 추가의 5' 서열을 함유하였다. 그 후, PCR 생성물을 하기와 같이 사카로마이세스 세레비지애에서 "갭 복구" 방법을 이용하여 클로닝하였다 (문헌[Ma, et al., Gene 58:201-216, 1987]). GPM1 프로모터 (서열 번호 72), 락토코쿠스 락티스 유래의 kivD 코딩 영역 (서열 번호 71), 및 ADH1 종결자 (서열 번호 73)를 함유하는 효모-이. 콜라이 셔틀 벡터 pRS425::GPM::kivD::ADH (미국 특허 공개 제2007/0092957 A1호, 실시예 17에 기재됨)를 BbvCI 및 PacI 제한 효소로 절단하여 kivD 코딩 영역을 방출하였다. 대략적으로 1 ㎍의 남아있는 벡터 단편을 1 ㎍의 sadB PCR 생성물과 함께 이용하여 에스. 세레비지애 스트레인 BY4741을 형질전환시켰다. 형질전환체를 류신이 결여된 합성 완전 배지에서 선별하였다. pRS425::GPM-sadB를 생성하는 적당한 재조합 사건(event)을 프라이머 N142 및 N459 (서열 번호 108 및 109)를 사용하여 PCR에 의해 확인하였다.The sadB coding region was PCR amplified from pCR4Blunt :: sadB. PCR primers contained an additional 5 'sequence that overlaps the yeast GPM1 promoter and ADH1 terminators (N583 and N584, provided as SEQ ID NOs: 76 and 77). PCR products were then cloned using the "gap repair" method in Saccharomyces cerevisiae as described below (Ma, et al., Gene 58: 201-216, 1987). Yeast containing a GPM1 promoter (SEQ ID NO: 72), a kivD coding region derived from Lactococcus lactis (SEQ ID NO: 71), and an ADH1 terminator (SEQ ID NO: 73). The E. coli shuttle vector pRS425 :: GPM :: kivD :: ADH (described in US Patent Publication 2007/0092957 A1, Example 17) was cleaved with BbvCI and PacI restriction enzymes to release the kivD coding region. Approximately 1 μg of the remaining vector fragment was used with 1 μg sadB PCR product. Serevisiae strain BY4741 was transformed. Transformants were selected in synthetic complete medium lacking leucine. Appropriate recombination events to generate pRS425 :: GPM-sadB were confirmed by PCR using primers N142 and N459 (SEQ ID NOs: 108 and 109).

pdc6:: PGPM1-sadB 통합 카세트의 제작 및 PDC6 결실:pdc6 :: Construction of PGPM1-sadB Integrated Cassette and PDC6 Deletion:

pdc6::PGPM1-sadB-ADH1t-URA3r 통합 카세트는 pRS425::GPM-sadB (서열 번호 78)로부터의 GPM-sadB-ADHt 세그먼트 (서열 번호 79)를 pUC19-URA3r로부터의 URA3r 유전자에 연결함으로써 만들었다. pUC19-URA3r (서열 번호80)은 75 bp의 상동성 반복 서열이 측면에 있는, pRS426 (ATCC 번호 77107)으로부터의 URA3 마커를 함유하여서 생체 내에서의 상동 재조합 및 URA3 마커의 제거를 허용한다. 퓨전(등록상표) DNA 폴리머라아제(미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.) 및 프라이머 114117-11A 내지 114117-11D (서열 번호 81, 82, 83, 및 84)와, 114117-13A 및 114117-13B (서열 번호 85 및 86) 를 이용하여, 주형으로서 pRS425::GPM-sadB 및 pUC19-URA3r 플라스미드 DNA를 사용하여 SOE PCR (문헌[Horton, et al., Gene 77:61-68, 1989]에 기재된 바와 같음)에 의해 상기 2개의 DNA 세그먼트를 연결시켰다.The pdc6 :: PGPM1-sadB-ADH1t-URA3r integration cassette was made by linking the GPM-sadB-ADHt segment (SEQ ID NO: 79) from pRS425 :: GPM-sadB (SEQ ID NO: 78) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO: 80) contains the URA3 marker from pRS426 (ATCC No 77107), flanked by a 75 bp homologous repeat sequence, allowing homologous recombination and removal of the URA3 marker in vivo. Fusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) And primers 114117-11A to 114117-11D (SEQ ID NOs: 81, 82, 83, and 84), and 114117-13A And SOE PCR using p111425 :: GPM-sadB and pUC19-URA3r plasmid DNA as templates using 114117-13B (SEQ ID NOs: 85 and 86) (Horton, et al., Gene 77: 61-68, The two DNA segments).

SOE PCR (114117-13A 및 114117-13B)을 위한 외부 프라이머는 각각 PDC6 프로모터 및 종결자의 상류 및 하류의 영역에 상동성인 5' 및 3'의 대략 50 bp 영역을 함유하였다. 완성된 카세트 PCR 단편으로 BY4700 (ATCC 번호 200866)을 형질전환시키고, 형질전환체는 표준 유전자 기술을 사용하여 30℃에서 2% 글루코스가 보충된 그리고 우라실은 결여된 합성 완전 배지 상에서 유지시켰다 (문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]). 형질전환체를 프라이머 112590-34G 및 112590-34H (서열 번호 87 및 88)와, 112590-34F 및 112590-49E (서열 번호 89 및 90)를 사용하여 PCR에 의해 스크리닝하여 PDC6 유전자좌에서의 통합을 확인하였으며, 이때 PDC6 코딩 영역은 결실되었다. URA3r 마커는 표준 프로토콜에 따라 30℃에서 2% 글루코스 및 5-FOA가 보충된 합성 완전 배지 상에 도말함으로써 재활용하였다. 5-FOA 플레이트로부터 SD-URA 배지 상으로 콜로니를 패칭하여 성장의 부재를 확인함으로써 마커 제거를 확인하였다. 생성된 동정된 스트레인은 다음의 유전자형을 갖는다: BY4700 pdc6::PGPM1-sadB-ADH1t.External primers for SOE PCR (114117-13A and 114117-13B) contained approximately 50 bp regions of 5 'and 3' homology to the regions upstream and downstream of the PDC6 promoter and terminator, respectively. BY4700 (ATCC No. 200866) was transformed with the completed cassette PCR fragment, and the transformants were maintained on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking uracil using standard genetic techniques (see [ Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]. Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs 87 and 88) and 112590-34F and 112590-49E (SEQ ID NOs 89 and 90) to confirm integration at the PDC6 locus. At this time, the PDC6 coding region was deleted. URA3r markers were recycled by plating onto synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth. The resulting identified strains have the following genotypes: BY4700 pdc6 :: PGPM1-sadB-ADH1t.

pdc1:: PPDC1-ilvD 통합 카세트의 제작 및 PDC1 결실:pdc1 :: Construction of PPDC1-ilvD Integration Cassette and PDC1 Deletion:

pdc1:: PPDC1-ilvD-FBA1t-URA3r 통합 카세트는 pLH468 (서열 번호 2)로부터의 ilvD-FBA1t 세그먼트 (서열 번호 91)를 퓨전(등록상표) DNA 폴리머라아제(미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.) 및 프라이머 114117-27A 내지 114117-27D (서열 번호 111, 112, 113, 및 114)를 이용하여, 주형으로서 pLH468 및 pUC19-URA3r 플라스미드 DNA를 사용하여 SOE PCR (문헌[Horton, et al., Gene 77:61-68, 1989]에 기재된 바와 같음)에 의해 pUC19-URA3r로부터의 URA3r 유전자에 연결시킴으로써 만들었다.pdc1 :: PPDC1-ilvD-FBA1t-URA3r integrated cassette was used to transfer the ilvD-FBA1t segment (SEQ ID NO: 91) from pLH468 (SEQ ID NO: 2) to a fusion DNA polymerase (Ipswich, Ipswich, MA, USA). BioLabs Inc.) and primers 114117-27A to 114117-27D (SEQ ID NOs: 111, 112, 113, and 114), and SOE PCR using pLH468 and pUC19-URA3r plasmid DNA as templates (Horton, et. al., Gene 77: 61-68, 1989), by linking to the URA3r gene from pUC19-URA3r.

SOE PCR (114117-27A 및 114117-27D)을 위한 외부 프라이머는 PDC1 프로모터의 하류의 영역 및 PDC1 코딩 서열의 하류의 영역에 상동성인 5' 및 3'의 대략 50 bp 영역을 함유하였다. 완성된 카세트 PCR 단편으로 BY4700 pdc6::PGPM1-sadB-ADH1t를 형질전환시키고, 형질전환체는 표준 유전자 기술을 사용하여 30℃에서 2% 글루코스가 보충된 그리고 우라실은 결여된 합성 완전 배지 상에서 유지시켰다 (문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]). 형질전환체를 프라이머 114117-36D 및 135 (서열 번호 92 및 93)와, 프라이머 112590-49E 및 112590-30F (서열 번호 90 및 94)를 사용하여 PCR에 의해 스크리닝하여 PDC1 유전자좌에서의 통합을 확인하였으며, 이때 PDC1 코딩 서열은 결실되었다. URA3r 마커는 표준 프로토콜에 따라 30℃에서 2% 글루코스 및 5-FOA가 보충된 합성 완전 배지 상에 도말함으로써 재활용하였다. 5-FOA 플레이트로부터 SD-URA 배지 상으로 콜로니를 패칭하여 성장의 부재를 확인함으로써 마커 제거를 확인하였다. 생성된 동정된 스트레인 "NYLA67"은 다음의 유전자형을 갖는다: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t.The outer primers for the SOE PCR (114117-27A and 114117-27D) contained approximately 50 bp regions of 5 'and 3' homology to the region downstream of the PDC1 promoter and the region downstream of the PDC1 coding sequence. BY4700 pdc6 :: PGPM1-sadB-ADH1t was transformed with the completed cassette PCR fragment, and the transformants were maintained on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking uracil using standard genetic techniques. (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 92 and 93) and primers 112590-49E and 112590-30F (SEQ ID NOs: 90 and 94) to confirm integration at the PDC1 locus. , Where the PDC1 coding sequence was deleted. URA3r markers were recycled by plating onto synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth. The resulting identified strain “NYLA67” has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t.

HIS3 결실HIS3 fruit

내인성 HIS3 코딩 영역을 결실시키기 위하여, his3::URA3r2 카세트를 URA3r2 주형 DNA (서열 번호 95)로부터 PCR 증폭시켰다. URA3r2는 500 bp의 상동성 반복 서열이 측면에 있는, pRS426 (ATCC 번호 77107) 으로부터의 URA3 마커를 함유하여서 생체 내에서의 상동 재조합 및 URA3 마커의 제거를 허용한다. PCR은 퓨전(등록상표) DNA 폴리머라아제 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.) 및 프라이머 114117-45A 및 114117-45B (서열 번호 96 및 97)를 사용하여 행하였으며, 이는 대략 2.3 kb의 PCR 생성물을 생성하였다. 각 프라이머의 HIS3 부분은 HIS3 프로모터의 상류의 5' 영역 및 코딩 영역의 하류의 3' 영역으로부터 유래되어, URA3r2 마커의 통합이 HIS3 코딩 영역의 대체로 이어지게 하였다. PCR 생성물로 표준 유전자 기술을 사용하여 NYLA67을 형질전환시켰고(문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]), 30℃에서 2% 글루코스로 보충된 그리고 우라실이 결여된 합성 완전 배지 상에서 형질전환체를 선별하였다. 30℃에서 2% 글루코스가 보충된 그리고 히스티딘이 결여된 합성 완전 배지 상에서 형질전환체를 레플리카(replica) 도말함으로써 형질전환체를 스크리닝하여, 올바른 통합을 확인하였다. 표준 프로토콜에 따라 30℃에서 2% 글루코스 및 5-FOA가 보충된 합성 완전 배지 상에 도말함으로써 URA3r 마커를 재활용하였다. 5-FOA 플레이트로부터 SD-URA 배지 상으로 콜로니를 패칭하여 성장의 부재를 확인함으로써 마커 제거를 확인하였다. NYLA73으로 불리우는 생성된 동정된 스트레인은 다음의 유전자형을 갖는다: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3.To delete the endogenous HIS3 coding region, the his3 :: URA3r2 cassette was PCR amplified from the URA3r2 template DNA (SEQ ID NO: 95). URA3r2 contains a URA3 marker from pRS426 (ATCC number 77107), flanked by a 500 bp homologous repeat sequence, allowing homologous recombination and removal of the URA3 marker in vivo. PCR was performed using Fusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) And primers 114117-45A and 114117-45B (SEQ ID NOs: 96 and 97). 2.3 kb of PCR product was generated. The HIS3 portion of each primer was derived from the 5 'region upstream of the HIS3 promoter and the 3' region downstream of the coding region, leading to the integration of the URA3r2 marker to replacement of the HIS3 coding region. NYLA67 was transformed using standard genetic techniques with PCR products (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and 2% glucose at 30 ° C. Transformants were selected on synthetic complete medium supplemented with and lacking uracil. Transformants were screened by replicating the transformants on synthetic complete medium supplemented with 2% glucose at 30 ° C. and lacking histidine to confirm correct integration. URA3r markers were recycled by plating onto synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth. The resulting identified strain called NYLA73 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3.

pdc5::kanMX 통합 카세트의 제작 및 PDC5 결실:Construction of the pdc5 :: kanMX Integrated Cassette and PDC5 Deletion:

pdc5::kanMX4 카세트는 퓨전(등록상표) DNA 폴리머라아제 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.) 및 프라이머 PDC5::KanMXF 및 PDC5::KanMXR (서열 번호 98 및 99)을 사용하여 스트레인 YLR134W 염색체 DNA (ATCC 번호 4034091)로부터 PCR 증폭시켰으며, 이는 대략 2.2 kb의 PCR 생성물을 생성하였다. 각 프라이머의 PDC5 부분은 PDC5 프로모터의 상류의 5' 영역 및 코딩 영역의 하류의 3' 영역으로부터 유래되어, kanMX4 마커의 통합이 PDC5 코딩 영역의 대체로 이어지게 하였다. 표준 유전자 기술을 사용하여 PCR 생성물로 NYLA73을 형질전환시켰고(문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]), 1% 에탄올 및 제네티신 (200 ㎍/mL)로 보충된 YP 배지 상에서 ℃에서 형질전환체를 선발하였다. 형질전환체를 프라이머 PDC5kofor 및 N175 (서열 번호 100 및 101)를 사용하여 PDC5 코딩 영역이 대체된 PDC 유전자좌에서의 올바른 통합을 확인하기 위하여 PCR에 의해 스크리닝하였다. 동정된 올바른 형질전환체는 다음의 유전자형을 갖는다: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 pdc5::kanMX4. 이 스트레인을 NYLA74로 명명하였다.The pdc5 :: kanMX4 cassette uses fusion DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers PDC5 :: KanMXF and PDC5 :: KanMXR (SEQ ID NOs: 98 and 99) By PCR amplification from strain YLR134W chromosomal DNA (ATCC No. 4034091), which produced approximately 2.2 kb of PCR product. The PDC5 portion of each primer was derived from the 5 'region upstream of the PDC5 promoter and the 3' region downstream of the coding region, resulting in integration of the kanMX4 marker leading to replacement of the PDC5 coding region. Standard genetic techniques were used to transform NYLA73 with PCR products (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), 1% ethanol and Geneti Transformants were selected at 캜 on YP medium supplemented with renal (200 μg / mL). Transformants were screened by PCR using primers PDC5kofor and N175 (SEQ ID NOs: 100 and 101) to confirm correct integration at the PDC locus where the PDC5 coding region was replaced. The correct transformant identified had the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 pdc5 :: kanMX4. This strain was named NYLA74.

플라스미드 벡터 pRS423::CUP1-alsS+FBA-budA 및 pRS426::FBA-budC+GPM-sadB로 NYLA74를 형질전환시켜 부탄다이올 생성 스트레인(NGCI-047)을 생성하였다.NYLA74 was transformed with the plasmid vectors pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB to generate a butanediol producing strain (NGCI-047).

플라스미드 벡터 pLH475-Z4B8 (서열 번호 140) 및 pLH468로 NYLA74를 형질전환시켜 아이소부탄올 생성 스트레인(NGCI-049)을 생성하였다.NYLA74 was transformed with plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 to produce isobutanol producing strain (NGCI-049).

HXK2HXK2 ( ( 헥소키나아제Hexokinase IIII )의 결실:Fruitful of):

hxk2::URA3r 카세트는 퓨전(등록상표) DNA 폴리머라아제 (미국 매사추세츠주 입스위치 소재의 뉴 잉글랜드 바이오랩스 인크.) 및 프라이머 384 및 385 (서열 번호 102 및 103)를 사용하여 URA3r2 주형 (상기에 기재됨)으로부터 PCR 증폭시켰으며, 이는 대략 2.3 kb의 PCR 생성물을 생성하였다. 각 프라이머의 HXK2 부분은 HXK2 프로모터의 상류의 5' 영역 및 코딩 영역의 하류의 3' 영역으로부터 유래되어, URA3r2 마커의 통합이 HXK2 코딩 영역의 대체로 이어지게 하였다. 표준 유전자 기술을 사용하여 PCR 생성물로 NYLA73을 형질전환시켰고(문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202]), 우라실이 결여된 그리고 2% 글루코스로 보충된 합성 완전 배지 상에서 30℃에서 형질전환체를 선발하였다. 형질전환체를 프라이머 N869 및 N871 (서열 번호 104 및 105)을 사용하여 HXK2 코딩 영역이 대체된 HXK2 유전자좌에서의 올바른 통합을 확인하기 위하여 PCR에 의해 스크리닝하였다. URA3r2 마커는 표준 프로토콜에 따라 30℃에서 2% 글루코스 및 5-FOA가 보충된 합성 완전 배지 상에 도말함으로써 재활용하였다. 마커 제거는 5-FOA 플레이트로부터 SD-URA 배지 상으로 콜로니를 패칭하여 성장의 부재를 확인함으로써, 그리고 프라이머 N946 및 N947 (서열 번호 106 및 107)을 사용하여 PCR을 하여 올바른 마커 제거를 확인함으로써 확인하였다. NYLA83으로 불리우는 생성된 동정된 스트레인은 다음의 유전자형을 갖는다: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.The hxk2 :: URA3r cassette is a URA3r2 template (described above) using a fusion DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers 384 and 385 (SEQ ID NOs: 102 and 103). PCR amplification), which produced approximately 2.3 kb of PCR product. The HXK2 portion of each primer was derived from the 5 'region upstream of the HXK2 promoter and the 3' region downstream of the coding region, resulting in the integration of the URA3r2 marker leading to replacement of the HXK2 coding region. Standard genetic techniques were used to transform NYLA73 with PCR products (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), and lack of uracil and 2 Transformants were selected at 30 ° C. on synthetic complete medium supplemented with% glucose. Transformants were screened by PCR using primers N869 and N871 (SEQ ID NOs: 104 and 105) to confirm correct integration at the HXK2 locus in which the HXK2 coding region was replaced. URA3r2 markers were recycled by plating onto synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates onto SD-URA medium to confirm the absence of growth, and by PCR using primers N946 and N947 (SEQ ID NOs 106 and 107) to confirm correct marker removal. It was. The resulting identified strain called NYLA83 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.

pdc5::kanMX 통합 카세트의 제작 및 PDC5 결실:Construction of the pdc5 :: kanMX Integrated Cassette and PDC5 Deletion:

pdc5::kanMX4 카세트를 상기에 설명한 바와 같이 PCR 증폭시켰다. 상기 PCR 단편으로 NYLA83을 형질전환시키고, 형질전환체를 상기에 설명한 바와 같이 선발하고 스크리닝하였다. NYLA84로 불리우는 동정된 올바른 형질전환체는 다음의 유전자형을 갖는다: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4.The pdc5 :: kanMX4 cassette was PCR amplified as described above. NYLA83 was transformed with the PCR fragment, and the transformants were selected and screened as described above. The correct transformant identified as NYLA84 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5 :: kanMX4.

표준 유전자 기술 (문헌[Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY])을 사용하여 플라스미드 벡터 pLH468 및 pLH532로 스트레인 NYLA84 (BY4700 pdc6::PGPM1-sadB-ADH1t pdc1::PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4)를 동시에 형질전환시켰으며, 생성된 "부탄올 생성 NYLA84"를 히스티딘 및 우라실이 결여된 그리고 1% 에탄올이 보충된 합성 완전 배지 상에서 30℃에서 유지하였다.Strain NYLA84 (BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1: :) into plasmid vectors pLH468 and pLH532 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY): PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5 :: kanMX4) were simultaneously transformed and the resulting “butanol producing NYLA84” was maintained at 30 ° C. on synthetic complete medium lacking histidine and uracil and supplemented with 1% ethanol.

발현 벡터 Expression vector pLH468pLH468

pLH468 플라스미드 (서열 번호2)를 효모에서의 DHAD, KivD 및 HADH 발현용으로 제작하였으며, 이는 본 명세서에 참고로 포함된 미국 특허 공개 제2009/0305363호에 개시되어 있다. pLH486을 하기를 함유하도록 제작하였다: DHAD의 발현을 위한, 사카로마이세스 세레비지애 FBA1 프로모터 (뉴클레오티드 2109 - 3105) 및 이에 이은 FBA1 종결자 (뉴클레오티드 4858 - 5857)로부터 발현되는 스트렙토코쿠스 뮤탄스 (뉴클레오티드 위치 3313-4849)로부터의 ilvD 유전자의 코딩 영역을 갖는 키메라 유전자; ADH의 발현을 위한, 사카로마이세스 세레비지애 GPM1 프로모터 (뉴클레오티드 7425-8181) 및 이에 이은 ADH1 종결자 (뉴클레오티드 5962-6277)로부터 발현되는 코돈 최적화된 말 간 알코올 데하이드로게나아제 (뉴클레오티드 6286-7413)의 코딩 영역을 갖는 키메라 유전자; 및 KivD의 발현을 위한, TDH3 프로모터 (뉴클레오티드 10896-11918)에 이은 TDH3 종결자 (뉴클레오티드 8237-9235)로부터 발현되는 락토코쿠스 락티스 (뉴클레오티드 9249-10895)로부터의 코돈-최적화된 kivD 유전자의 코딩 영역을 갖는 키메라 유전자.pLH468 plasmid (SEQ ID NO: 2) was constructed for DHAD, KivD and HADH expression in yeast, which is disclosed in US Patent Publication No. 2009/0305363, which is incorporated herein by reference. pLH486 was constructed to contain: Streptococcus mutans expressed from the Saccharomyces cerevisiae FBA1 promoter (nucleotides 2109-3105) followed by the FBA1 terminator (nucleotides 4858-5857) for expression of DHAD. A chimeric gene having the coding region of the ilvD gene from (nucleotide positions 3313-4849); For expression of ADH, a codon optimized horse liver alcohol dehydrogenase (nucleotide 6286-) expressed from a Saccharomyces cerevisiae GPM1 promoter (nucleotides 7425-8181) followed by an ADH1 terminator (nucleotides 5962-6277) Chimeric gene having the coding region of 7413; And encoding the codon-optimized kivD gene from Lactococcus lactis (nucleotides 9249-10895) expressed from the TDH3 promoter (nucleotides 10896-11918) followed by the TDH3 terminator (nucleotides 8237-9235) for expression of KivD. Chimeric genes with regions.

락토코쿠스 락티스 케토아이소발레레이트 데카르복실라아제(KivD) 코딩 영역 및 말 간 알코올 데하이드로게나아제(HADH)의 코딩 영역은 사카로마이세스 세레비지애에서의 발현을 위하여 최적화된 코돈을 기반으로 하여 디엔에이2.0, 인크.(DNA2.0, Inc.; 미국 캘리포니아주 멘로 파크 소재)가 합성하였으며 (각각 서열 번호 71 및 118), 이는 플라스미드 pKivDy-DNA2.0 및 pHadhy-DNA2.0의 형태로 제공되었다. 코딩된 단백질은 각각 서열 번호 117 및 119의 것이다. KivD 및 HADH에 대한 개개의 발현 벡터를 제작하였다. pLH467 (pRS426::PTDH3-kivDy-TDH3t)을 어셈블링하기 위하여, 벡터 pNY8 (서열 번호 121; pRS426.GPD-ald-GPDt로도 명명됨, 본 명세서에 참고로 포함된 미국 특허 공개 제2008/0182308호의 실시예 17에 기재되어 있음)을 AscI 및 SfiI 효소로 절단하여 GPD 프로모터 및 ald 코딩 영역이 절제되게 하였다. pNY8로부터의 TDH3 프로모터 단편 (서열 번호 122)을 5' 프라이머 OT1068 및 3' 프라이머 OT1067 (서열 번호 123 및 124)을 사용하여 PCR 증폭시켜 5' 말단에 AscI 부위, 그리고 3' 말단에 SpeI 부위를 부가하였다. AscI/SfiI로 절단한 pNY8 벡터 단편을 AscI 및 SpeI로 절단한 TDH3 프로모터 PCR 생성물과 라이게이션하였고, 코돈 최적화된 kivD 코딩 영역을 함유하는 SpeI-SfiI 단편을 벡터 pKivD-DNA2.0으로부터 단리하였다. 3중 라이게이션에 의해 벡터 pLH467 (pRS426::PTDH3-kivDy-TDH3t)을 생성하였다. 제한효소 맵핑 및 서열결정에 의해 pLH467을 확인하였다.The lactococcus lactis ketoisovalate decarboxylase (KivD) coding region and the coding region of hepatic alcohol dehydrogenase (HADH) are based on codons optimized for expression in Saccharomyces cerevisiae. DNA 2.0, Inc. (DNA2.0, Inc .; Menlo Park, Calif.) Was synthesized (SEQ ID NOs: 71 and 118, respectively), in the form of plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. Was provided. The encoded proteins are those of SEQ ID NOs: 117 and 119, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426 :: PTDH3-kivDy-TDH3t), vector pNY8 (SEQ ID NO: 121; also designated pRS426.GPD-ald-GPDt, of US Patent Publication No. 2008/0182308, incorporated herein by reference (Described in Example 17) were cleaved with AscI and SfiI enzymes so that the GPD promoter and ald coding region were excised. TDH3 promoter fragment (SEQ ID NO: 122) from pNY8 was PCR amplified using 5 'primer OT1068 and 3' primer OT1067 (SEQ ID NOs: 123 and 124) to add an AscI site at the 5 'end and a SpeI site at the 3' end. It was. PNY8 vector fragment digested with AscI / SfiI was ligated with TDH3 promoter PCR product digested with AscI and SpeI, and SpeI-SfiI fragments containing codon optimized kivD coding regions were isolated from vector pKivD-DNA2.0. Triple ligation produced the vector pLH467 (pRS426 :: PTDH3-kivDy-TDH3t). PLH467 was confirmed by restriction mapping and sequencing.

pLH435 (pRS425::PGPM1-Hadhy-ADH1t)는 본 명세서에 참고로 포함된 미국 가특허 출원 제61/058,970호의 실시예 3에 설명되어 있는 벡터 pRS425::GPM-sadB (서열 번호 78)로부터 유래시켰다. pRS425::GPM-sadB는 GPM1 프로모터 (서열 번호 72), 아크로모박터 자일로속시단스의 부탄올 데하이드로게나아제로부터의 코딩 영역 (sadB; DNA: 서열 번호 69; 단백질: 서열 번호70: 미국 특허 공개 제2009/0269823호에 개시됨), 및 ADH1 종결자 (서열 번호 73)를 함유하는 키메라 유전자를 포함하는 pRS425 벡터 (ATCC 번호 77106)이다. pRS425::GPMp-sadB는 각각 sadB 코딩 영역의 5' 및 3' 말단에 BbvI 및 PacI 부위를 함유한다. 프라이머 OT1074 및 OT1075 (서열 번호 126 및 127)를 사용하여 부위 지정 돌연변이 유발(site-directed mutagenesis)에 의해 sadB 코딩 영역의 5' 말단에 NheI 부위를 부가하여 벡터 pRS425-GPMp-sadB-NheI을 생성하였으며, 이를 서열결정에 의해 확인하였다. pRS425::PGPM1-sadB-NheI을 NheI 및 PacI로 절단하여 sadB 코딩 영역을 제거하고, 벡터 pHadhy-DNA2.0으로부터의 코돈 최적화 HADH 코딩 영역을 함유하는 NheI-PacI 단편과 라이게이션시켜 pLH435를 생성하였다.pLH435 (pRS425 :: PGPM1-Hadhy-ADH1t) was derived from the vector pRS425 :: GPM-sadB (SEQ ID NO: 78) described in Example 3 of US Provisional Patent Application 61 / 058,970, which is incorporated herein by reference. . pRS425 :: GPM-sadB is the coding region from butanol dehydrogenase of the GPM1 promoter (SEQ ID NO: 72), Acromobacter xyloxoxydans (sadB; DNA: SEQ ID NO: 69; Protein: SEQ ID NO: 70: US patent) PRS425 vector (ATCC No. 77106) comprising a chimeric gene containing ADH1 terminator (SEQ ID NO: 73), and disclosed in Publication 2009/0269823. pRS425 :: GPMp-sadB contains BbvI and PacI sites at the 5 'and 3' ends of the sadB coding region, respectively. The vector pRS425-GPMp-sadB-NheI was generated by adding the NheI site to the 5 'end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NOs 126 and 127). This was confirmed by sequencing. pRS425 :: PGPM1-sadB-NheI was cleaved with NheI and PacI to remove sadB coding region and ligated with NheI-PacI fragment containing codon optimized HADH coding region from vector pHadhy-DNA2.0 to generate pLH435 .

단일 벡터에서 KivD 및 HADH 발현 카세트를 합하기 위하여, 효모 벡터 pRS411 (ATCC 번호 87474)을 SacI 및 NotI로 절단하고, 3중 라이게이션 반응으로, PGPM1-Hadhy-ADH1t 카세트를 함유하는 pLH435로부터의 SalI-NotI 단편과 함께, PTDH3-kivDy-TDH3t 카세트를 함유하는 pLH467로부터의 SacI-SalI 단편과 라이게이션시켰다. 이로써 벡터 pRS411::PTDH3-kivDy-PGPM1-Hadhy (pLH441)를 수득하였으며, 이를 제한효소 맵핑으로 확인하였다.To combine the KivD and HADH expression cassettes in a single vector, the yeast vector pRS411 (ATCC No. 87474) was cleaved with SacI and NotI, and in a triple ligation reaction, SalI- from pLH435 containing the PGPM1-Hadhy-ADH1t cassette. Along with the NotI fragment, the SacI-SalI fragment from pLH467 containing the PTDH3-kivDy-TDH3t cassette was ligated. This gave vector pRS411 :: PTDH3-kivDy-PGPM1-Hadhy (pLH441), which was confirmed by restriction mapping.

더 낮은 아이소부탄올 경로 내의 세 유전자, ilvD, kivDy, 및 Hadhy의 동시 발현 벡터를 생성하기 위하여, 미국 특허 공개 제2010/0081154호에 개시되어 있는 pRS423 FBA ilvD(스트렙(Strep)) (서열 번호 128)를 IlvD 유전자의 공급원으로서 사용하였다. 이 셔틀 벡터에는 이. 콜라이에서의 유지를 위한 F1 복제 기원(nt 1423 내지 1879)이, 그리고 효모에서의 복제를 위한 2마이크론 기원(nt 8082 내지 9426)이 함유되어 있다. 상기 벡터는 FBA1 프로모터 (nt 2111 to 3108; 서열 번호 120) 및 FBA 종결자 (nt 4861 to 5860; 서열 번호 129)를 갖는다. 또한, 이는 효모에서의 선별을 위한 His 마커 (nt 504 내지 1163), 및 이. 콜라이에서의 선별을 위한 앰피실린 내성 마커(nt 7092 내지 7949)를 갖고 있다. 스트렙토코쿠스 뮤탄스 UA159 (ATCC 번호 700610) 유래의 ilvD 코딩 영역 (nt 3116 to 4828; 서열 번호 115; 단백질: 서열 번호 116)은 발현을 위한 키메라 유전자를 형성하도록 FBA 프로모터와 FBA 종결자 사이에 있다. 게다가, ilvD 코딩 영역 (뉴클레오티드 4829-4849)에 융합된 루미오 태그(lumio tag)가 있다.PRS423 FBA ilvD (Strep) disclosed in US Patent Publication No. 2010/0081154 (SEQ ID NO: 128) to generate co-expression vectors of the three genes, ilvD, kivDy, and Hadhy, in the lower isobutanol pathway. Was used as the source of the IlvD gene. This shuttle vector has a tooth. F1 origin of replication (nt 1423-1879) for maintenance in E. coli and 2 micron origin (nt 8082-9426) for replication in yeast. The vector has an FBA1 promoter (nt 2111 to 3108; SEQ ID NO: 120) and a FBA terminator (nt 4861 to 5860; SEQ ID NO: 129). In addition, it is His marker for selection in yeast (nt 504-1163), and E. coli. Ampicillin resistance markers (nt 7092-7949) for selection in E. coli. The ilvD coding region from Streptococcus mutans UA159 (ATCC No. 700610) (nt 3116 to 4828; SEQ ID NO: 115; Protein: SEQ ID NO: 116) is between the FBA promoter and the FBA terminator to form a chimeric gene for expression . In addition, there is a lumio tag fused to the ilvD coding region (nucleotides 4829-4849).

제1 단계는 pRS423 FBA ilvD(스트렙) (pRS423-FBA(SpeI)-IlvD(스트렙토코커스 뮤탄스)-루미오로도 불리움)를 SacI 및 SacII (T4 DNA 폴리머라아제를 사용해 SacII 부위가 블런트 말단화된 것)와 함께 선형화해서, 총 길이가 9,482 bp인 벡터를 제공하는 것이었다. 제2 단계는 SacI 및 KpnI (KpnI 부위는 T4 DNA 폴리머라아제를 사용해 블런트 말단화됨)를 이용해 pLH441로부터 kivDy-hADHy 카세트를 단리하여, 6,063 bp 단편을 제공하는 것이었다. 이 단편을 pRS423-FBA(SpeI)-IlvD(스트렙토코터스 뮤탄스)-루미오 유래의 9,482 bp 벡터 단편과 라이게이션시켰다. 이는 벡터 pLH468 (pRS423::PFBA1-ilvD(스트렙) 루미오-FBA1t-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t)을 생성하였고, 이를 제한효소 맵핑 및 서열결정에 의해 확인하였다.The first step consists of pac423 FBA ilvD (strep) (also called pRS423-FBA (SpeI) -IlvD (also called Streptococcus mutans) -Lumio) blunt ended SacII sites using SacI and SacII (T4 DNA polymerase). Linearized to provide a vector with a total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 using SacI and KpnI (KpnI site blunt terminated using T4 DNA polymerase) to provide a 6,063 bp fragment. This fragment was ligated with a 9,482 bp vector fragment from pRS423-FBA (SpeI) -IlvD (Streptococcus mutans) -Lumio. This produced the vector pLH468 (pRS423 :: PFBA1-ilvD (strep) Lumio-FBA1t-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.

pLH532 제작pLH532 production

pLH532 플라스미드 (서열 번호 130)를 효모 내에서의 ALS 및 KARI 발현용으로 제작하였다. pLH532는 하기 키메라 유전자를 함유하는 pHR81 벡터 (ATCC 번호 87541)이다: 1) CUP1 프로모터 (서열 번호 139), 바실루스 섭틸리스 유래의 아세토락테이트 신타아제 코딩 영역 (AlsS; 서열 번호 137; 단백질: 서열 번호 138) 및 CYC1 종결자2 (서열 번호 133); 2) ILV5 프로모터 (서열 번호 134), Pf5.IlvC 코딩 영역 (서열 번호 132) 및 ILV5 종결자 (서열 번호 135); 및 3) FBA1 프로모터 (서열 번호 136), 에스. 세레비지애 KARI 코딩 영역 (ILV5; 서열 번호 131); 및 CYC1 종결자.pLH532 plasmid (SEQ ID NO: 130) was constructed for expression of ALS and KARI in yeast. pLH532 is a pHR81 vector (ATCC No. 87541) containing the following chimeric genes: 1) CUP1 promoter (SEQ ID NO: 139), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NO: 137; protein: sequence 138) and CYC1 terminator 2 (SEQ ID NO: 133); 2) ILV5 promoter (SEQ ID NO: 134), Pf5.IlvC coding region (SEQ ID NO: 132) and ILV5 terminator (SEQ ID NO: 135); And 3) FBA1 promoter (SEQ ID NO: 136), S. a. Cerevisiae KARI coding region (ILV5; SEQ ID NO: 131); And CYC1 terminator.

Pf5.IlvC 코딩 영역은 슈도모나스 플루오레센스로부터 유래된 KARI을 코딩하는 서열이며, 이는 본 명세서에 참고로 포함된 미국 특허 공개 제2009/0163376호에 기재되었다.The Pf5.IlvC coding region is a sequence encoding KARI derived from Pseudomonas fluorescens, which was described in US Patent Publication No. 2009/0163376, which is incorporated herein by reference.

Pf5.IlvC 코딩 영역을 사카로마이세스 세레비지애에서의 발현에 대하여 최적화된 코돈을 기반으로 하여 디엔에이2.0, 인크. (미국 캘리포니아주 멘로 파크 소재; 서열 번호 132)에 의해 합성하였다.Pf5.IlvC coding region based on codon optimized for expression in Saccharomyces cerevisiae DNA 2.0, Inc. (Menlo Park, CA; SEQ ID NO: 132).

pYZ090 제작pYZ090 production

pYZ090 (서열 번호 1은 pHR81 (ATCC 번호 87541) 골격을 기반으로 하며, ALS의 발현을 위한, 효모 CUP1 프로모터 (뉴클레오티드 2-449) 및 이에 이은 CYC1 종결자 (뉴클레오티드 2181-2430)로부터 발현되는 바실루스 섭틸리스 (뉴클레오티드 위치 457-2172)로부터의 alsS 유전자의 코딩 영역을 갖는 키메라 유전자, 및 KARI의 발현을 위한, 효모 ILV5 프로모터 (2433-3626) 및 이에 이은 ILV5 종결자 (뉴클레오티드 4682-5304)로부터 발현되는 락토코쿠스 락티스 (뉴클레오티드 3634-4656)로부터의 ilvC 유전자의 코딩 영역을 갖는 키메라 유전자를 함유하도록 제작하였다.pYZ090 (SEQ ID NO: 1 is based on the pHR81 (ATCC No. 87541) skeleton and is a Bacillus subexpressed from the yeast CUP1 promoter (nucleotides 2-449) followed by the CYC1 terminator (nucleotides 2181-2430) for expression of ALS Expression from the yeast ILV5 promoter (2433-3626) followed by the ILV5 terminator (nucleotides 4682-5304) for expression of the chimeric gene with the coding region of the alsS gene from Tillis (nucleotide positions 457-2172), and KARI Was constructed to contain a chimeric gene with the coding region of the ilvC gene from Lactococcus lactis (nucleotides 3634-4656).

pYZ067 제작by pYZ067

pYZ067을 하기 키메라 유전자를 함유하도록 제작하였다: 1) 다이하이드록시산 데하이드라타아제 (DHAD)의 발현을 위한, 효모 FBA1 프로모터 (뉴클레오티드 1161-2250), 이어서 FBA 종결자 (뉴클레오티드 4005-4317)로부터 발현되는 에스. 뮤탄스 UA159 (뉴클레오티드 위치 2260-3971)로부터의 ilvD 유전자의 코딩 영역, 2) 알코올 데하이드로게나아제의 발현을 위한, 효모 GPM 프로모터 (뉴클레오티드 5819-6575), 이어서 ADH1 종결자 (뉴클레오티드 4356-4671)로부터 발현되는 말 간 ADH (뉴클레오티드 4680-5807)의 코딩 영역, 및 3) 케토아이소발레레이트 데카르복실라아제 발현을 위한, 효모 TDH3 프로모터 (뉴클레오티드 8830-9493), 이어서 TDH3 종결자 (뉴클레오티드 5682-7161)로부터 발현되는 락토코쿠스 락티스 (뉴클레오티드 7175-8821) 유래의 KivD 유전자의 코딩 영역.pYZ067 was constructed to contain the following chimeric genes: 1) Yeast FBA1 promoter (nucleotides 1161-2250) followed by FBA terminator (nucleotides 4005-4317) for expression of dihydroxy acid dehydratase (DHAD) Expressed from S. Coding region of the ilvD gene from Mutans UA159 (nucleotide positions 2260-3971), 2) Yeast GPM promoter (nucleotides 5819-6575), followed by ADH1 terminator (nucleotides 4356-4671) for expression of alcohol dehydrogenases Coding region of hepatic ADH (nucleotides 4680-5807) expressed from, and 3) yeast TDH3 promoter (nucleotides 8830-9493) followed by TDH3 terminator (nucleotides 5682-7161) for ketoisovalerate decarboxylase expression Coding region of the KivD gene derived from Lactococcus lactis (nucleotides 7175-8821).

pRS423::CUP1-alsS+FBA-budA 및 pRS426::FBA-budC+GPM-sadB 및 pLH475-Z4B8의 제작Construction of pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB and pLH475-Z4B8

pRS423::CUP1-alsS+FBA-budA 및 pRS426::FBA-budC+GPM-sadB 및 pLH475-Z4B8의 제작은 본 명세서에 참고로 포함된 미국 특허 공개 제2009/0305363호에 기재되어 있다.The construction of pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB and pLH475-Z4B8 is described in US Patent Publication No. 2009/0305363, which is incorporated herein by reference.

실시예Example

하기 비제한적 실시예는 본 발명을 추가로 예시할 것이다. 하기 실시예는 공급재료로서의 옥수수, 그리고 옥수수 지질의 효소적 가수분해에 의해 수득한 ISPR 추출제로서의 COFA를 포함하지만, 본 발명으로부터 벗어나지 않고서 다른 바이오매스 공급원을 바이오매스 오일의 효소적 가수분해 및 공급재료용으로 사용할 수 있음을 이해하여야 한다.The following non-limiting examples will further illustrate the present invention. The following examples include COFA as a feedstock and COFA as an ISPR extractant obtained by enzymatic hydrolysis of corn lipids, but other biomass sources can be enzymatically hydrolyzed and fed biomass oil without departing from the present invention. It should be understood that it can be used for materials.

본 명세서에 사용되는 바와 같이, 사용한 약어의 의미는 하기와 같았다: "g"는 그램을 의미하며, "㎏"는 킬로그램을 의미하고, "L"은 리터를 의미하며, "mL"은 밀리리터를 의미하고, "㎕"은 마이크로리터를 의미하며, "mL/L"은 리터 당 밀리리터를 의미하고, "mL/min"은 분 당 밀리리터를 의미하며, "DI"는 탈이온화를 의미하고, "uM"은 마이크로미터를 의미하며, "㎚"은 나노미터를 의미하고, "w/v"는 중량/부피를 의미하며, "OD"는 광학 밀도를 의미하고, "OD₆₀₀"은 600 nM의 파장에서의 광학 밀도를 의미하며, "dcw"는 건조 세포 중량을 의미하고, "rpm"은 분 당 회전수를 의미하며, "℃"는 섭씨 온도를 의미하고, "℃/min"은 분 당 섭씨 온도를 의미하며, "slpm"은 분 당 표준 리터를 의미하고, "ppm"은 백만분율을 의미하며, "pdc"는 효소 번호가 뒤따르는 피루베이트 데카르복실라아제 효소를 의미한다.As used herein, the meaning of the abbreviations used was as follows: "g" means grams, "kg" means kilograms, "L" means liters, and "mL" means milliliters. "Μl" means microliters, "mL / L" means milliliters per liter, "mL / min" means milliliters per minute, "DI" means deionization, " uM "means micrometer," nm "means nanometer," w / v "means weight / volume," OD "means optical density," OD ₆₀₀ "means 600 nM Means optical density at wavelength, "dcw" means dry cell weight, "rpm" means revolutions per minute, "° C" means degrees Celsius, and "° C / min" per minute Mean Celsius, "slpm" means standard liters per minute, "ppm" means parts per million, "pdc" is pyruvate decarboxylase followed by enzyme number It means an enzyme.

일반 방법General method

시드 플라스크 성장Seed flask growth

pdc1이 결실되고, pdc5가 결실되고, pdc6이 결실된, 탄수화물 공급원으로부터 아이소부탄올을 생성하도록 조작한 사카로마이세스 세레비지애 스트레인을, 냉동 배양물로부터 시드 플라스크에서 0.55-1.1 g/L의 dcw (OD600 1.3-2.6 - 서모 헬리오스(Thermo Helios) α 서모 피셔 사이언티픽 인크.(Thermo Fisher Scientific Inc.), 미국 매사추세츠주 월섬 소재)로 성장시켰다. 상기 배양물을 300 rpm으로 회전하는 인큐베이터에서 26℃에서 성장시켰다. 냉동 배양물은 이전에 - 80℃에서 보관하였다. 제1 시드 플라스크 배지의 조성은 다음과 같았다:A Saccharomyces cerevisiae strain engineered to produce isobutanol from a carbohydrate source, deleted pdc1, deleted pdc5, deleted pdc6, and 0.55-1.1 g / L dcw in a seed flask from frozen culture (OD600 1.3-2.6-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Mass.). The culture was grown at 26 ° C. in an incubator rotating at 300 rpm. Frozen cultures were previously stored at -80 ° C. The composition of the first seed flask medium was as follows:

3.0 g/L의 덱스트로스3.0 g / L dextrose

3.0 g/L의 무수 에탄올3.0 g / L anhydrous ethanol

3.7 g/L의 포메디움(ForMedium™) 신테틱 컴플리트 아미노 애시드(Synthetic Complete Amino Acid) (카이저(Kaiser) 드롭-아웃(Drop-Out): HIS를 포함하지 않으며, URA를 포함하지 않음 (참조 번호: DSCK162CK)3.7 g / L ForMedium ™ Synthetic Complete Amino Acid (Kaiser Drop-Out: Does not contain HIS, does not contain URA (reference number) DSCK162CK)

6.7 g/L의 디프코(Difco) 효모 질소 베이스, 아미노산을 포함하지 않음 (번호: 291920)6.7 g / L of Difco yeast nitrogen base, does not contain amino acids (No .: 291920)

제1 시드 플라스크 배양물로부터의 12 밀리리터를 2 L 플라스크로 옮기고, 300 rpm으로 회전하는 인큐베이터에서 30℃에서 성장시켰다. 제2 시드 플라스크는 하기 배지 220 mL을 갖는다:Twelve milliliters from the first seed flask culture were transferred to a 2 L flask and grown at 30 ° C. in an incubator rotating at 300 rpm. The second seed flask has 220 mL of the following medium:

30.0 g/L의 덱스트로스30.0 g / L dextrose

5.0 g/L의 무수 에탄올5.0 g / L anhydrous ethanol

3.7 g/L의 포메디움™ 신테틱 컴플리트 아미노 애시드 (카이저) 드롭-아웃:3.7 g / L Pomedium ™ Synthetic Complete Amino Acid (Kaizer) Drop-Out:

HIS를 포함하지 않으며, URA를 포함하지 않음 (참조 번호: DSCK162CK)Does not contain HIS, does not contain URA (Reference: DSCK162CK)

6.7 g/L의 디프코 효모 질소 베이스, 아미노산을 포함하지 않음 (번호: 291920)6.7 g / L Diphco Yeast Nitrogen Base, does not contain amino acids (No .: 291920)

pH 5.5-6.0으로 적정한 0.2 M MES 완충제0.2 M MES buffer titrated to pH 5.5-6.0

상기 배양물을 0.55-1.1 g/L의 dcw (OD₆₀₀ 1.3-2.6)로 성장시켰다. 200 g/L 펩톤 및 100 g/L의 효모 추출물을 함유하는 용액 30 mL의 첨가물을 이 세포 농도에서 첨가하였다. 그 후, 0.2 uM 필터로 살균한 코그니스(Cognis)의 90-95% 올레일 알코올 300 mL의 첨가물을 상기 플라스크에 첨가하였다. 상기 배양물을 계속하여 > 4 g/L의 dcw (OD₆₀₀ > 10)로 성장시킨 후 수확하고, 발효물에 첨가하였다.The culture was grown to 0.55-1.1 g / L dcw (OD ₆₀₀ 1.3-2.6). Addition of 30 mL of solution containing 200 g / L peptone and 100 g / L yeast extract was added at this cell concentration. An addition of 300 mL of Cognis' 90-95% oleyl alcohol sterilized with a 0.2 uM filter was then added to the flask. The culture was subsequently grown to> 4 g / L dcw (OD ₆₀₀ > 10), harvested and added to the fermentation.

발효 준비Fermentation Preparation

초기 발효 용기 준비Initial Fermentation Vessel Preparation

유리 재킷(glass jacked), 2 L 발효 용기 (독일 괴팅겐 소재의 사르토리우스 아게(Sartorius AG))를 액화 중량의 66%가 되도록 가정용수로 충전시켰다. pH 탐침자 (해밀턴 이지펌 플러스(Hamilton Easyferm Plus) K8, 부품 번호: 238627, 스위스 보나두즈 소재의 해밀턴 보나두즈 아게(Hamilton Bonaduz AG))를, 사르토리우스 DCU-3 컨트롤 타워 캘리브레이션(Control Tower Calibration) 메뉴를 통하여 보정하였다. 영점을 pH=7로 보정하였다. 그 범위(span)를 pH=4로 보정하였다. 그 후, 탐침자를 스테인리스강 헤드 플레이트를 통하여 발효 용기 내에 넣었다. 용존 산소 탐침자 (pO₂ 탐침자)도 상기 헤드 플레이트를 통하여 발효 용기 내에 넣었다. 영양소, 시드 배양물, 추출 용매 및 베이스의 전달에 사용되는 튜브류를 상기 헤드 플레이트에 부착시키고, 그 단부를 포일로 감쌌다. 전체 발효 용기를 스테리스(Steris) (미국 오하이오주 멘토르 소재의 스테리스 코포레이션(Steris Corporation)) 오토클레이브 내에 넣고, 액체 사이클에서 30분 동안 살균하였다.A glass jacked, 2 L fermentation vessel (Sartorius AG, Göttingen, Germany) was filled with household water to 66% of the liquefied weight. pH probe (Hamilton Easyferm Plus K8, part number: 238627, Hamilton Bonaduz AG, Bonaduz, Switzerland), and the Stortorius DCU-3 Control Tower Calibration ) Through the menu. The zero point was corrected to pH = 7. The span was corrected to pH = 4. The probe was then placed into the fermentation vessel through a stainless steel head plate. Dissolved oxygen probe (pO ₂ probe) was also placed into the fermentation vessel through the head plate. Tubes used for the delivery of nutrients, seed cultures, extraction solvents and bases were attached to the head plate and the ends were wrapped with foil. The entire fermentation vessel was placed in a Steris (Steris Corporation, Mentor, Ohio) autoclave and sterilized for 30 minutes in a liquid cycle.

발효 용기를 오토클레이브로부터 꺼내고, 로드셀(load cell) 상에 두었다. 재킷수 공급부 및 리턴 라인(return line)을 각각 가정용수 및 청결한 배수관에 연결하였다. 응축기 냉각수 유입 및 유출 라인을 7℃에서 작동하는 6-L 재순환 항온조(recirculating temperature bath)에 연결하였다. 발효 용기로부터 가스를 이송시키는 배기 라인을 서모 질량 분광계 (프리마 dB(Prima dB), 미국 매사추세츠주 월섬 소재의 서모 피셔 사이언티픽 인크.)에 연결된 이송 라인에 연결하였다. 스파저(sparger) 라인을 가스 공급 라인에 연결하였다. 영양소, 추출 용매, 시드 배양물, 및 베이스 첨가용 튜브류를 펌프를 통하여 배수관에 연결하거나 또는 클램프로 폐쇄시켰다.The fermentation vessel was removed from the autoclave and placed on a load cell. The jacket water supply and return line were connected to household water and clean drain, respectively. The condenser coolant inlet and outlet lines were connected to a 6-L recirculating temperature bath operating at 7 ° C. An exhaust line for delivering gas from the fermentation vessel was connected to a transfer line connected to a thermomass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Mass.). A sparger line was connected to the gas supply line. Nutrients, extraction solvents, seed cultures, and base addition tubes were connected to the drainpipe via pumps or closed with clamps.

발효 용기 온도를 열전쌍 및 가정용수 순환 루프를 이용하여 55℃로 제어하였다. 습윤 옥수수알 (#2 옐로우 덴트(yellow dent))을 1.0 ㎜ 스크린을 갖춘 해머 밀을 이용하여 분쇄하고, 그 후, 생성된 분쇄 통옥수수알을 액화 반응 매스의 29-30% (건조 옥수수 고형물 중량)인 충전율로 발효 용기에 첨가하였다.Fermentation vessel temperature was controlled to 55 ° C. using thermocouples and domestic water circulation loops. Wet corn kernels (# 2 yellow dent) are ground using a hammer mill with a 1.0 mm screen, and the resulting ground corn kernels are then 29-30% of the liquefied mass (dry corn solids weight). Was added to the fermentation vessel at a filling rate.

액화 전 리파아제 처리Lipase treatment before liquefaction

리파아제 효소 원액을 10 ppm의 최종 리파아제 농도로 발효 용기에 첨가하였다. 발효 용기를 55℃, 300 rpm, 및 0.3 slpm N₂ 오버레이(overlay)에서 >6시간 동안 유지하였다. 리파아제 처리가 완료된 후, 하기(액화)에 설명되는 바와 같이 액화를 실시하였다.Lipase enzyme stock was added to the fermentation vessel at a final lipase concentration of 10 ppm. The fermentation vessel was maintained at 55 ° C., 300 rpm, and 0.3 slpm N ₂ overlay for> 6 hours. After the lipase treatment was completed, liquefaction was performed as described below (liquefaction).

액화liquefaction

발효 용기를 300-1200 rpm에서 혼합하면서 알파-아밀라아제를 발효 용기에 그의 사양서에 따라 첨가하였으며, 이때 가정용 살균 N₂를 스파저를 통하여 0.3 slpm으로 첨가하였다. 온도 설정점을 55℃로부터 85℃로 변화시켰다. 온도가 > 80℃일 때, 액화 쿡 타임(cook time)을 시작하고, 액화 사이클을 > 80℃에서 90 내지 120분 동안 유지하였다. 액화 사이클이 완료된 후 발효 용기 온도 설정점을 30℃의 발효 온도로 설정하였다. N₂를 스파저로부터 상부 공간으로 재전송하여 화학적 소포제를 첨가하지 않고서 발포를 방지하였다.Alpha-amylase was added to the fermentation vessel according to its specifications while mixing the fermentation vessel at 300-1200 rpm, at which time domestic sterilization N ₂ was added at 0.3 slpm via a sparger. The temperature set point was changed from 55 ° C to 85 ° C. When the temperature was> 80 ° C, the liquefaction cook time was started and the liquefaction cycle was maintained at> 80 ° C for 90-120 minutes. The fermentation vessel temperature set point was set to a fermentation temperature of 30 ° C. after the liquefaction cycle was completed. N ₂ was retransmitted from the sparger to the headspace to prevent foaming without the addition of chemical antifoam.

액화 후 리파아제 처리Lipase treatment after liquefaction

액화 사이클이 완료된 후 (액화), 발효 용기 온도를 30℃ 대신 55℃로 설정하였다. 필요할 때 산 또는 염기를 볼루스 첨가함으로써 pH를 수동으로 pH=5.8로 제어하였다. 리파아제 효소 원액을 10 ppm의 최종 리파아제 농도로 발효 용기에 첨가하였다. 발효 용기를 55℃, 300 rpm, 및 0.3 slpm의 N₂ 오버레이에서 >6시간 동안 유지하였다. 리파아제 처리가 완료된 후, 발효 용기 온도를 30℃로 설정하였다.After the liquefaction cycle was completed (liquefaction), the fermentation vessel temperature was set to 55 ° C instead of 30 ° C. PH was manually controlled to pH = 5.8 by bolus addition of acid or base when needed. Lipase enzyme stock was added to the fermentation vessel at a final lipase concentration of 10 ppm. The fermentation vessels were maintained for> 6 hours at an N ₂ overlay of 55 ° C., 300 rpm, and 0.3 slpm. After the lipase treatment was completed, the fermentation vessel temperature was set to 30 ° C.

리파아제 열 불활성화 처리 (열 불활성화 처리 방법)Lipase heat inactivation treatment (heat inactivation treatment method)

발효 용기 온도를 > 80℃에서 > 15분 동안 유지하여 리파아제를 불활성화시켰다. 열 불활성화 처리가 완료된 후, 발효 용기 온도를 30℃로 설정하였다.The lipase was inactivated by maintaining the fermentation vessel temperature at> 80 ° C for> 15 minutes. After the heat inactivation treatment was completed, the fermentation vessel temperature was set to 30 ° C.

접종 전 영양소 첨가Addition of nutrients before inoculation

접종 직전에 에탄올 (6.36 mL/L, 접종 후 부피, 200 프루프(proof), 무수)을 발효 용기에 첨가하였다. 접종 직전에 티아민을 20 ㎎/L의 최종 농도로 첨가하고, 100 ㎎/L의 니코틴산을 또한 첨가하였다.Immediately before inoculation, ethanol (6.36 mL / L, volume after inoculation, 200 proof, anhydrous) was added to the fermentation vessel. Thiamine was added at a final concentration of 20 mg / L immediately before inoculation, and 100 mg / L nicotinic acid was also added.

접종 전 Before vaccination 올레일Orail 알코올 또는 옥수수유 지방산의 첨가 Addition of Alcohol or Corn Oil Fatty Acids

접종 직후 1 L/L (접종 후 부피)의 올레일 알코올 또는 옥수수유 지방산을 첨가하였다.Immediately after inoculation, 1 L / L (volume after inoculation) of oleyl alcohol or corn oil fatty acid was added.

발효 용기 접종Fermentation vessel inoculation

N₂를 발효 용기에 첨가하면서 발효 용기 pO₂ 탐침자를 영점으로 보정하였다. 발효 용기 pO₂ 탐침자를 그 범위로 보정하였으며, 이때 살균 공기를 300 rpm으로 살포하였다. 제2 시드 플라스크 후 발효 용기에 > 4 g/L의 dcw를 접종하였다. 진탕 플라스크를 인큐베이터/진탕기로부터 5분 동안 꺼내 놓아서 올레일 알코올 상 및 수성 상이 상 분리되게 하였다. 수성 상 (110 mL) 을 살균, 접종 병으로 옮겼다. 접종물을 연동 펌프를 통하여 발효 용기 내로 펌핑하였다.The fermentation vessel pO ₂ probe was calibrated to zero with N ₂ added to the fermentation vessel. The fermentation vessel pO ₂ probe was calibrated to that range, with sterile air sparged at 300 rpm. The fermentation vessel was inoculated with> 4 g / L dcw after the second seed flask. The shake flask was removed from the incubator / shaker for 5 minutes to allow phase separation of the oleyl alcohol phase and the aqueous phase. The aqueous phase (110 mL) was transferred to a sterile, inoculated bottle. The inoculum was pumped into the fermentation vessel through a peristaltic pump.

발효 용기 작동 조건Fermentation vessel operating conditions

발효 용기를 전체 성장 및 생성 단계에 있어서 30℃에서 작동시켰다. pH를 어떠한 산도 첨가하지 않고서 5.7 내지 5.9의 pH로부터 5.2의 제어 설정점으로 낮추었다. 수산화암모늄을 이용하여 pH를 나머지의 성장 및 생성 단계에 있어서 pH=5.2로 제어하였다. 나머지의 성장 및 생성 단계에 있어서 살균 공기를 스파저를 통하여 0.3 slpm으로 발효 용기에 첨가하였다. pO₂를 단지 교반 제어를 이용하여 사르토리우스 DCU-3 컨트롤 박스(Control Box) PID 제어 루프에 의해 3.0%로 제어되도록 설정하였으며, 이때 교반기 최소치는 300 rpm으로 설정하고 최대치는 2000 rpm으로 설정하였다. 글루코스를, α-아밀라아제 (글루코아밀라아제)의 첨가에 의해 동시적 당화 및 발효의 액화 옥수수 매시를 통하여 공급하였다. 전분이 당화에 이용가능한 한, 글루코스는 과량으로 (1-50 g/L) 유지되었다.The fermentation vessel was operated at 30 ° C. for the entire growth and production stage. The pH was lowered from a pH of 5.7 to 5.9 to a control set point of 5.2 without adding any acid. Ammonium hydroxide was used to control the pH to pH = 5.2 for the remaining growth and production steps. For the remaining growth and production stages, sterile air was added to the fermentation vessel at 0.3 slpm through a sparger. pO ₂ was set to be controlled to 3.0% by the Sartorius DCU-3 Control Box PID control loop using only agitation control, with the stirrer minimum set at 300 rpm and the maximum set at 2000 rpm. . Glucose was fed through the liquefied corn mash of simultaneous saccharification and fermentation by the addition of α-amylase (glucoamylase). As long as starch was available for saccharification, glucose was maintained in excess (1-50 g / L).

분석적Analytical

가스 분석Gas analysis

공정 공기를 서모 프리마 (미국 매사추세츠주 월섬 소재의 서모 피셔 사이언티픽 인크.) 질량 분광계에서 분석하였다. 이는 살균한 후 각각의 발효 용기에 첨가된 바로 그 공정 공기였다. 각각의 발효 용기의 배출 가스(off-gas)를 동일한 질량 분광계에서 분석하였다. 이러한 서모 프리마 dB는 매 월요일 아침 6:00 am에 보정 점검을 작동시킨다. 보정 점검은 질량 분광계와 관련된 가스 웍스(Gas Works) v1.0 (미국 매사추세츠주 월섬 소재의 서모 피셔 사이언티픽 인크.) 소프트웨어를 통하여 일정을 잡았다. 가스 보정은 다음과 같았다:Process air was analyzed on a Thermo Prima (Thermo Fisher Scientific Inc., Waltham, Mass.) Mass spectrometer. This was the same process air added to each fermentation vessel after sterilization. The off-gas of each fermentation vessel was analyzed on the same mass spectrometer. This thermo prima dB activates the calibration check at 6:00 am every Monday morning. Calibration checks were scheduled through the Gas Works v1.0 (Thermo Fisher Scientific Inc., Waltham, Mass.) Software associated with the mass spectrometer. The gas calibration was as follows:

이산화탄소를 병에 담긴 다른 공지된 가스를 이용하여 보정 사이클 동안 5% 및 15%에서 점검하였다. 산소를 병에 담긴 다른 공지된 가스를 이용하여 15%에서 점검하였다. 각 발효 용기의 배출 가스의 분석을 기반으로 하여, 질량 분광계의 몰 분율 분석 및 발효 용기 내로의 가스 유량 (매스 유동 제어기)을 이용함으로써 배출 가스 내로 호흡되는 이산화탄소, 소비된 산소 및 스트리핑된 아이소부탄올의 양을 측정하였다. 시간 당 가스 배출 속도를 계산하고, 이어서 발효 동안의 배출 속도를 적분하였다.Carbon dioxide was checked at 5% and 15% during the calibration cycle using other known gases in bottles. Oxygen was checked at 15% using other known gases in bottles. Based on the analysis of the exhaust gas of each fermentation vessel, the molar fraction analysis of the mass spectrometer and the gas flow rate into the fermentation vessel (mass flow controller) of carbon dioxide, oxygen consumed and stripped isobutanol breathed into the exhaust gas The amount was measured. The gas discharge rate per hour was calculated and then the discharge rate during fermentation was integrated.

바이오매스 측정Biomass Measurement

1X PBS를 이용하여 NaCl (VWR BDH8721-0) 중 0.4% 트리판 블루(Trypan Blue)를 1:5로 희석시킨 것에 의해 0.08% 트리판 블루 용액을 제조하였다. 1.0 mL 샘플을 발효 용기로부터 꺼내고, 1.5 mL 에펜도르프 원심분리 튜브 내에 넣고, 에펜도르프, 5415C에서 14,000 rpm에서 5분 동안 원심분리하였다. 원심분리 후, 20-200 ㎕ 바이오히트(BioHit) 피펫 팁을 갖춘 m200 배리어블 채널 바이오히트 피펫을 이용하여 상부 용매 층을 제거하였다. 용매 층과 수성 층 사이의 층을 제거하지 않도록 주의하였다. 일단 용매 층을 제거하였으면, 샘플을 2700 rpm으로 설정한 볼텍스-지니(Vortex-Genie)(등록상표)를 이용하여 재현탁시켰다.A 0.08% Trypan Blue solution was prepared by diluting 1: 5 of 0.4% Trypan Blue in NaCl (VWR BDH8721-0) using IX PBS. The 1.0 mL sample was removed from the fermentation vessel and placed in a 1.5 mL Eppendorf centrifuge tube and centrifuged at 14,000 rpm for 5 minutes at Eppendorf, 5415C. After centrifugation, the upper solvent layer was removed using an m200 barrierable channel bioheat pipette with a 20-200 μl BioHit pipette tip. Care was taken not to remove the layer between the solvent layer and the aqueous layer. Once the solvent layer was removed, the samples were resuspended using Vortex-Genie® set at 2700 rpm.

일련의 희석이 혈구계수기 계수에 이상적인 농도의 준비를 위하여 필요하였다. OD가 10일 경우, 1:20의 희석을 실시하여 0.5의 OD를 달성하는데, 상기 0.5의 OD는 스퀘어 당 계수될 세포의 이상적인 양, 20개 내지 30개를 제공할 것이다. 옥수수 고형물로 인한 희석에서의 부정확성을 감소시키기 위하여, 절단된 100-1000 ㎕ 바이오히트 피펫 팁을 이용한 다중 희석이 필요하였다. 대략적으로, 1 cm를 상기 팁에서 절단 제거하여 개구를 늘렸으며, 이는 팁이 막히지 않도록 하였다. 1:20의 최종 희석에 있어서, 발효 샘플과 0.9% NaCl 용액의 초기 1:1 희석물을 제조하였다. 그 후, 이전의 용액(즉, 초기 1:1 희석물)과 0.9% NaCl 용액의 1:1 희석물(제2 희석물)을 생성하고, 이어서 제2 희석물과 트리판 블루 용액의 1:5 희석물을 생성하였다. 샘플들을 각각의 희석 사이에 와동시키고, 절단한 팁을 0.9% NaCl 및 트리판 블루 용액 내에 넣어서 헹구었다.A series of dilutions were required for the preparation of concentrations ideal for hemocytometer counts. When the OD is 10, a 1:20 dilution is performed to achieve an OD of 0.5, which will provide an ideal amount of cells to be counted, 20 to 30, per square square. In order to reduce inaccuracies in dilution due to corn solids, multiple dilutions with cut 100-1000 μl bioheat pipette tips were required. Roughly, 1 cm was cut away from the tip to increase the opening, which prevented the tip from clogging. For a final dilution of 1:20, an initial 1: 1 dilution of the fermentation sample and 0.9% NaCl solution was prepared. Thereafter, a 1: 1 dilution (second dilution) of the previous solution (ie, the initial 1: 1 dilution) and 0.9% NaCl solution was generated, followed by 1: 1 of the second dilution and trypan blue solution. 5 dilutions were generated. Samples were vortexed between each dilution and the cut tips were rinsed in 0.9% NaCl and trypan blue solution.

커버 슬립을 혈구계수기 (하우저 사이언티픽 브라이트-라인(Hausser Scientific Bright-Line) 1492)의 상부에 조심스럽게 두었다. 2-20 ㎕ 바이오히트 피펫 팁을 갖춘 m20 배리어블 채널 바이오히트 피펫을 이용하여 분취물 (10 ㎕)을 최종 트리판 블루 희석물에서 꺼내고, 이를 혈구계수기 내로 주입하였다. 혈구계수기를 40배 배율의 자이스 악시오스코프(Zeis Axioskop) 40 현미경 상에 두었다. 중심 사분면을 25개의 스퀘어(square)로 나누고, 그 후 둘 모두의 챔버 내의 4개의 코너 및 중심 스퀘어를 계수하고 기록하였다. 둘 모두의 챔버를 계수한 후, 평균을 취하고, 이것에 희석 계수 (20)을 곱하고, 그 후 혈구계수기 내의 사분면 내의 스퀘어의 수에 대한 25를 곱하고, 그 후 계수된 사분면의 부피인 0.0001 mL로 나누었다. 이 계산의 총계는 mL 당 세포의 수이다.The cover slip was carefully placed on top of the hemocytometer (Hausser Scientific Bright-Line 1492). Aliquots (10 μl) were removed from the final trypan blue dilution using an m20 barrierable channel bioheat pipette with a 2-20 μl bioheat pipette tip and injected into the hemocytometer. Hemocytometer was placed on a Zeiss Axioskop 40 microscope at 40x magnification. The central quadrant was divided into 25 squares, and then the four corners and central squares in both chambers were counted and recorded. After counting both chambers, take the average, multiply this by the dilution factor (20), then multiply by 25 for the number of squares in the quadrant in the hemocytometer, and then to 0.0001 mL, the volume of the quadrant counted Divided. The total of this calculation is the number of cells per mL.

수성 상 중 발효 생성물의 LC 분석LC Analysis of Fermentation Product in Aqueous Phase

프로세싱을 위하여 준비가 될 때까지 샘플을 냉장시켰다. 샘플을 냉장고로부터 꺼내어 실온에 도달되게 하였다 (약 1시간). 100-1000 ㎕ 바이오히트 피펫 팁을 갖춘 m1000 배리어블 채널 바이오히트 피펫을 이용하여 대략 300 ㎕의 샘플을 0.2 um 원심분리 필터 (나노셉(Nanosep)(등록상표) MF 변형 나일론 원심분리 필터) 내로 옮기고, 그 후 에펜도르프, 5415C를 이용하여 14,000 rpm에서 5분 동안 원심분리하였다. 대략 200 ㎕의 여과 샘플을 중합체 피트(feet)를 포함하는 250 ㎕ 유리 바이알 인서트를 갖춘 1.8 자동 샘플러 바이알 내로 옮겼다. PTFE 격벽이 있는 나사 뚜껑을 사용하여 바이알의 뚜껑을 덮은 후 샘플을 2700 rpm으로 설정한 볼텍스-지니(등록상표)를 이용하여 와동시켰다.Samples were refrigerated until ready for processing. The sample was removed from the refrigerator and allowed to reach room temperature (about 1 hour). Approximately 300 μl of the sample was transferred into a 0.2 um centrifugal filter (Nanosep® MF modified nylon centrifugal filter) using an m1000 barrierable channel bioheat pipette with a 100-1000 μl bioheat pipette tip. Then, it was centrifuged for 5 minutes at 14,000 rpm using Eppendorf, 5415C. Approximately 200 μl of filtration sample was transferred into 1.8 auto sampler vials with 250 μl glass vial inserts containing polymer feet. The lid of the vial was capped using a screw cap with PTFE septum and the sample was vortexed using a Vortex-Genie® set at 2700 rpm.

그 후, 바이너리, 등용매 펌프, 진공 탈기 장치, 가열 컬럼 구획, 샘플러 냉각 시스템, UV DAD 검출기 및 RI 검출기를 갖춘 애질런트(Agilent) 1200 시리즈 LC에서 샘플을 진행시켰다. 사용한 컬럼은 바이오-라드 케이션(Bio-Rad Cation) H 리필(refill), 30X4.6 가드 컬럼을 갖춘 아미넥스(Aminex) HPX-87H, 300 X 7.8이었다. 컬럼 온도는 40℃였으며, 이때 이동상은 40분 동안 0.6 mL/min의 유량의 0.01 N 황산이었다. 결과는 표 1에 나타나 있다.The samples were then run on an Agilent 1200 Series LC with binary, isocratic pump, vacuum degasser, heated column compartment, sampler cooling system, UV DAD detector and RI detector. Columns used were Bio-Rad Cation H refill, Aminex HPX-87H with 30 × 4.6 guard column, 300 × 7.7. The column temperature was 40 ° C. where the mobile phase was 0.01 N sulfuric acid at a flow rate of 0.6 mL / min for 40 minutes. The results are shown in Table 1.

용매 상 중 발효 생성물의 Of the fermentation product in the solvent phase GCGC 분석 analysis

프로세싱을 위하여 준비가 될 때까지 샘플을 냉장시켰다. 샘플을 냉장고로부터 꺼내어 실온에 도달되게 하였다 (약 1시간). 100-1000 ㎕ 바이오히트 피펫 팁을 갖춘 m1000 배리어블 채널 바이오히트 피펫을 이용하여 대략 150 ㎕의 샘플을, 중합체 피트를 포함하는 250 ㎕ 유리 바이알 인서트를 갖춘 1.8 자동 샘플러 바이알 내로 옮겼다. PTFE 격벽이 있는 나사 뚜껑을 사용하여 바이알의 뚜껑을 덮었다.Samples were refrigerated until ready for processing. The sample was removed from the refrigerator and allowed to reach room temperature (about 1 hour). Approximately 150 μl of samples were transferred into 1.8 auto sampler vials with 250 μl glass vial inserts containing polymer pits using an m1000 barrierable channel bioheat pipette with 100-1000 μl bioheat pipette tips. The lid of the vial was covered using a screw cap with a PTFE bulkhead.

그 후, 7683B 주입기 및 G2614A 자동 샘플러를 갖춘 애질런트 7890A GC에서 샘플을 진행시켰다. 컬럼은 HP-이노왁스(InnoWax) 컬럼 (30 m × 0.32 ㎜ ID, 0.25 ㎛ 필름)이었다. 캐리어 가스는 일정 헤드 압력을 이용하여 45℃에서 측정된 유량이 1.5 mL/min인 헬륨이었으며; 주입기 분할비(injector split)는 225℃에서 1:50이었으며; 오븐 온도는, 29분의 실행 시간에 있어서, 1.5분 동안 45℃; 0분 동안 10℃/min으로 45℃에서 160℃; 그 후 14분 동안 35℃/min으로 230℃였다. 불꽃 이온화 검출을 40 mL/min의 헬륨 메이크업 가스를 이용하여 260℃에서 사용하였다. 결과는 표 2에 나타나 있다.The sample was then run on an Agilent 7890A GC equipped with a 7683B injector and a G2614A autosampler. The column was an HP-InnoWax column (30 m × 0.32 mm ID, 0.25 μm film). The carrier gas was helium with a flow rate of 1.5 mL / min measured at 45 ° C. using constant head pressure; The injector split was 1:50 at 225 ° C .; The oven temperature was 45 ° C. for 1.5 minutes at a run time of 29 minutes; 45 ° C. to 160 ° C. at 10 ° C./min for 0 minutes; It was then 230 ° C. at 35 ° C./min for 14 minutes. Flame ionization detection was used at 260 ° C. with 40 mL / min of helium makeup gas. The results are shown in Table 2.

지방산 부틸 에스테르에 대하여 분석한 샘플을 7683B 주입기 및 G2614A 자동 샘플러를 갖춘 애질런트 6890 GC에서 진행시켰다. 컬럼은 HP-DB-FFAP 컬럼 (15 m × 0.53 ㎜ ID (메가보어(Megabore)), 1 마이크로미터 필름 두께의 컬럼 (30 m × 0.32 ㎜ ID, 0.25 ㎛ 필름)이었다. 캐리어 가스는 일정 헤드 압력을 이용하여 45℃에서 측정된 유량이 3.7 mL/min인 헬륨이었으며; 주입기 분할비는 225℃에서 1:50이었으며; 오븐 온도는, 26분의 실행 시간에 있어서, 2.0분 동안 100℃; 10℃/min으로 100℃에서 250℃; 그 후 9분 동안 250℃였다. 불꽃 이온화 검출을 40 mL/min의 헬륨 메이크업 가스를 이용하여 300℃에서 사용하였다. 하기 GC 표준물 (뉴첵 프렙(Nu-Chek Prep); 미국 미네소타주 엘리시안 소재)을 사용하여 지방산 아이소부틸 에스테르 생성물의 아이덴티티(identity)를 확인하였다: 아이소부틸 팔미테이트, 아이소부틸 스테아레이트, 아이소부틸 올레에이트, 아이소부틸 리놀레에이트, 아이소부틸 리놀레네이트, 아이소부틸 아라키데이트.Samples analyzed for fatty acid butyl esters were run on an Agilent 6890 GC equipped with a 7683B injector and a G2614A autosampler. The column was an HP-DB-FFAP column (15 m × 0.53 mm ID (Megabore), 1 micron film thickness column (30 m × 0.32 mm ID, 0.25 μm film). The carrier gas was a constant head pressure. Helium with a flow rate of 3.7 mL / min measured at 45 ° C .; injector split ratio was 1:50 at 225 ° C .; oven temperature was 100 ° C. for 2.0 minutes at a run time of 26 minutes; 10 ° C. 100 ° C./min at 250 ° C .; then 250 ° C. for 9 minutes. Flame ionization detection was used at 300 ° C. with 40 mL / min of helium make-up gas. The following GC standards (Nu-Chek Prep); Elysian, Minnesota) was used to identify the identity of fatty acid isobutyl ester products: isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, isobutyl Linoleate, child Butyl Araki date.

실시예 1 내지 14에는 청구된 방법에 사용할 수 있는 다양한 발효 조건들이 기재되어 있다. 일례로서, 일부 배양물들을 액화 전 리파아제 처리에 처하고, 다른 것들을 액화 후 리파아제 처리에 처하였다. 다른 실시예에서, 발효물을 열 불활성화 처리에 처하였다. 발효 후, 유효 아이소부탄올 타이터(effective isobutanol titer; Eff Iso 타이터)를 측정하였으며, 즉, 수성 부피 1리터 당 생성된 아이소부탄올의 총 그램을 측정하였다. 결과는 표 3 나타나 있다.Examples 1 to 14 describe various fermentation conditions that can be used in the claimed method. As an example, some cultures were subjected to lipase treatment before liquefaction and others were subjected to lipase treatment after liquefaction. In another example, the fermentation was subjected to heat inactivation treatment. After fermentation, the effective isobutanol titer (Eff Iso titer) was measured, ie the total gram of isobutanol produced per liter of aqueous volume. The results are shown in Table 3.

예 1 - (대조군)Example 1-(control)

실험 식별자 2010Y014는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 접종 전 영양소 첨가에 대한 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐(butanologen)은 NGCI-070이었다.Experiment identifier 2010Y014 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, method for nutrient addition before inoculation, fermentation vessel inoculation method, method for fermentation vessel operating conditions, and all analytical methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 2Example 2

실험 식별자 2010Y015는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 리파아제 처리 방법, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y015 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, method for fermentation vessel operating conditions, and all Analytical Methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 3Example 3

실험 식별자 2010Y016은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 리파아제 처리 방법, 접종 전 영양소 첨가 방법 - 에탄올 배제를 예외로 함 - , 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experimental identifier 2010Y016 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, nutrient addition method before inoculation-excluding ethanol exclusion-fermentation vessel inoculation method, fermentation vessel Methods for operating conditions, and all analytical methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 4Example 4

실험 식별자 2010Y017은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 열 불활성화 처리 방법, 접종 전 영양소 첨가 방법 - 에탄올 배제를 예외로 함 - , 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experimental identifier 2010Y017 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, heat inactivation treatment method after liquefaction, nutrient addition method before inoculation-with the exception of ethanol exclusion, fermentation vessel inoculation method, Methods for fermentation vessel operating conditions, and all analytical methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 5Example 5

실험 식별자 2010Y018은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 리파아제 처리 방법 - 액화 후 단지 7.2 ppm의 리파아제를 첨가하는 것을 예외로 함 - , 액화 후 열 불활성화 처리 방법, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y018 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, with the exception of adding only 7.2 ppm lipase after liquefaction, heat inactivation after liquefaction Treatment method, nutrient addition method before inoculation, fermentation vessel inoculation method, method for fermentation vessel operating conditions, and all analytical methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 6 - (대조군)Example 6-(control)

실험 식별자 2010Y019는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 열 불활성화 처리 방법, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y019 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, thermal inactivation method after liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, method for fermentation vessel operating conditions, And all analysis methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 7 - (대조군)Example 7-(control)

실험 식별자 2010Y021은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 전 리파아제 처리 방법, 액화 방법, 액화 동안 열 불활성화 처리, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y021 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat inactivation treatment during liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operation Methods for conditions, and all methods of analysis. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 8Example 8

실험 식별자 2010Y022는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y022 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, nutrient addition method before inoculation, fermentation vessel inoculation method, method for fermentation vessel operating conditions, and all analytical methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 9Example 9

실험 식별자 2010Y023은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 리파아제 처리 방법, 열 불활성화 처리를 하지 않음, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 조(crude) 옥수수유로부터 만들어진 옥수수유 지방산을 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y023 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, no heat inactivation treatment, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel Methods for operating conditions, and all analytical methods. Corn oil fatty acids made from crude corn oil were added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 10Example 10

실험 식별자 2010Y024는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 전 리파아제 처리 방법, 액화 방법, 액화 동안 열 불활성화 처리, 접종 전 영양소 첨가 방법 - 에탄올 첨가를 하지 않은 것을 예외로 함 - , 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 올레일 알코올은 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment Identifier 2010Y024 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat inactivation during liquefaction, nutrient addition method before inoculation-with no exception of ethanol addition -Fermentation vessel inoculation method, fermentation vessel operating conditions, and all analytical methods. Oleyl alcohol was added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 11Example 11

실험 식별자 2010Y029는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 전 리파아제 처리 방법, 액화 방법, 액화 동안 열 불활성화 처리, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 조 옥수수유로부터 만들어진 옥수수유 지방산을 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y029 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat inactivation treatment during liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operation Methods for conditions, and all methods of analysis. Corn oil fatty acids made from crude corn oil were added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 12Example 12

실험 식별자 2010Y030은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 전 리파아제 처리 방법, 액화 방법, 액화 동안 열 불활성화 처리, 접종 전 영양소 첨가 방법 - 에탄올 첨가를 하지 않은 것을 예외로 함 - , 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 조 옥수수유로부터 만들어진 옥수수유 지방산을 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y030 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat inactivation treatment during liquefaction, nutrient addition method before inoculation-except no ethanol addition. -Fermentation vessel inoculation method, fermentation vessel operating conditions, and all analytical methods. Corn oil fatty acids made from crude corn oil were added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 13 - (대조군)Example 13-(control)

실험 식별자 2010Y031은 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 리파아제 처리 방법, 열 불활성화 처리를 하지 않음, 접종 전 영양소 첨가 방법 - 에탄올 첨가를 하지 않은 것을 예외로 함 - , 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 조 옥수수유로부터 만들어진 옥수수유 지방산을 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment Identifier 2010Y031 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, no heat inactivation method, nutrient addition method before inoculation-no ethanol addition Fermentation vessel inoculation method, fermentation vessel operating conditions, and all analytical methods. Corn oil fatty acids made from crude corn oil were added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 14Example 14

실험 식별자 2010Y032는 하기를 포함하였다: 시드 플라스크 성장 방법, 초기 발효 용기 준비 방법, 액화 방법, 액화 후 리파아제 처리 방법, 열 불활성화 처리를 하지 않음, 접종 전 영양소 첨가 방법, 발효 용기 접종 방법, 발효 용기 작동 조건에 대한 방법, 및 모든 분석 방법들. 조 옥수수유로부터 만들어진 옥수수유 지방산을 접종 후 0.1 내지 1.0시간 사이에 단일 배치로 첨가하였다. 부탄올로겐은 NGCI-070이었다.Experiment identifier 2010Y032 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, no heat inactivation treatment, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel Methods for operating conditions, and all analytical methods. Corn oil fatty acids made from crude corn oil were added in a single batch between 0.1 and 1.0 hours after inoculation. Butanologen was NGCI-070.

예 15Example 15

실험 식별자는 GLNOR432A였다. 냉동 바이알로부터 NGCI-047 (부탄다이올 생산자)을 250 mL 플라스크 내에서 25 mL 배지에서 대략 1의 OD로 성장시켰다. 예비 시드 배양물을 2 L 플라스크로 옮기고, 1.7 내지 1.8의 OD로 성장시켰다. 둘 모두의 플라스크를 위한 배지는 하기의 것이었다:The experiment identifier was GLNOR432A. NGCI-047 (butanediol producer) was grown from frozen vials to an OD of approximately 1 in 25 mL medium in a 250 mL flask. The preliminary seed culture was transferred to a 2 L flask and grown to an OD of 1.7 to 1.8. The medium for both flasks was as follows:

3.0 g/L의 덱스트로스3.0 g / L dextrose

3.0 g/L의 무수 에탄올3.0 g / L anhydrous ethanol

6.7 g/L의 디프코(Difco) 효모 질소 베이스, 아미노산을 포함하지 않음 (번호: 291920)6.7 g / L of Difco yeast nitrogen base, does not contain amino acids (No .: 291920)

1.4 g/L의 효모 드롭아웃 믹스(Dropout Mix) (시그마(Sigma) Y2001)1.4 g / L Yeast Dropout Mix (Sigma Y2001)

10 mL/L의 1% (w/v) L-류신 원액PP "Normal" 11>2 mL/L의 1% (w/v) L-트립토판 원액10 mL / L 1% (w / v) L-Leucine Stock Solution PP "Normal" 11> 2 mL / L 1% (w / v) L-Tryptophan Stock Solution

1 L, 애플리콘(Applikon) 발효 용기에 60 mL의 시드 플라스크를 접종하였다. 발효 용기는 700 mL의 하기 살균 배지를 함유하였다:1 L, Applikon fermentation vessels were inoculated with 60 mL seed flasks. The fermentation vessel contained 700 mL of the following sterilization medium:

20.0 g/L의 덱스트로스20.0 g / L dextrose

8.0 mL/L의 무수 에탄올8.0 mL / L anhydrous ethanol

6.7 g/L의 디프코(Difco) 효모 질소 베이스, 아미노산을 포함하지 않음 (번호: 291920)6.7 g / L of Difco yeast nitrogen base, does not contain amino acids (No .: 291920)

2.8 g/L의 효모 드롭아웃 믹스 (시그마 Y2001)2.8 g / L Yeast Dropout Mix (Sigma Y2001)

20 mL/L의 1% (w/v) L-류신 원액20 mL / L of 1% (w / v) L-leucine stock solution

4 mL/L의 1% (w/v) L-트립토판 원액4 mL / L of 1% (w / v) L-Tryptophan stock solution

0.5 mL의 시그마 204 소포제0.5 mL sigma 204 antifoam

1:1::트윈(Tween) 80:에탄올 중 1% (w/v)의 에르게스테롤 용액 0.8 mL/L1: 1 :: Tween 80: 0.8 mL / L of a 1% (w / v) ersterol solution in ethanol

잔류 글루코스를 50% (w/w) 글루코스 용액에 의해 과량으로 유지하였다. 발효 용기의 용존 산소 농도를 교반 제어에 의해 30%로 제어하였다. pH를 pH=5.5로 제어하였다. 발효 용기에 0.3 slpm의 살균 실내 공기를 살포하였다. 온도를 30℃로 제어하였다.Residual glucose was maintained in excess by 50% (w / w) glucose solution. The dissolved oxygen concentration of the fermentation vessel was controlled to 30% by stirring control. The pH was controlled to pH = 5.5. The fermentation vessel was sprayed with 0.3 slpm of sterile room air. The temperature was controlled to 30 ° C.

예 16Example 16

실험 식별자는 GLNOR434A였다. 이 예는 3 g의 올레산을 첨가하고 3 g의 팔미트산을 첨가한 후 접종한 것을 제외하고는 예 15와 동일하였다. NGCI-047 (부탄다이올 생산자)이 생촉매였다.The experiment identifier was GLNOR434A. This example was the same as Example 15 except that 3 g of oleic acid was added and 3 g of palmitic acid was added before inoculation. NGCI-047 (producer of butanediol) was a biocatalyst.

도 6은 지방산을 수용하는 발효 용기 내에서 리터 당 더 많은 그램의 글루코스가 소비되었음을 나타낸다. 정사각형은 올레산 및 팔미트산을 수용한 발효 용기를 나타낸다. 원은 어떠한 가외의 지방산도 수용하지 않은 발효 용기를 나타낸다.6 shows that more grams of glucose were consumed per liter in fermentation vessels containing fatty acids. Squares represent fermentation vessels containing oleic acid and palmitic acid. The circles represent fermentation vessels that do not contain any extra fatty acids.

예 17Example 17

실험 식별자는 GLNOR435A였다. 이 예는 이것에 NGCI-049 (아이소부탄올 생산자)를 접종한 것을 제외하고는 예 15와 동일하였다.The experiment identifier was GLNOR435A. This example was the same as Example 15 except that it was inoculated with NGCI-049 (isobutanol producer).

예 18Example 18

실험 식별자는 GLNOR437A였다. 이 예는 이것에 NGCI-049 (아이소부탄올 생산자)를 접종한 것을 제외하고는 예 16과 동일하였다.The experiment identifier was GLNOR437A. This example was the same as Example 16 except that it was inoculated with NGCI-049 (isobutanol producer).

도 7은 지방산을 수용하는 발효 용기 내에서 리터 당 더 많은 그램의 글루코스가 소비되었음을 나타낸다. 정사각형은 올레산 및 팔미트산을 수용한 발효 용기를 나타낸다. 원은 어떠한 가외의 지방산도 수용하지 않은 발효 용기를 나타낸다.7 shows that more grams of glucose were consumed per liter in fermentation vessels containing fatty acids. Squares represent fermentation vessels containing oleic acid and palmitic acid. The circles represent fermentation vessels that do not contain any extra fatty acids.

예 19Example 19

실험 식별자는 090420_3212였다. 이 실험은 이것에 부탄올로겐 NYLA84 (아이소부탄올 생산자)를 접종한 것을 제외하고는 예 15와 유사하게 실행하였다. 이 발효를 1 L 사르토리우스 발효 용기에서 실행하였다.The experiment identifier was 090420_3212. This experiment was performed similarly to Example 15 except that it was inoculated with butanologen NYLA84 (isobutanol producer). This fermentation was carried out in a 1 L Sartorius fermentation vessel.

예 20Example 20

실험 식별자는 2009Y047이었다. 이 실험은 이것에 부탄올로겐 NYLA84 (아이소부탄올 생산자)를 접종한 것을 제외하고는 예 16과 유사하게 실행하였다. 이 발효를 1 L 사르토리우스 발효에서 실행하였다.The experiment identifier was 2009Y047. This experiment was performed similarly to Example 16 except that it was inoculated with butanologen NYLA84 (isobutanol producer). This fermentation was carried out in 1 L Sartorius fermentation.

도 8은 지방산을 수용하는 발효 용기 내에서 리터 당 더 많은 그램의 글루코스가 소비되었음을 나타낸다. 정사각형은 올레산 및 팔미트산을 수용한 발효 용기를 나타낸다. 원은 어떠한 가외의 지방산도 수용하지 않은 발효 용기를 나타낸다. 예 15 내지 20의 결과가 표 4에 나타나 있으며, 이는 +/- 지방산 첨가, 최대 광학 밀도, 및 글루코스 소비량 (g/L)을 나타낸다.8 shows that more grams of glucose were consumed per liter in a fermentation vessel containing fatty acids. Squares represent fermentation vessels containing oleic acid and palmitic acid. The circles represent fermentation vessels that do not contain any extra fatty acids. The results of Examples 15-20 are shown in Table 4, which shows the +/- fatty acid addition, maximum optical density, and glucose consumption (g / L).

예 21Example 21

올레일 알코올을 사용한 원위치 생성물 제거에 의한 동시적 당화 및 발효를 위한 액화 옥수수 매시의 리파아제 처리Lipase treatment of liquefied corn mash for simultaneous saccharification and fermentation by in situ product removal with oleyl alcohol

예 1, 2 및 3에서 상기에 설명한 바와 같이 실행한 발효로부터 취한 올레일 알코올 및 브로쓰의 샘플들을 문헌[E. G. Bligh and W. J. Dyer, Canadian Journal of Biochemistry and Physiology, 37:911-17, 1959, 이하, 참고 문헌 1]에 설명된 방법에 따라 지질(지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량%, 및 유리 지방산(FFA, 지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량%에 대하여 분석하였다. 세 발효 각각을 위하여 제조한 액화 옥수수 매시를, 리폴라아제(등록상표) 100 L (노보자임즈) (30 중량%의 분쇄 옥수수알을 함유하는 액화 반응 매스 1 ㎏ 당 10 ppm의 리폴라아제(등록상표) 전체 용해성 단백질 (BCA 단백질 분석, 시그마 알드리치(Sigma Aldrich))로 처리 후 지질 중량% 및 FFA 중량%에 대하여 또한 분석하였다. 예 1 (대조군)에서는 리파아제를 액화 옥수수 매시에 첨가하지 않았으며, 리파아제로 처리한 액화 옥수수 매시를 함유하는 예 2 및 예 3에서 설명한 발효물 (리파아제의 열 불활성화는 하지 않음)은 에탄올을 예 3에서 설명한 발효물에 첨가하지 않은 것을 제외하고는 동일하였다.Samples of oleyl alcohol and broth taken from fermentation carried out as described above in Examples 1, 2 and 3 are described in E. G. Bligh and WJ Dyer, Canadian Journal of Biochemistry and Physiology, 37: 911-17, 1959, hereinafter, in weight percent of lipids (fatty acid methyl esters, derivatized as FAME), and Analysis was made on the weight percent of free fatty acids (FFA, fatty acid methyl esters, derivatized as FAME). The liquefied corn mash prepared for each of the three fermentations was subjected to 10 ppm lipolase per kilogram of liquefaction mass containing 100 L (Novozymes) (30 wt% ground corn kernels). (0110) Lipid weight and FFA weight percentage after treatment with total soluble protein (BCA protein assay, Sigma Aldrich) were also analyzed: in Example 1 (control) no lipase was added to the liquefied corn mash. The fermentation products described in Examples 2 and 3 (without heat inactivation of lipases) containing liquefied corn mash treated with lipase were the same except that ethanol was not added to the fermentation product described in Example 3.

예 2 및 예 3에서 설명한 바와 같이 실행한 발효를 위하여 제조한 리파아제 처리된 액화 옥수수 매시에서의 FFA (%)는 리파아제 처리를 하지 않았을 경우 (예 1)의 31%와 비교하여 각각 88% 및 89%였다. 70 h (실행 마지막(end of run; EOR))에, 예 2 및 예 3에서 설명한 바와 같이 실행한 발효들의 OA 상 중 FFA 농도는 각각 14% 및 20%였으며, (옥수수유 지방산 메틸 에스테르 유도체로서 측정한) 지질의 상응하는 증가를 GC/MS로 결정하였더니 이는 OA에 의한 COFA의 리파아제 촉매된 에스테르화로 인한 것이었으며, 여기서 COFA는 처음에 액화 옥수수 매시 중 옥수수유의 리파아제 촉매된 가수분해에 의해 생성시켰다. 결과는 표 5에 나타나 있다.FFA (%) in lipase treated liquefied corn mash prepared for fermentation performed as described in Examples 2 and 3 was 88% and 89, respectively, compared to 31% of lipase-treated (Example 1) Was%. At 70 h (end of run (EOR)), the FFA concentrations in the OA phase of the fermentations run as described in Examples 2 and 3 were 14% and 20%, respectively (as corn oil fatty acid methyl ester derivatives). The corresponding increase in lipid) was determined by GC / MS, which was due to lipase catalyzed esterification of COFA by OA, where COFA was initially produced by lipase catalyzed hydrolysis of corn oil in a liquefied corn mash. I was. The results are shown in Table 5.

예 22Example 22

옥수수유 유리 지방산의 올레일 알코올 에스테르의 생성을 제한하기 위한 리파아제 처리 액화 옥수수 매시 중 리파아제의 열 불활성화Heat inactivation of lipase in lipase treated liquefied corn mash to limit the production of oleyl alcohol esters of corn oil free fatty acids

수돗물 (918.4 g)을 재킷 2-L 수지 케틀(kettle)에 첨가하고, 그 후 474.6 g의 습윤 중량 (417.6 g의 건조 중량)의 분쇄 통옥수수알 (해머 밀 상의 1.0 ㎜ 스크린)을 교반하면서 첨가하였다. 상기 혼합물을 300 rpm으로 교반하면서 55℃로 가열하고, 2 N 황산을 이용하여 pH를 5.8로 조정하였다. 상기 혼합물에 0.672 g의 스페자임(Spezyme)(등록상표)-FRED L (미국 캘리포니아주 팔로 알토 소재의 제넨코르(Genencor)(등록상표))을 함유하는 14.0 g의 수성 용액을 첨가하고, 상기 혼합물의 온도를 85℃로 증가시켰으며, 이때 600 rpm에서 교반시키고 pH를 5.8로 하였다. 85℃에서 120분 후, 상기 혼합물을 50℃로 냉각시키고, 생성된 액화 옥수수 매시의 분취물 45.0 mL을 50 mL 폴리프로필렌 원심분리 튜브로 옮기고, -80℃에서 냉동 보관하였다.Tap water (918.4 g) is added to the jacket 2-L resin kettle followed by addition of 474.6 g of wet weight (dry weight of 417.6 g) of ground whole corn kernels (1.0 mm screen on hammer mill) with stirring. It was. The mixture was heated to 55 ° C. with stirring at 300 rpm and the pH was adjusted to 5.8 with 2 N sulfuric acid. To the mixture was added 14.0 g of an aqueous solution containing 0.672 g of Spzyme®-FRED L (Genencor®, Palo Alto, Calif.), And the mixture The temperature of was increased to 85 ° C. at which time it was stirred at 600 rpm and the pH was set at 5.8. After 120 minutes at 85 ° C., the mixture was cooled to 50 ° C. and 45.0 mL of the resulting liquefied corn mash was transferred to a 50 mL polypropylene centrifuge tube and stored frozen at −80 ° C.

제1 반응에서, 상기에 설명한 바와 같이 제조한 50 g의 액화 옥수수 매시를 55℃에서 6시간 동안 10 ppm의 리폴라아제(등록상표) 100 L (노보자임즈)과 혼합시키고, 85℃에서의 1시간 동안의 리파아제 불활성화 없이, 상기 혼합물을 30℃로 냉각시켰다. 제2 반응에서, 50 g의 액화 옥수수 매시를 55℃에서 6시간 동안 10 ppm의 리폴라아제(등록상표)와 혼합하고, 85℃로 1시간 동안 가열하고 (리파아제 불활성화), 그 후 30℃로 냉각시켰다. 제3 반응에서, 리파아제를 첨가하지 않은 50 g의 액화 옥수수 매시를 55℃에서 6시간 동안 혼합하고, 85℃에서의 1시간 동안의 가열 없이, 상기 혼합물을 30℃로 냉각시키고, 38 g의 올레일 알코올을 첨가하고, 생성된 혼합물을 30℃에서 73시간 동안 교반시켰다. 제4 반응에서, 리파아제를 첨가하지 않은 50 g의 액화 옥수수 매시를 55℃에서 6시간 동안 혼합하고, 그 후 85℃로 1시간 동안 가열하고, 그 후 30℃로 냉각시켰다. 4가지의 반응 혼합물 각각을 6시간에 샘플링하고, 그 후 38 g의 올레일 알코올을 첨가하고, 생성된 혼합물을 30℃에서 교반시키고, 25시간 및 73시간에 샘플링하였다. 샘플들 (액화 매시 및 올레일 알코올(OA) 둘 모두)을 참고 문헌 1에 설명된 방법에 따라 지질(지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량%, 및 유리 지방산(FFA, 지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량%에 대하여 분석하였다.In a first reaction, 50 g of liquefied corn mash prepared as described above was mixed with 10 ppm of Lipolase® 100 L (Novozymes) at 55 ° C. for 6 hours and at 85 ° C. The mixture was cooled to 30 ° C. without lipase inactivation for 1 hour. In the second reaction, 50 g of liquefied corn mash is mixed with 10 ppm of Lipolase® at 55 ° C. for 6 hours, heated to 85 ° C. for 1 hour (lipase inactivation), then 30 ° C. Cooled to. In the third reaction, 50 g of liquefied corn mash without addition of lipase was mixed at 55 ° C. for 6 hours, the mixture was cooled to 30 ° C. without heating at 85 ° C. for 1 hour, and 38 g of oleic acid. One alcohol was added and the resulting mixture was stirred at 30 ° C. for 73 h. In the fourth reaction, 50 g of liquefied corn mash without addition of lipase was mixed at 55 ° C. for 6 hours, then heated to 85 ° C. for 1 hour, and then cooled to 30 ° C. Each of the four reaction mixtures was sampled at 6 hours, then 38 g of oleyl alcohol were added and the resulting mixture was stirred at 30 ° C. and sampled at 25 and 73 hours. Samples (both liquefied mash and oleyl alcohol (OA)) were weight percent lipids (fatty acid methyl ester, derivatized as FAME) according to the method described in reference 1, and free fatty acids (FFA, fatty acid methyl ester, Analyzed by weight percent of derivatized as FAME).

OA 첨가 전 리파아제를 열 불활성화시켜 실행한 제2 반응의 OA 상 중 FFA (%)는, 리파아제 처리 액화 옥수수 매시 중 리파아제를 열 불활성화시키지 않았을 때 (제1 반응) 각각 25시간 및 73시간에서 단지 40%의 FFA 및 21%의 FFA인 것과 비교하여, 25시간에서 99%, 그리고 73시간에서 95%였다. FFA (%)의 유의한 변화는 리파아제를 첨가하지 않은 두 대조 반응물에서 전혀 관찰되지 않았다. 결과는 표 6에 나타나 있다.The FFA (%) in the OA phase of the second reaction carried out by thermally inactivating the lipase prior to OA addition was at 25 and 73 hours, respectively, when the lipase was not thermally inactivated in the lipase treated liquefied corn mash (first reaction), respectively. Compared with only 40% FFA and 21% FFA, it was 99% at 25 hours and 95% at 73 hours. No significant change in FFA (%) was observed at all in the two control reactions without the addition of lipase. The results are shown in Table 6.

예 23Example 23

올레일 알코올을 사용한 원위치 생성물 제거에 의한 동시적 당화 및 발효를 위한 리파아제 처리 액화 옥수수 매시 중 리파아제의 열 불활성화Thermal inactivation of lipase in lipase treated liquefied corn mash for simultaneous saccharification and fermentation by in situ product removal with oleyl alcohol

세 발효를 예 4, 5 및 6에서 상기에 설명한 바와 같이 실행하였다. 발효 전에 예 4 및 예 6에서의 액화 옥수수 매시에 리파아제를 첨가하지 않았으며, 예 5에서 설명한 발효에서의 액화 옥수수 매시의 리파아제 처리 (7.2 ppm의 리폴라아제(등록상표) 전체 용해성 단백질을 사용함) 직후에 (리파아제를 완전히 불활성화시키기 위하여) 열 불활성화 처리를 하고, 후속적으로 그에 이어서 접종 전 영양소 첨가를 하고 발효시켰다. 예 4 및 예 6에서 설명한 바와 같이 실행한 발효를 위하여 리파아제 처리 없이 제조한 액화 옥수수 매시 중 FFA (%) 는 리파아제를 처리한 경우의 89%와 비교하여 각각 31% 및 34%였다. 표 10에 열거된 발효들의 코스에 걸쳐, OA 상 중 FFA의 농도는 열 불활성화 리파아제를 함유하는 것을 비롯한 세 발효물 중 어떠한 것에서도 감소하지 않았다. 예 5에 따라 실행한 발효 (발효 전 리파아제를 열 불활성화시킴)의 OA 상 중 FFA (%)는 액화 옥수수 매시를 리파아제로 처리하지 않은 남아있는 두 발효물 (예 4 및 예 6)에 있어서 FFA가 단지 33%인 것과 비교하여, 70시간 (실행의 마지막; EOR)에서 95%였다. 결과는 표 7에 나타나 있다.Three fermentations were performed as described above in Examples 4, 5 and 6. No lipase was added to the liquefied corn mash in Examples 4 and 6 prior to fermentation, and lipase treatment of the liquefied corn mash in the fermentation described in Example 5 (using 7.2 ppm of Lipolase® whole soluble protein) Immediately following the heat inactivation treatment (to completely inactivate the lipase), followed by subsequent nutrient addition and fermentation following inoculation. FFA (%) in liquefied corn mash prepared without lipase treatment for fermentation performed as described in Examples 4 and 6 was 31% and 34%, respectively, compared to 89% with lipase treatment. Over the course of the fermentations listed in Table 10, the concentration of FFA in the OA phase did not decrease in any of the three fermentations, including those containing heat inactivated lipases. The FFA (%) in the OA phase of the fermentation (heat inactivation of lipase prior to fermentation) performed according to Example 5 was the FFA for the remaining two fermentations (Examples 4 and 6) that did not liquefy the liquefied corn mash. Was 95% at 70 hours (end of run; EOR), compared to only 33%. The results are shown in Table 7.

예 24Example 24

액화 전 분쇄 통옥수수알의 리파아제 처리Lipase treatment of ground whole corn kernels before liquefaction

수돗물 (1377.6 g)을 2개의 재킷 2 L 수지 케틀에 첨가하고, 그 후 711.9 g의 습윤 중량 (625.8 g의 건조 중량)의 분쇄 통옥수수알 (해머 밀 상의 1.0 ㎜ 스크린)을 교반하면서 각각의 케틀에 첨가하였다. 각각의 혼합물을 300 rpm으로 교반하면서 55℃로 가열하고, 2 N 황산을 이용하여 pH를 5.8로 조정하였다. 각각의 혼합물에 1.008 g의 스페자임(등록상표)-FRED L (미국 캘리포니아주 팔로 알토 소재의 제넨코르(등록상표))을 함유하는 21.0 g의 수성 용액을 첨가하였다. 그 후, 하나의 혼합물에 리폴라아제(등록상표) 100L 용액 (21 ㎎의 전체 용해성 단백질, 10 ppm의 리파아제 최종 농도)의 수성 용액 10.5 mL을 첨가하고, 제2 혼합물에 리폴라아제(등록상표) 100L 용액 (2.1 ㎎의 전체 용해성 단백질, 1.0 ppm의 리파아제 최종 농도)의 수성 용액 1.05 mL을 첨가하였다. 샘플들을 55℃에서 1시간, 2시간, 4시간 및 6시간에 각각의 반응 혼합물로부터 꺼내고, 그 후 혼합물의 온도를 600 rpm으로 교반하면서 pH를 5.8로 하여 85℃로 증가시키고, 혼합물이 처음에 85℃에 도달했을 때 샘플을 취하였다. 85℃에서 120분 후, 샘플을 취하고, 상기 혼합물을 50℃로 냉각시키고, 생성된 액화 옥수수 매시의 최종 샘플을 50 mL 폴리프로필렌 원심분리 튜브로 옮기고, 모든 샘플을 -80℃에서 냉동 보관하였다.Tap water (1377.6 g) is added to two jacketed 2 L resin kettles, each of which is then stirred with stirring of 711.9 g of wet weight (625.8 g of dry weight) of ground whole corn kernels (1.0 mm screen on hammer mill). Was added. Each mixture was heated to 55 ° C. with stirring at 300 rpm and the pH was adjusted to 5.8 with 2 N sulfuric acid. To each mixture was added 21.0 g of an aqueous solution containing 1.008 g of Speczyme®-FRED L (Genencor®, Palo Alto, Calif.). Then, to one mixture, 10.5 mL of an aqueous solution of 100L solution of Lipolase® (21 mg total soluble protein, 10 ppm lipase final concentration) was added, and to the second mixture, Lipolase® ) 1.05 mL of an aqueous solution of 100 L solution (2.1 mg total soluble protein, 1.0 ppm lipase final concentration) was added. Samples were removed from each reaction mixture at 55 ° C. for 1 hour, 2 hours, 4 hours and 6 hours, after which the temperature of the mixture was increased to 85 ° C. with a pH of 5.8 with stirring at 600 rpm and the mixture initially Samples were taken when they reached 85 ° C. After 120 minutes at 85 ° C., samples were taken, the mixture was cooled to 50 ° C. and the final sample of the resulting liquefied corn mash was transferred to a 50 mL polypropylene centrifuge tube and all samples were stored frozen at −80 ° C.

두 별도의 반응에서, 상기에 설명한 바와 같이 제조한, 10 ppm 리파아제로 처리한 액화 옥수수 매시의 50 g 샘플 또는 1.0 ppm 리파아제로 처리한 액화 옥수수 매시의 55 g 샘플을 30℃에서 20시간 동안 올레일 알코올(OA) (38 g)과 혼합하고, 그 후 각각의 반응 혼합물 중 OA 및 액화 매시를 원심분리에 의해 분리하고, 각각의 상을 참고 문헌 1에 의해 설명된 방법에 따라 지질 (지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량% 및 유리 지방산 (FFA, 지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량%에 대하여 분석하였다. 액화 동안의 10 ppm 리파아제의 열 불활성화를 이용하여 제조한 액화 매시/OA 혼합물의 OA 상 중 FFA (%)는, 액화 동안의 1.0 ppm 리파아제의 열 불활성화를 이용하여 제조한 액화 매시/OA 혼합물의 OA 상 중 FFA가 단지 62%라는 것과 비교하여 20시간에서 98%였다. 결과는 표 8에 나타나 있다.In two separate reactions, 50 g sample of liquefied corn mash treated with 10 ppm lipase or 55 g sample of liquefied corn mash treated with 1.0 ppm lipase, prepared as described above, was oleyl at 30 ° C. for 20 hours. Mixed with alcohol (OA) (38 g), and then OA and liquefied mash in each reaction mixture are separated by centrifugation, and each phase is lipid (fatty acid methyl ester according to the method described by reference 1). , By weight percent of derivatized as FAME and by weight percent of free fatty acid (FFA, fatty acid methyl ester, derivatized as FAME). The FFA (%) in the OA phase of the liquefied mash / OA mixture prepared using thermal inactivation of 10 ppm lipase during liquefaction is the liquefied mash / OA mixture prepared using thermal inactivation of 1.0 ppm lipase during liquefaction. Of the OA phases were 98% at 20 hours compared to only 62%. The results are shown in Table 8.

예 25Example 25

액화 전 분쇄 통옥수수알의 처리에 대한 리파아제 스크리닝Lipase screening for treatment of ground whole corn kernels before liquefaction

수돗물 (67.9 g) 및 분쇄 통옥수수알 (35.1 g의 습윤 중량, 해머 밀을 이용하여 1.0 ㎜ 스크린에 의해 분쇄)을 함유하는 7가지의 반응 혼합물 - pH 5.8 - 을 마개를 막은 플라스크에서 55℃에서 교반하였다. 3 mL 샘플 (t = 0 h)을 각각의 플라스크로부터 꺼내고, 상기 샘플을 즉시 드라이아이스에서 냉동시키고, 그 후 하기 리파아제들 (노보자임즈), 즉 리폴라아제9등록상표) 100 L, 리펙스(등록상표) 100L, 리포클린(등록상표) 2000T, 리포자임(등록상표) CALB L, 노보자임(등록상표) CALA L, 및 팔라타아제 20000L 중 하나의 1 ㎎의 전체 용해성 단백질 (반응 혼합물 중 10 ppm의 최종 농도)을 함유하는 대략 0.5 mL의 10 mM 인산나트륨 완충제 (pH 7.0)를 각각의 플라스크 중 하나에 첨가하고, 일곱 번째 플라스크에는 리파아제를 첨가하지 않았다. 생성된 혼합물들을 마개를 막은 플라스크에서 55℃에서 교반시키고, 3 mL 샘플들을 1시간, 2시간, 4시간 및 6시간에 각각의 반응 혼합물로부터 꺼내고, 참고 문헌 1에 의해 설명된 방법에 따라 지질 (지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량% 및 유리 지방산 (FFA, 지방산 메틸 에스테르, FAME로서 유도체화됨)의 중량%에 대하여 분석할 때까지 드라이아이스에서 즉시 냉동시키고, 유리 지방산 함량 (%)을 지질의 전체 합한 농도에 대하여 계산하고, 유리 지방산을 각각의 샘플에 대하여 결정하였다. 결과는 표 9에 나타나 있다.Seven reaction mixtures-pH 5.8-containing tap water (67.9 g) and ground whole corn kernels (wet weight of 35.1 g, ground by a 1.0 mm screen using a hammer mill)-pH 5.8-at 55 ° C. in a stoppered flask Stirred. 3 mL sample (t = 0 h) was taken out of each flask, and the sample was immediately frozen in dry ice, then 100 L of the following lipases (Novozymes), ie Lipolase 9®, Repex 1 mg total soluble protein of one of 100L, Lipocrine® 2000T, Lipozyme® CALB L, Novozyme® CALA L, and palatase 20000L (10 in reaction mixture) Approximately 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0) containing ppm) was added to one of each flask and no lipase was added to the seventh flask. The resulting mixtures were stirred at 55 ° C. in a stoppered flask, 3 mL samples were removed from each reaction mixture at 1 h, 2 h, 4 h and 6 h and lipids were prepared according to the method described by reference 1 Frozen immediately on dry ice until analyzed by weight percent of fatty acid methyl ester, derivatized as FAME) and weight percent of free fatty acid (FFA, fatty acid methyl ester, derivatized as FAME) and free fatty acid content (%) Calculations were made for the total combined concentration of lipids and free fatty acids were determined for each sample. The results are shown in Table 9.

예 26Example 26

올레일 알코올을 사용한 원위치 생성물 제거에 의한 동시적 당화 및 발효 전의 분쇄 통옥수수알의 리파아제 처리Simultaneous Saccharification by In Situ Removal of Oleyl Alcohol and Lipase Treatment of Grinded Whole Corn Kernels Before Fermentation

세 발효를 예 7, 8 및 10에서 상기에 설명한 바와 같이 실행하였다. 예 7 및 예 10에 설명한 바와 같이 실행한 발효에 있어서, 리파아제 (10 ppm의 리폴라아제(등록상표) 전체 용해성 단백질)를 분쇄 옥수수의 현탁물에 첨가하고, 액화 전 55℃에서 6시간 동안 가열하여 열 불활성화 리파아제를 함유하는 액화 옥수수 매시를 생성하였다. 예 8에서 설명한 발효를 위한 액화 옥수수 매시를 제조하기 위하여 사용한 분쇄 옥수수의 현탁물에 리파아제를 첨가하지 않았지만, 상기 현탁물은 액화 전에 55℃에서 동일 가열 단계에 처하였다. 예 7 및 예 10에서 설명한 바와 같이 실행한 발효를 위하여 제조한 리파아제 처리된 액화 옥수수 매시에서의 FFA (%)는 리파아제 처리를 하지 않았을 경우 (예 8)의 41%와 비교하여 각각 83% 및 86%였다. 발효들의 코스에 걸쳐, FFA의 농도는 열 불활성화 리파아제를 함유하는 것을 비롯한 발효물들 중 어떠한 것에서도 감소하지 않았다. 예 7 및 예 10에 따라 실행한 발효 (발효 전 리파아제를 열 불활성화시킴)의 OA 상 중 FFA (%)는 분쇄 통옥수수알을 액화 전 리파아제로 처리하지 않은 예 8에 따라 실행한 발효물의 경우의 단지 49%의 FFA와 비교하여, 70시간 (실행의 마지막; EOR)에서 97%였다. 결과는 표 10에 나타나 있다.Three fermentations were performed as described above in Examples 7, 8 and 10. In the fermentation carried out as described in Examples 7 and 10, lipase (10 ppm of Lipolase® total soluble protein) was added to the suspension of ground corn and heated at 55 ° C. for 6 hours before liquefaction. To produce a liquefied corn mash containing heat inactivated lipase. No lipase was added to the suspension of ground corn used to prepare the liquefied corn mash for the fermentation described in Example 8, but the suspension was subjected to the same heating step at 55 ° C. before liquefaction. The FFA (%) in lipase treated liquefied corn mash prepared for fermentation performed as described in Examples 7 and 10 was 83% and 86, respectively, compared to 41% of lipase-treated (Example 8) Was%. Over the course of the fermentations, the concentration of FFA did not decrease in any of the fermentations, including those containing heat inactivated lipases. FFA (%) in the OA phase of the fermentation (heat inactivation of lipase prior to fermentation) carried out according to Examples 7 and 10 was for fermentation carried out according to Example 8, in which the milled corn kernels were not treated with lipase before liquefaction. 97% at 70 hours (end of run; EOR), compared to only 49% of FFA. The results are shown in Table 10.

예 27Example 27

옥수수유 지방산(COFA)을 사용한 원위치 생성물 제거에 의한 동시적 당화 및 발효를 위한 분쇄 통옥수수알 또는 액화 옥수수 매시의 리파아제 처리Lipase treatment of ground whole corn or liquefied corn mash for simultaneous saccharification and fermentation by in situ product removal using corn oil fatty acid (COFA)

5개의 발효를 예 9, 11, 12, 13, 및 14에서 상기에 설명한 바와 같이 실행하였다. 예 9, 13 및 14에서 설명한 바와 같이 실행한 발효에 있어서, 리파아제 (10 ppm의 리폴라아제(등록상표) 전체 용해성 단백질)를 액화 후 첨가하였으며, 리파아제의 열 불활성화는 없었다. 예 9 및 예 14에서 설명한 바와 같이 실행한 발효에서는 접종 전에 5 g/L의 에탄올을 첨가하였으며, 반면에 예 13에서 설명한 바와 같이 실행한 발효에서는 에탄올을 첨가하지 않았다. 예 11 및 예 12에서 설명한 바와 같이 실행한 발효에서는 액화 전에 분쇄 옥수수의 현탁물에 10 ppm의 리폴라아제(등록상표) 전체 용해성 단백질을 첨가하는 것을 이용하였으며, 이는 액화 동안 리파아제의 열 불활성화로 이어졌다. 예 11에서 설명한 바와 같이 실행한 발효에서는 접종 전에 5 g/L의 에탄올을 첨가하였으며, 반면에 예 12에서 설명한 바와 같이 실행한 발효에서는 에탄올을 첨가하지 않았다. 활성 리파아제를 함유하는 발효물들의 COFA 상에 존재하는 아이소부탄올(i-BuOH)의 최종 전체 그램은 불활성 리파아제를 함유하는 발효물들의 COFA에 존재하는 i-BuOH의 최종 전체 그램보다 유의하게 더 컸다. 활성 리파아제를 함유하는 발효 브로쓰에 존재하는 아이소부탄올(i-BuOH)의 최종 전체 그램은 불활성 리파아제를 함유하는 발효 브로쓰에 존재하는 i-BuOH의 최종 전체 그램보다 단지 약간 더 적어서, i-BuOH의 전체 생성량 (유리 i-BuOH와 COFA의 아이소부틸 에스테르 (FABE)를 합한 것으로서)은 열 불활성화 리파아제의 존재 하에 수득되는 것과 비교하여 활성 리파아제의 존재 하에서 유의하게 더 컸다. 그 결과가 표 11 및 표 12에 나타나 있다.Five fermentations were performed as described above in Examples 9, 11, 12, 13, and 14. In the fermentation carried out as described in Examples 9, 13 and 14, lipase (10 ppm of Lipolase® total soluble protein) was added after liquefaction and there was no thermal inactivation of the lipase. In the fermentation run as described in Examples 9 and 14, 5 g / L ethanol was added before inoculation, while in the fermentation run as described in Example 13, no ethanol was added. In the fermentation run as described in Examples 11 and 12, it was used to add 10 ppm of Lipolase® total soluble protein to suspension of ground corn prior to liquefaction, which was caused by thermal inactivation of lipase during liquefaction. Followed. In the fermentation run as described in Example 11, 5 g / L of ethanol was added before inoculation, while in the fermentation run as described in Example 12, no ethanol was added. The final total grams of isobutanol (i-BuOH) present on the COFAs of the fermentations containing active lipase were significantly greater than the final total grams of i-BuOH present in the COFA of fermentations containing inactive lipases. The final total grams of isobutanol (i-BuOH) present in the fermentation broth containing active lipase are only slightly less than the final total grams of i-BuOH present in the fermentation broth containing inactive lipase, i-BuOH The total yield of (as a combination of free i-BuOH and isobutyl ester of COFA (FABE)) was significantly greater in the presence of active lipase compared to that obtained in the presence of heat inactivated lipase. The results are shown in Tables 11 and 12.

본 발명의 다양한 실시 형태들을 상기에 기재하였지만, 이들은 단지 예로서 제시되었으며, 한정하는 것이 아님을 이해해야 한다. 형태 및 상세 사항에서의 다양한 변화가 본 발명의 사상 및 범주로부터 벗어나지 않고서 본 발명에서 이루어질 수 있음이 관련 기술 분야의 숙련자에게 명백할 것이다. 따라서, 본 발명의 폭 및 범주는 임의의 상기 예시적인 실시 형태들에 의해 제한되어서는 안되며, 단지 하기 특허청구범위 및 그 등가물에 따라 정의되어야 한다.While various embodiments of the invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to those skilled in the art that various changes in form and detail may be made in the present invention without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention should not be limited by any of the above exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

본 명세서에서 언급된 모든 간행물, 특허 및 특허 출원은 본 발명이 속하는 기술 분야의 숙련자의 기술 수준을 나타내는 것으로서, 이는 마치 각각의 개별 간행물, 특허 또는 특허 출원이 구체적으로 그리고 개별적으로 참고로 포함되는 것으로 나타내어지는 것처럼 동일한 정도로 본 명세서에 참고로 포함된다.All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, as if each individual publication, patent or patent application was specifically and individually incorporated by reference. Incidentally incorporated herein by reference to the same extent as indicated.

SEQUENCE LISTING <110> Butamax(TM) Advanced Biofuels <120> SUPPLEMENTATION OF FATTY ACIDS FOR IMPROVING ALCOHOL PRODUCTIVITY <130> CL4727 <150> US 61/368,451 <151> 2010-07-28 <160> 145 <170> PatentIn version 3.5 <210> 1 <211> 11844 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 1 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt cagcttccat ttgttgaaac atatcaagct 1200 gccggtaccc tttctagaga tttagaggat caatattttg gccgtatcgg tttgttccgc 1260 aaccagcctg gcgatttact gctagagcag gcagatgttg ttctgacgat cggctatgac 1320 ccgattgaat atgatccgaa attctggaat atcaatggag accggacaat tatccattta 1380 gacgagatta tcgctgacat tgatcatgct taccagcctg atcttgaatt gatcggtgac 1440 attccgtcca cgatcaatca tatcgaacac gatgctgtga aagtggaatt tgcagagcgt 1500 gagcagaaaa tcctttctga tttaaaacaa tatatgcatg aaggtgagca ggtgcctgca 1560 gattggaaat cagacagagc gcaccctctt gaaatcgtta aagagttgcg taatgcagtc 1620 gatgatcatg ttacagtaac ttgcgatatc ggttcgcacg ccatttggat gtcacgttat 1680 ttccgcagct acgagccgtt aacattaatg atcagtaacg gtatgcaaac actcggcgtt 1740 gcgcttcctt gggcaatcgg cgcttcattg gtgaaaccgg gagaaaaagt ggtttctgtc 1800 tctggtgacg gcggtttctt attctcagca atggaattag agacagcagt tcgactaaaa 1860 gcaccaattg tacacattgt atggaacgac agcacatatg acatggttgc attccagcaa 1920 ttgaaaaaat ataaccgtac atctgcggtc gatttcggaa atatcgatat cgtgaaatat 1980 gcggaaagct tcggagcaac tggcttgcgc gtagaatcac cagaccagct ggcagatgtt 2040 ctgcgtcaag gcatgaacgc tgaaggtcct gtcatcatcg atgtcccggt tgactacagt 2100 gataacatta atttagcaag tgacaagctt ccgaaagaat tcggggaact catgaaaacg 2160 aaagctctct agttaattaa tcatgtaatt agttatgtca cgcttacatt cacgccctcc 2220 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 2280 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt 2340 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt 2400 tttgggacgc tcgaaggctt taatttgcgg gcggccgcac ctggtaaaac ctctagtgga 2460 gtagtagatg taatcaatga agcggaagcc aaaagaccag agtagaggcc tatagaagaa 2520 actgcgatac cttttgtgat ggctaaacaa acagacatct ttttatatgt ttttacttct 2580 gtatatcgtg aagtagtaag tgataagcga atttggctaa gaacgttgta agtgaacaag 2640 ggacctcttt tgcctttcaa aaaaggatta aatggagtta atcattgaga tttagttttc 2700 gttagattct gtatccctaa ataactccct tacccgacgg gaaggcacaa aagacttgaa 2760 taatagcaaa cggccagtag ccaagaccaa ataatactag agttaactga tggtcttaaa 2820 caggcattac gtggtgaact ccaagaccaa tatacaaaat atcgataagt tattcttgcc 2880 caccaattta aggagcctac atcaggacag tagtaccatt cctcagagaa gaggtataca 2940 taacaagaaa atcgcgtgaa caccttatat aacttagccc gttattgagc taaaaaacct 3000 tgcaaaattt cctatgaata agaatacttc agacgtgata aaaatttact ttctaactct 3060 tctcacgctg cccctatctg ttcttccgct ctaccgtgag aaataaagca tcgagtacgg 3120 cagttcgctg tcactgaact aaaacaataa ggctagttcg aatgatgaac ttgcttgctg 3180 tcaaacttct gagttgccgc tgatgtgaca ctgtgacaat aaattcaaac cggttatagc 3240 ggtctcctcc ggtaccggtt ctgccacctc caatagagct cagtaggagt cagaacctct 3300 gcggtggctg tcagtgactc atccgcgttt cgtaagttgt gcgcgtgcac atttcgcccg 3360 ttcccgctca tcttgcagca ggcggaaatt ttcatcacgc tgtaggacgc aaaaaaaaaa 3420 taattaatcg tacaagaatc ttggaaaaaa aattgaaaaa ttttgtataa aagggatgac 3480 ctaacttgac tcaatggctt ttacacccag tattttccct ttccttgttt gttacaatta 3540 tagaagcaag acaaaaacat atagacaacc tattcctagg agttatattt ttttacccta 3600 ccagcaatat aagtaaaaaa ctgtttaaac agtatggcag ttacaatgta ttatgaagat 3660 gatgtagaag tatcagcact tgctggaaag caaattgcag taatcggtta tggttcacaa 3720 ggacatgctc acgcacagaa tttgcgtgat tctggtcaca acgttatcat tggtgtgcgc 3780 cacggaaaat cttttgataa agcaaaagaa gatggctttg aaacatttga agtaggagaa 3840 gcagtagcta aagctgatgt tattatggtt ttggcaccag atgaacttca acaatccatt 3900 tatgaagagg acatcaaacc aaacttgaaa gcaggttcag cacttggttt tgctcacgga 3960 tttaatatcc attttggcta tattaaagta ccagaagacg ttgacgtctt tatggttgcg 4020 cctaaggctc caggtcacct tgtccgtcgg acttatactg aaggttttgg tacaccagct 4080 ttgtttgttt cacaccaaaa tgcaagtggt catgcgcgtg aaatcgcaat ggattgggcc 4140 aaaggaattg gttgtgctcg agtgggaatt attgaaacaa cttttaaaga agaaacagaa 4200 gaagatttgt ttggagaaca agctgttcta tgtggaggtt tgacagcact tgttgaagcc 4260 ggttttgaaa cactgacaga agctggatac gctggcgaat tggcttactt tgaagttttg 4320 cacgaaatga aattgattgt tgacctcatg tatgaaggtg gttttactaa aatgcgtcaa 4380 tccatctcaa atactgctga gtttggcgat tatgtgactg gtccacggat tattactgac 4440 gaagttaaaa agaatatgaa gcttgttttg gctgatattc aatctggaaa atttgctcaa 4500 gatttcgttg atgacttcaa agcggggcgt ccaaaattaa tagcctatcg cgaagctgca 4560 aaaaatcttg aaattgaaaa aattggggca gagctacgtc aagcaatgcc attcacacaa 4620 tctggtgatg acgatgcctt taaaatctat cagtaaggcc ctgcaggcct atcaagtgct 4680 ggaaactttt tctcttggaa tttttgcaac atcaagtcat agtcaattga attgacccaa 4740 tttcacattt aagatttttt ttttttcatc cgacatacat ctgtacacta ggaagccctg 4800 tttttctgaa gcagcttcaa atatatatat tttttacata tttattatga ttcaatgaac 4860 aatctaatta aatcgaaaac aagaaccgaa acgcgaataa ataatttatt tagatggtga 4920 caagtgtata agtcctcatc gggacagcta cgatttctct ttcggttttg gctgagctac 4980 tggttgctgt gacgcagcgg cattagcgcg gcgttatgag ctaccctcgt ggcctgaaag 5040 atggcgggaa taaagcggaa ctaaaaatta ctgactgagc catattgagg tcaatttgtc 5100 aactcgtcaa gtcacgtttg gtggacggcc cctttccaac gaatcgtata tactaacatg 5160 cgcgcgcttc ctatatacac atatacatat atatatatat atatatgtgt gcgtgtatgt 5220 gtacacctgt atttaatttc cttactcgcg ggtttttctt ttttctcaat tcttggcttc 5280 ctctttctcg agcggaccgg atcctccgcg gtgccggcag atctatttaa atggcgcgcc 5340 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 5400 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 5460 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 5520 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 5580 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 5640 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 5700 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 5760 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 5820 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 5880 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 5940 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 6000 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 6060 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 6120 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 6180 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 6240 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 6300 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 6360 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 6420 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 6480 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 6540 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 6600 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 6660 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 6720 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 6780 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 6840 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 6900 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 6960 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 7020 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 7080 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 7140 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 7200 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 7260 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 7320 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 7380 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 7440 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7500 tgattacgcc aagctttttc tttccaattt tttttttttc gtcattataa aaatcattac 7560 gaccgagatt cccgggtaat aactgatata attaaattga agctctaatt tgtgagttta 7620 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat 7680 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg 7740 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg 7800 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa 7860 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa 7920 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata 7980 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt 8040 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg 8100 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat 8160 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg 8220 cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt 8280 ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac 8340 gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg 8400 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt 8460 atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc 8520 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc 8580 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa 8640 aaaaagaata aaaaaaaaat gatgaattga aaagcttgca tgcctgcagg tcgactctag 8700 tatactccgt ctactgtacg atacacttcc gctcaggtcc ttgtccttta acgaggcctt 8760 accactcttt tgttactcta ttgatccagc tcagcaaagg cagtgtgatc taagattcta 8820 tcttcgcgat gtagtaaaac tagctagacc gagaaagaga ctagaaatgc aaaaggcact 8880 tctacaatgg ctgccatcat tattatccga tgtgacgctg catttttttt tttttttttt 8940 tttttttttt tttttttttt tttttttttt ttttgtacaa atatcataaa aaaagagaat 9000 ctttttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac 9060 tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat 9120 cttcaatggc tttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag 9180 acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagcg gaaccatggc 9240 atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg 9300 gcaacaaacc caaggagcct gggataacgg aggcttcatc ggagatgata tcaccaaaca 9360 tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg 9420 cagaatcaat caattgatgt tgaactttca atgtagggaa ttcgttcttg atggtttcct 9480 ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa 9540 taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca 9600 cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc 9660 tcttaccaaa gtaaatacct cccactaatt ctctaacaac aacgaagtca gtacctttag 9720 caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc 9780 ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa 9840 cactaccggt accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct 9900 tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata gcagcaccac 9960 caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa 10020 gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga 10080 tcttcttagg ggcagacatt acaatggtat atccttgaaa tatatataaa aaaaaaaaaa 10140 aaaaaaaaaa aaaaaaatgc agcttctcaa tgatattcga atacgctttg aggagataca 10200 gcctaatatc cgacaaactg ttttacagat ttacgatcgt acttgttacc catcattgaa 10260 ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg aacctgtata 10320 ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc ctggagaaac 10380 tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt 10440 ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc attttgtaga 10500 acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 10560 agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 10620 gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10680 tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10740 tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10800 actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10860 gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10920 tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10980 aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 11040 gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 11100 ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 11160 aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 11220 gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 11280 ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 11340 attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 11400 tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 11460 ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 11520 gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 11580 tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 11640 gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11700 tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11760 ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatatgc 11820 atagtaccga gaaactagag gatc 11844 <210> 2 <211> 15539 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1680 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1740 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1800 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1860 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 1980 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 2040 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac 2100 ggcgcgccac tggtagagag cgactttgta tgccccaatt gcgaaacccg cgatatcctt 2160 ctcgattctt tagtacccga ccaggacaag gaaaaggagg tcgaaacgtt tttgaagaaa 2220 caagaggaac tacacggaag ctctaaagat ggcaaccagc cagaaactaa gaaaatgaag 2280 ttgatggatc caactggcac cgctggcttg aacaacaata ccagccttcc aacttctgta 2340 aataacggcg gtacgccagt gccaccagta ccgttacctt tcggtatacc tcctttcccc 2400 atgtttccaa tgcccttcat gcctccaacg gctactatca caaatcctca tcaagctgac 2460 gcaagcccta agaaatgaat aacaatactg acagtactaa ataattgcct acttggcttc 2520 acatacgttg catacgtcga tatagataat aatgataatg acagcaggat tatcgtaata 2580 cgtaatagct gaaaatctca aaaatgtgtg ggtcattacg taaataatga taggaatggg 2640 attcttctat ttttcctttt tccattctag cagccgtcgg gaaaacgtgg catcctctct 2700 ttcgggctca attggagtca cgctgccgtg agcatcctct ctttccatat ctaacaactg 2760 agcacgtaac caatggaaaa gcatgagctt agcgttgctc caaaaaagta ttggatggtt 2820 aataccattt gtctgttctc ttctgacttt gactcctcaa aaaaaaaaat ctacaatcaa 2880 cagatcgctt caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa 2940 ttttatccct catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga 3000 cataatactt ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc 3060 tttttctttt gtcatatata accataacca agtaatacat attcaaacta gtatgactga 3120 caaaaaaact cttaaagact taagaaatcg tagttctgtt tacgattcaa tggttaaatc 3180 acctaatcgt gctatgttgc gtgcaactgg tatgcaagat gaagactttg aaaaacctat 3240 cgtcggtgtc atttcaactt gggctgaaaa cacaccttgt aatatccact tacatgactt 3300 tggtaaacta gccaaagtcg gtgttaagga agctggtgct tggccagttc agttcggaac 3360 aatcacggtt tctgatggaa tcgccatggg aacccaagga atgcgtttct ccttgacatc 3420 tcgtgatatt attgcagatt ctattgaagc agccatggga ggtcataatg cggatgcttt 3480 tgtagccatt ggcggttgtg ataaaaacat gcccggttct gttatcgcta tggctaacat 3540 ggatatccca gccatttttg cttacggcgg aacaattgca cctggtaatt tagacggcaa 3600 agatatcgat ttagtctctg tctttgaagg tgtcggccat tggaaccacg gcgatatgac 3660 caaagaagaa gttaaagctt tggaatgtaa tgcttgtccc ggtcctggag gctgcggtgg 3720 tatgtatact gctaacacaa tggcgacagc tattgaagtt ttgggactta gccttccggg 3780 ttcatcttct cacccggctg aatccgcaga aaagaaagca gatattgaag aagctggtcg 3840 cgctgttgtc aaaatgctcg aaatgggctt aaaaccttct gacattttaa cgcgtgaagc 3900 ttttgaagat gctattactg taactatggc tctgggaggt tcaaccaact caacccttca 3960 cctcttagct attgcccatg ctgctaatgt ggaattgaca cttgatgatt tcaatacttt 4020 ccaagaaaaa gttcctcatt tggctgattt gaaaccttct ggtcaatatg tattccaaga 4080 cctttacaag gtcggagggg taccagcagt tatgaaatat ctccttaaaa atggcttcct 4140 tcatggtgac cgtatcactt gtactggcaa aacagtcgct gaaaatttga aggcttttga 4200 tgatttaaca cctggtcaaa aggttattat gccgcttgaa aatcctaaac gtgaagatgg 4260 tccgctcatt attctccatg gtaacttggc tccagacggt gccgttgcca aagtttctgg 4320 tgtaaaagtg cgtcgtcatg tcggtcctgc taaggtcttt aattctgaag aagaagccat 4380 tgaagctgtc ttgaatgatg atattgttga tggtgatgtt gttgtcgtac gttttgtagg 4440 accaaagggc ggtcctggta tgcctgaaat gctttccctt tcatcaatga ttgttggtaa 4500 agggcaaggt gaaaaagttg cccttctgac agatggccgc ttctcaggtg gtacttatgg 4560 tcttgtcgtg ggtcatatcg ctcctgaagc acaagatggc ggtccaatcg cctacctgca 4620 aacaggagac atagtcacta ttgaccaaga cactaaggaa ttacactttg atatctccga 4680 tgaagagtta aaacatcgtc aagagaccat tgaattgcca ccgctctatt cacgcggtat 4740 ccttggtaaa tatgctcaca tcgtttcgtc tgcttctagg ggagccgtaa cagacttttg 4800 gaagcctgaa gaaactggca aaaaatgttg tcctggttgc tgtggttaag cggccgcgtt 4860 aattcaaatt aattgatata gttttttaat gagtattgaa tctgtttaga aataatggaa 4920 tattattttt atttatttat ttatattatt ggtcggctct tttcttctga aggtcaatga 4980 caaaatgata tgaaggaaat aatgatttct aaaattttac aacgtaagat atttttacaa 5040 aagcctagct catcttttgt catgcactat tttactcacg cttgaaatta acggccagtc 5100 cactgcggag tcatttcaaa gtcatcctaa tcgatctatc gtttttgata gctcattttg 5160 gagttcgcga ttgtcttctg ttattcacaa ctgttttaat ttttatttca ttctggaact 5220 cttcgagttc tttgtaaagt ctttcatagt agcttacttt atcctccaac atatttaact 5280 tcatgtcaat ttcggctctt aaattttcca catcatcaag ttcaacatca tcttttaact 5340 tgaatttatt ctctagctct tccaaccaag cctcattgct ccttgattta ctggtgaaaa 5400 gtgatacact ttgcgcgcaa tccaggtcaa aactttcctg caaagaattc accaatttct 5460 cgacatcata gtacaatttg ttttgttctc ccatcacaat ttaatatacc tgatggattc 5520 ttatgaagcg ctgggtaatg gacgtgtcac tctacttcgc ctttttccct actcctttta 5580 gtacggaaga caatgctaat aaataagagg gtaataataa tattattaat cggcaaaaaa 5640 gattaaacgc caagcgttta attatcagaa agcaaacgtc gtaccaatcc ttgaatgctt 5700 cccaattgta tattaagagt catcacagca acatattctt gttattaaat taattattat 5760 tgatttttga tattgtataa aaaaaccaaa tatgtataaa aaaagtgaat aaaaaatacc 5820 aagtatggag aaatatatta gaagtctata cgttaaacca cccgggcccc ccctcgaggt 5880 cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag 5940 agcggccgct ctagaactag taccacaggt gttgtcctct gaggacataa aatacacacc 6000 gagattcatc aactcattgc tggagttagc atatctacaa ttgggtgaaa tggggagcga 6060 tttgcaggca tttgctcggc atgccggtag aggtgtggtc aataagagcg acctcatgct 6120 atacctgaga aagcaacctg acctacagga aagagttact caagaataag aattttcgtt 6180 ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata acttatttaa 6240 taataaaaat cataaatcat aagaaattcg cttactctta attaatcaaa aagttaaaat 6300 tgtacgaata gattcaccac ttcttaacaa atcaaaccct tcattgattt tctcgaatgg 6360 caatacatgt gtaattaaag gatcaagagc aaacttcttc gccataaagt cggcaacaag 6420 ttttggaaca ctatccttgc tcttaaaacc gccaaatata gctcccttcc atgtacgacc 6480 gcttagcaac agcataggat tcatcgacaa attttgtgaa tcaggaggaa cacctacgat 6540 cacactgact ccatatgcct cttgacagca ggacaacgca gttaccatag tatcaagacg 6600 gcctataact tcaaaagaga aatcaactcc accgtttgac atttcagtaa ggacttcttg 6660 tattggtttc ttataatctt gagggttaac acattcagta gccccgacct ccttagcttt 6720 tgcaaatttg tccttattga tgtctacacc tataatcctc gctgcgcctg cagctttaca 6780 ccccataata acgcttagtc ctactcctcc taaaccgaat actgcacaag tcgaaccctg 6840 tgtaaccttt gcaactttaa ctgcggaacc gtaaccggtg gaaaatccgc accctatcaa 6900 gcaaactttt tccagtggtg aagctgcatc gattttagcg acagatatct cgtccaccac 6960 tgtgtattgg gaaaatgtag aagtaccaag gaaatggtgt ataggtttcc ctctgcatgt 7020 aaatctgctt gtaccatcct gcatagtacc tctaggcata gacaaatcat ttttaaggca 7080 gaaattaccc tcaggatgtt tgcagactct acacttacca cattgaggag tgaacagtgg 7140 gatcacttta tcaccaggac gaacagtggt aacaccttca cctatggatt caacgattcc 7200 ggcagcctcg tgtcccgcga ttactggcaa aggagtaact agagtgccac tcaccacatg 7260 gtcgtcggat ctacagattc cggtggcaac catcttgatt ctaacctcgt gtgcttttgg 7320 tggcgctact tctacttctt ctatgctaaa cggctttttc tcttcccaca aaactgccgc 7380 tttacactta ataactttac cggctgttga catcctcagc tagctattgt aatatgtgtg 7440 tttgtttgga ttattaagaa gaataattac aaaaaaaatt acaaaggaag gtaattacaa 7500 cagaattaag aaaggacaag aaggaggaag agaatcagtt cattatttct tctttgttat 7560 ataacaaacc caagtagcga tttggccata cattaaaagt tgagaaccac cctccctggc 7620 aacagccaca actcgttacc attgttcatc acgatcatga aactcgctgt cagctgaaat 7680 ttcacctcag tggatctctc tttttattct tcatcgttcc actaaccttt ttccatcagc 7740 tggcagggaa cggaaagtgg aatcccattt agcgagcttc ctcttttctt caagaaaaga 7800 cgaagcttgt gtgtgggtgc gcgcgctagt atctttccac attaagaaat ataccataaa 7860 ggttacttag acatcactat ggctatatat atatatatat atatatgtaa cttagcacca 7920 tcgcgcgtgc atcactgcat gtgttaaccg aaaagtttgg cgaacacttc accgacacgg 7980 tcatttagat ctgtcgtctg cattgcacgt cccttagcct taaatcctag gcgggagcat 8040 tctcgtgtaa ttgtgcagcc tgcgtagcaa ctcaacatag cgtagtctac ccagtttttc 8100 aagggtttat cgttagaaga ttctcccttt tcttcctgct cacaaatctt aaagtcatac 8160 attgcacgac taaatgcaag catgcggatc ccccgggctg caggaattcg atatcaagct 8220 tatcgatacc gtcgactggc cattaatctt tcccatatta gatttcgcca agccatgaaa 8280 gttcaagaaa ggtctttaga cgaattaccc ttcatttctc aaactggcgt caagggatcc 8340 tggtatggtt ttatcgtttt atttctggtt cttatagcat cgttttggac ttctctgttc 8400 ccattaggcg gttcaggagc cagcgcagaa tcattctttg aaggatactt atcctttcca 8460 attttgattg tctgttacgt tggacataaa ctgtatacta gaaattggac tttgatggtg 8520 aaactagaag atatggatct tgataccggc agaaaacaag tagatttgac tcttcgtagg 8580 gaagaaatga ggattgagcg agaaacatta gcaaaaagat ccttcgtaac aagattttta 8640 catttctggt gttgaaggga aagatatgag ctatacagcg gaatttccat atcactcaga 8700 ttttgttatc taattttttc cttcccacgt ccgcgggaat ctgtgtatat tactgcatct 8760 agatatatgt tatcttatct tggcgcgtac atttaatttt caacgtattc tataagaaat 8820 tgcgggagtt tttttcatgt agatgatact gactgcacgc aaatataggc atgatttata 8880 ggcatgattt gatggctgta ccgataggaa cgctaagagt aacttcagaa tcgttatcct 8940 ggcggaaaaa attcatttgt aaactttaaa aaaaaaagcc aatatcccca aaattattaa 9000 gagcgcctcc attattaact aaaatttcac tcagcatcca caatgtatca ggtatctact 9060 acagatatta catgtggcga aaaagacaag aacaatgcaa tagcgcatca agaaaaaaca 9120 caaagctttc aatcaatgaa tcgaaaatgt cattaaaata gtatataaat tgaaactaag 9180 tcataaagct ataaaaagaa aatttattta aatgcaagat ttaaagtaaa ttcacggccc 9240 tgcaggcctc agctcttgtt ttgttctgca aataacttac ccatcttttt caaaacttta 9300 ggtgcaccct cctttgctag aataagttct atccaataca tcctatttgg atctgcttga 9360 gcttctttca tcacggatac gaattcattt tctgttctca caattttgga cacaactctg 9420 tcttccgttg ccccgaaact ttctggcagt tttgagtaat tccacatagg aatgtcatta 9480 taactctggt tcggaccatg aatttccctc tcaaccgtgt aaccatcgtt attaatgata 9540 aagcagattg ggtttatctt ctctctaatg gctagtccta attcttggac agtcagttgc 9600 aatgatccat ctccgataaa caataaatgt ctagattctt tatctgcaat ttggctgcct 9660 agagctgcgg ggaaagtgta tcctatagat ccccacaagg gttgaccaat aaaatgtgat 9720 ttcgatttca gaaatataga tgaggcaccg aagaaagaag tgccttgttc agccacgatc 9780 gtctcattac tttgggtcaa attttcgaca gcttgccaca gtctatcttg tgacaacagc 9840 gcgttagaag gtacaaaatc ttcttgcttt ttatctatgt acttgccttt atattcaatt 9900 tcggacaagt caagaagaga tgatatcagg gattcgaagt cgaaattttg gattctttcg 9960 ttgaaaattt taccttcatc gatattcaag gaaatcattt tattttcatt aagatggtga 10020 gtaaatgcac ccgtactaga atcggtaagc tttacaccca acataagaat aaaatcagca 10080 gattccacaa attccttcaa gtttggctct gacagagtac cgttgtaaat ccccaaaaat 10140 gagggcaatg cttcatcaac agatgattta ccaaagttca aagtagtaat aggtaactta 10200 gtctttgaaa taaactgagt aacagtcttc tctaggccga acgatataat ttcatggcct 10260 gtgattacaa ttggtttctt ggcattcttc agactttcct gtattttgtt cagaatctct 10320 tgatcagatg tattcgacgt ggaattttcc ttcttaagag gcaaggatgg tttttcagcc 10380 ttagcggcag ctacatctac aggtaaattg atgtaaaccg gctttctttc ctttagtaag 10440 gcagacaaca ctctatcaat ttcaacagtt gcattctcgg ctgtcaataa agtcctggca 10500 gcagtaaccg gttcgtgcat cttcataaag tgcttgaaat caccatcagc caacgtatgg 10560 tgaacaaact taccttcgtt ctgcactttc gaggtaggag atcccacgat ctcaacaaca 10620 ggcaggttct cagcatagga gcccgctaag ccattaactg cggataattc gccaacacca 10680 aatgtagtca agaatgccgc agcctttttc gttcttgcgt acccgtcggc catataggag 10740 gcatttaact cattagcatt tcccacccat ttcatatctt tgtgtgaaat aatttgatct 10800 agaaattgca aattgtagtc acctggtact ccgaatattt cttctatacc taattcgtgt 10860 aatctgtcca acagatagtc acctactgta tacattttgt ttactagttt atgtgtgttt 10920 attcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 10980 tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 11040 cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 11100 tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 11160 cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 11220 ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 11280 cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 11340 tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 11400 cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 11460 cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 11520 ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaactga 11580 aaaagcgtgt tttttattca aaatgattct aactccctta cgtaatcaag gaatcttttt 11640 gccttggcct ccgcgtcatt aaacttcttg ttgttgacgc taacattcaa cgctagtata 11700 tattcgtttt tttcaggtaa gttcttttca acgggtctta ctgatgaggc agtcgcgtct 11760 gaacctgtta agaggtcaaa tatgtcttct tgaccgtacg tgtcttgcat gttattagct 11820 ttgggaattt gcatcaagtc ataggaaaat ttaaatcttg gctctcttgg gctcaaggtg 11880 acaaggtcct cgaaaatagg gcgcgcccca ccgcggtgga gctccagctt ttgttccctt 11940 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 12000 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 12060 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 12120 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 12180 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 12240 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 12300 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 12360 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 12420 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 12480 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 12540 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 12600 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 12660 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 12720 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 12780 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 12840 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 12900 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 12960 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 13020 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 13080 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 13140 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 13200 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 13260 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 13320 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 13380 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 13440 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 13500 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 13560 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 13620 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 13680 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 13740 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 13800 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 13860 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 13920 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 13980 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 14040 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 14100 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca 14160 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga 14220 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta 14280 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt 14340 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt 14400 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact 14460 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc 14520 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga 14580 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa 14640 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg 14700 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc 14760 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat 14820 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta 14880 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga 14940 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt 15000 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct 15060 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga 15120 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg 15180 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat 15240 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg 15300 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc 15360 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc 15420 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat catactaaga 15480 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 15539 <210> 3 <211> 4586 <212> DNA <213> Template <400> 3 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 60 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 120 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 180 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 240 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 300 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 360 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 420 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 480 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 540 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 600 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 660 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 720 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 780 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 840 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 900 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 960 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1020 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1080 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1140 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1200 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1260 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1320 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1380 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1440 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1500 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 1560 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 1620 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 1680 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 1740 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 1800 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 1860 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1920 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1980 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2040 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2100 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2160 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2220 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2280 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2340 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2400 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2460 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 2520 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 2580 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 2640 attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgcat tgcggattac 2700 gtattctaat gttcagataa cttcgtatag catacattat acgaagttat ctagggattc 2760 ataaccattt tctcaatcga attacacaga acacaccgta caaacctctc tatcataact 2820 acttaatagt cacacacgta ctcgtctaaa tacacatcat cgtcctacaa gttcatcaaa 2880 gtgttggaca gacaactata ccagcatgga tctcttgtat cggttctttt ctcccgctct 2940 ctcgcaataa caatgaacac tgggtcaatc atagcctaca caggtgaaca gagtagcgtt 3000 tatacagggt ttatacggtg attcctacgg caaaaatttt tcatttctaa aaaaaaaaag 3060 aaaaattttt ctttccaacg ctagaaggaa aagaaaaatc taattaaatt gatttggtga 3120 ttttctgaga gttccctttt tcatatatcg aattttgaat ataaaaggag atcgaaaaaa 3180 tttttctatt caatctgttt tctggtttta tttgatagtt tttttgtgta ttattattat 3240 ggattagtac tggtttatat gggtttttct gtataacttc tttttatttt agtttgttta 3300 atcttatttt gagttacatt atagttccct aactgcaaga gaagtaacat taaaactcga 3360 gatgggtaag gaaaagactc acgtttcgag gccgcgatta aattccaaca tggatgctga 3420 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 3480 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 3540 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc 3600 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 3660 cggcaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 3720 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa 3780 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga 3840 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 3900 gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 3960 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 4020 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 4080 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 4140 gtttcatttg atgctcgatg agtttttcta agtttaactt gatactacta gattttttct 4200 cttcatttat aaaatttttg gttataattg aagctttaga agtatgaaaa aatccttttt 4260 tttcattctt tgcaaccaaa ataagaagct tcttttattc attgaaatga tgaatataaa 4320 cctaacaaaa gaaaaagact cgaatatcaa acattaaaaa aaaataaaag aggttatctg 4380 ttttcccatt tagttggagt ttgcattttc taatagatag aactctcaat taatgtggat 4440 ttagtttctc tgttcgtttt tttttgtttt gttctcactg tatttacatt tctatttagt 4500 atttagttat tcatataatc tataacttcg tatagcatac attatacgaa gttatccagt 4560 gatgatacaa cgagttagcc aaggtg 4586 <210> 4 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 4 ttccggtttc tttgaaattt ttttgattcg gtaatctccg agcagaagga gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 5 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 5 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta caccttggct 60 aactcgttgt atcatcactg g 81 <210> 6 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 6 gcctcgagtt ttaatgttac ttctcttgca gttaggga 38 <210> 7 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 gctaaattcg agtgaaacac aggaagacca g 31 <210> 8 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 8 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 9 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 9 ttggttgggg gaaaaagagg caacaggaaa gatcagaggg ggaggggggg ggagagtgtc 60 accttggcta actcgttgta tcatcactgg 90 <210> 10 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 10 tcggtgcggg cctcttcgct a 21 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 11 aatgtgagtt agctcactca t 21 <210> 12 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 12 aattggatcc ggcgcgccgt ttaaacggcc ggccaatgtg gctgtggttt cagggtc 57 <210> 13 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 13 aatttctaga ttaattaagc ggccgcaagg ccatgaagct ttttctttc 49 <210> 14 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 14 ttctcgacgt gggccttttt cttg 24 <210> 15 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 15 tgcagcttta aataatcggt gtcactactt tgccttcgtt tatcttgcc 49 <210> 16 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 16 gagcaggcaa gataaacgaa ggcaaagtag tgacaccgat tatttaaag 49 <210> 17 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 tatggaccct gaaaccacag ccacattgta accaccacga cggttgttg 49 <210> 18 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 18 tttagcaaca accgtcgtgg tggttacaat gtggctgtgg tttcagggt 49 <210> 19 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 19 ccagaaaccc tatacctgtg tggacgtaag gccatgaagc tttttcttt 49 <210> 20 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 20 attggaaaga aaaagcttca tggccttacg tccacacagg tatagggtt 49 <210> 21 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 21 cataagaaca cctttggtgg ag 22 <210> 22 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 22 aggattatca ttcataagtt tc 22 <210> 23 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 23 ttcttggagc tgggacatgt ttg 23 <210> 24 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 24 tgatgatatt tcataaataa tg 22 <210> 25 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 25 atgcgtccat ctttacagtc ctg 23 <210> 26 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 26 tacgtacgga ccaatcgaag tg 22 <210> 27 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 27 aattcgtttg agtacactac taatggcttt gttggcaata tgtttttgc 49 <210> 28 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 28 atatagcaaa aacatattgc caacaaagcc attagtagtg tactcaaac 49 <210> 29 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 29 tatggaccct gaaaccacag ccacattctt gttatttata aaaagacac 49 <210> 30 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 30 ctcccgtgtc tttttataaa taacaagaat gtggctgtgg tttcagggt 49 <210> 31 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 31 taccgtaggc gtccttagga aagatagaag gccatgaagc tttttcttt 49 <210> 32 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 32 attggaaaga aaaagcttca tggccttcta tctttcctaa ggacgccta 49 <210> 33 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 33 ttattgtttg gcatttgtag c 21 <210> 34 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 34 ccaagcatct cataaaccta tg 22 <210> 35 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 35 tgtgcagatg cagatgtgag ac 22 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 36 agttattgat accgtac 17 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 37 cgagataccg taggcgtcc 19 <210> 38 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 38 ttatgtatgc tcttctgact tttc 24 <210> 39 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 39 aataattaga gattaaatcg ctcatttttt gccagtttct tcaggcttc 49 <210> 40 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 40 agcctgaaga aactggcaaa aaatgagcga tttaatctct aattattag 49 <210> 41 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 41 tatggaccct gaaaccacag ccacattttt caatcattgg agcaatcat 49 <210> 42 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 42 taaaatgatt gctccaatga ttgaaaaatg tggctgtggt ttcagggtc 49 <210> 43 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 43 accgtaggtg ttgtttggga aagtggaagg ccatgaagct ttttctttc 49 <210> 44 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 44 ttggaaagaa aaagcttcat ggccttccac tttcccaaac aacacctac 49 <210> 45 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 45 ttattgctta gcgttggtag cag 23 <210> 46 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 46 tttttggtgg ttccggcttc c 21 <210> 47 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 47 aaagttggca tagcggaaac tt 22 <210> 48 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 48 gtcattgaca ccatct 16 <210> 49 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 49 agagataccg taggtgttg 19 <210> 50 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 50 aattggcgcg ccatgaaagc tctggtttat cac 33 <210> 51 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 51 tgaatcatga gttttatgtt aattagctca ggcagcgcct gcgttcgag 49 <210> 52 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 52 atcctctcga acgcaggcgc tgcctgagct aattaacata aaactcatg 49 <210> 53 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 53 aattgtttaa acaagtaaat aaattaatca gcat 34 <210> 54 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 54 acacaataca ataacaagaa gaacaaaatg aaagctctgg tttatcacg 49 <210> 55 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 55 agcgtataca tctgttggga aagtagaagg ccatgaagct ttttctttc 49 <210> 56 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 56 ttggaaagaa aaagcttcat ggccttctac tttcccaaca gatgtatac 49 <210> 57 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 57 ttattgttta gcgttagtag cg 22 <210> 58 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 58 cataatcaat ctcaaagaga acaacacaat acaataacaa gaagaacaaa atgaaagctc 60 tggtttatca cg 72 <210> 59 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 59 taggcataat caccgaagaa g 21 <210> 60 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 60 aaaatggtaa gcagctgaaa g 21 <210> 61 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 61 agttgttaga actgttg 17 <210> 62 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 62 gacgatagcg tatacatct 19 <210> 63 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 63 cttagcctct agccatagcc at 22 <210> 64 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 64 ttagttttgc tggccgcatc ttc 23 <210> 65 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 65 cccattaata tactattgag a 21 <210> 66 <211> 7523 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 66 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 60 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga gccggaagca 120 taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt gcgttgcgct 180 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 240 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 300 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 360 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 420 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 480 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 540 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 600 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 660 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 720 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 780 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 840 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 900 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 960 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1020 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1080 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1140 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1200 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1260 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1320 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1380 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1440 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1500 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1560 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1620 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1680 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1740 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1800 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1860 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1920 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1980 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2040 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2100 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2160 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg 2220 cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg 2280 agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc 2340 tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga 2400 atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa 2460 gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca 2520 aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac 2580 tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt 2640 ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg 2700 cattttttca agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac 2760 tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt 2820 ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt 2880 cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga 2940 taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg 3000 ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg 3060 tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt 3120 tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata 3180 ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct 3240 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct 3300 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa 3360 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat 3420 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct 3480 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat 3540 attggatcat ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 3600 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3660 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3720 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3780 ttgtactgag agtgcaccat aaattcccgt tttaagagct tggtgagcgc taggagtcac 3840 tgccaggtat cgtttgaaca cggcattagt cagggaagtc ataacacagt cctttcccgc 3900 aattttcttt ttctattact cttggcctcc tctagtacac tctatatttt tttatgcctc 3960 ggtaatgatt ttcatttttt tttttcccct agcggatgac tctttttttt tcttagcgat 4020 tggcattatc acataatgaa ttatacatta tataaagtaa tgtgatttct tcgaagaata 4080 tactaaaaaa tgagcaggca agataaacga aggcaaagat gacagagcag aaagccctag 4140 taaagcgtat tacaaatgaa accaagattc agattgcgat ctctttaaag ggtggtcccc 4200 tagcgataga gcactcgatc ttcccagaaa aagaggcaga agcagtagca gaacaggcca 4260 cacaatcgca agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg 4320 ctctggccaa gcattccggc tggtcgctaa tcgttgagtg cattggtgac ttacacatag 4380 acgaccatca caccactgaa gactgcggga ttgctctcgg tcaagctttt aaagaggccc 4440 tactggcgcg tggagtaaaa aggtttggat caggatttgc gcctttggat gaggcacttt 4500 ccagagcggt ggtagatctt tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa 4560 gggagaaagt aggagatctc tcttgcgaga tgatcccgca ttttcttgaa agctttgcag 4620 aggctagcag aattaccctc cacgttgatt gtctgcgagg caagaatgat catcaccgta 4680 gtgagagtgc gttcaaggct cttgcggttg ccataagaga agccacctcg cccaatggta 4740 ccaacgatgt tccctccacc aaaggtgttc ttatgtagtg acaccgatta tttaaagctg 4800 cagcatacga tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta 4860 tacgaacagt atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg 4920 aacgaggcgc gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac 4980 ggatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 5040 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 5100 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 5160 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 5220 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 5280 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 5340 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 5400 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 5460 ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca 5520 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 5580 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 5640 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc 5700 cccccctcga ggtattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc 5760 tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca 5820 ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt 5880 ggcagtaacc tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg 5940 ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt 6000 ttgatctatt aacagatata taaatggaaa agctgcataa ccactttaac taatactttc 6060 aacattttca gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg 6120 ttaatatacc tctatacttt aacgtcaagg agaaaaatgt ccaatttact gcccgtacac 6180 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg 6240 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt 6300 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct 6360 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc 6420 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt 6480 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt 6540 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc 6600 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat 6660 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact 6720 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt 6780 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct 6840 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc 6900 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact 6960 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga 7020 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag 7080 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg 7140 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagga gtaagcgaat 7200 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 7260 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 7320 taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 7380 ggcatgccga gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa 7440 ctccagcaat gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg 7500 tggtccgcca ccgcggtgga gct 7523 <210> 67 <211> 15456 <212> DNA <213> Artificial sequence <220> <223> Template <400> 67 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca 60 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag 120 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca 180 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag 240 cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg 300 tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt 360 cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata 420 gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag 480 tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca 540 ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat 600 gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc 660 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa 720 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 780 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac 840 tatgcggcat cagagcagat tgtactgaga gtgcaccata aattcccgtt ttaagagctt 900 ggtgagcgct aggagtcact gccaggtatc gtttgaacac ggcattagtc agggaagtca 960 taacacagtc ctttcccgca attttctttt tctattactc ttggcctcct ctagtacact 1020 ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct agcggatgac 1080 tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta tataaagtaa 1140 tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga aggcaaagat 1200 gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc agattgcgat 1260 ctctttaaag ggtggtcccc tagcgataga gcactcgatc ttcccagaaa aagaggcaga 1320 agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag gtatagggtt 1380 tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa tcgttgagtg 1440 cattggtgac ttacacatag acgaccatca caccactgaa gactgcggga ttgctctcgg 1500 tcaagctttt aaagaggccc taggggccgt gcgtggagta aaaaggtttg gatcaggatt 1560 tgcgcctttg gatgaggcac tttccagagc ggtggtagat ctttcgaaca ggccgtacgc 1620 agttgtcgaa cttggtttgc aaagggagaa agtaggagat ctctcttgcg agatgatccc 1680 gcattttctt gaaagctttg cagaggctag cagaattacc ctccacgttg attgtctgcg 1740 aggcaagaat gatcatcacc gtagtgagag tgcgttcaag gctcttgcgg ttgccataag 1800 agaagccacc tcgcccaatg gtaccaacga tgttccctcc accaaaggtg ttcttatgta 1860 gtgacaccga ttatttaaag ctgcagcata cgatatatat acatgtgtat atatgtatac 1920 ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac aaggtaatgc 1980 atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt ttgctttttc 2040 tttttttttc tcttgaactc gacggatcta tgcggtgtga aataccgcac agatgcgtaa 2100 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 2160 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 2220 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 2280 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 2340 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 2400 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 2460 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 2520 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 2580 tcgccattca ggctgcgcaa ctgttgggaa gggcgcggtg cgggcctctt cgctattacg 2640 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 2700 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg 2760 cgaattgggt accgggcccc ccctcgaggt cgacggcgcg ccactggtag agagcgactt 2820 tgtatgcccc aattgcgaaa cccgcgatat ccttctcgat tctttagtac ccgaccagga 2880 caaggaaaag gaggtcgaaa cgtttttgaa gaaacaagag gaactacacg gaagctctaa 2940 agatggcaac cagccagaaa ctaagaaaat gaagttgatg gatccaactg gcaccgctgg 3000 cttgaacaac aataccagcc ttccaacttc tgtaaataac ggcggtacgc cagtgccacc 3060 agtaccgtta cctttcggta tacctccttt ccccatgttt ccaatgccct tcatgcctcc 3120 aacggctact atcacaaatc ctcatcaagc tgacgcaagc cctaagaaat gaataacaat 3180 actgacagta ctaaataatt gcctacttgg cttcacatac gttgcatacg tcgatataga 3240 taataatgat aatgacagca ggattatcgt aatacgtaat agctgaaaat ctcaaaaatg 3300 tgtgggtcat tacgtaaata atgataggaa tgggattctt ctatttttcc tttttccatt 3360 ctagcagccg tcgggaaaac gtggcatcct ctctttcggg ctcaattgga gtcacgctgc 3420 cgtgagcatc ctctctttcc atatctaaca actgagcacg taaccaatgg aaaagcatga 3480 gcttagcgtt gctccaaaaa agtattggat ggttaatacc atttgtctgt tctcttctga 3540 ctttgactcc tcaaaaaaaa aaatctacaa tcaacagatc gcttcaatta cgccctcaca 3600 aaaacttttt tccttcttct tcgcccacgt taaattttat ccctcatgtt gtctaacgga 3660 tttctgcact tgatttatta taaaaagaca aagacataat acttctctat caatttcagt 3720 tattgttctt ccttgcgtta ttcttctgtt cttctttttc ttttgtcata tataaccata 3780 accaagtaat acatattcaa actagtatga ctgacaaaaa aactcttaaa gacttaagaa 3840 atcgtagttc tgtttacgat tcaatggtta aatcacctaa tcgtgctatg ttgcgtgcaa 3900 ctggtatgca agatgaagac tttgaaaaac ctatcgtcgg tgtcatttca acttgggctg 3960 aaaacacacc ttgtaatatc cacttacatg actttggtaa actagccaaa gtcggtgtta 4020 aggaagctgg tgcttggcca gttcagttcg gaacaatcac ggtttctgat ggaatcgcca 4080 tgggaaccca aggaatgcgt ttctccttga catctcgtga tattattgca gattctattg 4140 aagcagccat gggaggtcat aatgcggatg cttttgtagc cattggcggt tgtgataaaa 4200 acatgcccgg ttctgttatc gctatggcta acatggatat cccagccatt tttgcttacg 4260 gcggaacaat tgcacctggt aatttagacg gcaaagatat cgatttagtc tctgtctttg 4320 aaggtgtcgg ccattggaac cacggcgata tgaccaaaga agaagttaaa gctttggaat 4380 gtaatgcttg tcccggtcct ggaggctgcg gtggtatgta tactgctaac acaatggcga 4440 cagctattga agttttggga cttagccttc cgggttcatc ttctcacccg gctgaatccg 4500 cagaaaagaa agcagatatt gaagaagctg gtcgcgctgt tgtcaaaatg ctcgaaatgg 4560 gcttaaaacc ttctgacatt ttaacgcgtg aagcttttga agatgctatt actgtaacta 4620 tggctctggg aggttcaacc aactcaaccc ttcacctctt agctattgcc catgctgcta 4680 atgtggaatt gacacttgat gatttcaata ctttccaaga aaaagttcct catttggctg 4740 atttgaaacc ttctggtcaa tatgtattcc aagaccttta caaggtcgga ggggtaccag 4800 cagttatgaa atatctcctt aaaaatggct tccttcatgg tgaccgtatc acttgtactg 4860 gcaaaacagt cgctgaaaat ttgaaggctt ttgatgattt aacacctggt caaaaggtta 4920 ttatgccgct tgaaaatcct aaacgtgaag atggtccgct cattattctc catggtaact 4980 tggctccaga cggtgccgtt gccaaagttt ctggtgtaaa agtgcgtcgt catgtcggtc 5040 ctgctaaggt ctttaattct gaagaagaag ccattgaagc tgtcttgaat gatgatattg 5100 ttgatggtga tgttgttgtc gtacgttttg taggaccaaa gggcggtcct ggtatgcctg 5160 aaatgctttc cctttcatca atgattgttg gtaaagggca aggtgaaaaa gttgcccttc 5220 tgacagatgg ccgcttctca ggtggtactt atggtcttgt cgtgggtcat atcgctcctg 5280 aagcacaaga tggcggtcca atcgcctacc tgcaaacagg agacatagtc actattgacc 5340 aagacactaa ggaattacac tttgatatct ccgatgaaga gttaaaacat cgtcaagaga 5400 ccattgaatt gccaccgctc tattcacgcg gtatccttgg taaatatgct cacatcgttt 5460 cgtctgcttc taggggagcc gtaacagact tttggaagcc tgaagaaact ggcaaaaaat 5520 gttgtcctgg ttgctgtggt taagcggccg cgttaattca aattaattga tatagttttt 5580 taatgagtat tgaatctgtt tagaaataat ggaatattat ttttatttat ttatttatat 5640 tattggtcgg ctcttttctt ctgaaggtca atgacaaaat gatatgaagg aaataatgat 5700 ttctaaaatt ttacaacgta agatattttt acaaaagcct agctcatctt ttgtcatgca 5760 ctattttact cacgcttgaa attaacggcc agtccactgc ggagtcattt caaagtcatc 5820 ctaatcgatc tatcgttttt gatagctcat tttggagttc gcgattgtct tctgttattc 5880 acaactgttt taatttttat ttcattctgg aactcttcga gttctttgta aagtctttca 5940 tagtagctta ctttatcctc caacatattt aacttcatgt caatttcggc tcttaaattt 6000 tccacatcat caagttcaac atcatctttt aacttgaatt tattctctag ctcttccaac 6060 caagcctcat tgctccttga tttactggtg aaaagtgata cactttgcgc gcaatccagg 6120 tcaaaacttt cctgcaaaga attcaccaat ttctcgacat catagtacaa tttgttttgt 6180 tctcccatca caatttaata tacctgatgg attcttatga agcgctgggt aatggacgtg 6240 tcactctact tcgccttttt ccctactcct tttagtacgg aagacaatgc taataaataa 6300 gagggtaata ataatattat taatcggcaa aaaagattaa acgccaagcg tttaattatc 6360 agaaagcaaa cgtcgtacca atccttgaat gcttcccaat tgtatattaa gagtcatcac 6420 agcaacatat tcttgttatt aaattaatta ttattgattt ttgatattgt ataaaaaaac 6480 caaatatgta taaaaaaagt gaataaaaaa taccaagtat ggagaaatat attagaagtc 6540 tatacgttaa accacccggg ccccccctcg aggtcgacgg tatcgataag cttgatatcg 6600 aattcctgca gcccggggga tccactagtt ctagagcggc cgctctagaa ctagtaccac 6660 aggtgttgtc ctctgaggac ataaaataca caccgagatt catcaactca ttgctggagt 6720 tagcatatct acaattgggt gaaatgggga gcgatttgca ggcatttgct cggcatgccg 6780 gtagaggtgt ggtcaataag agcgacctca tgctatacct gagaaagcaa cctgacctac 6840 aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat 6900 ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa 6960 ttcgcttact cttaattaat caggcagcgc ctgcgttcga gaggatgatc ttcatcgcct 7020 tctccttggc gccattgagg aatacctgat aggcgtgctc gatctcggcc agctcgaagc 7080 gatgggtaat catcttcttc aacggaagct tgtcggtcga ggcgaccttc atcagcatgg 7140 gcgtcgtgtt cgtgttcacc agtcccgtgg tgatcgtcag gttcttgatc cagagcttct 7200 gaatctcgaa gtcaaccttg acgccatgca cgccgacgtt ggcgatgtgc gcgccgggct 7260 tgacgatctc ctggcagatg tcccaagtcg ccggtatgcc caccgcctcg atcgcaacat 7320 cgactccctc tgccgcaatc ctatgcacgg cttcgacaac gttctccgtg ccggagttga 7380 tggtgtgcgt tgccccgagc tccttggcga gctggaggcg attctcgtcc atgtcgatca 7440 cgatgatggt cgagggggag tagaactggg cggtcaacag tacggacatg ccgacggggc 7500 ccgcgccgac aatagccacc gcatcgcccg gctggacatt cccatactgg acgccgattt 7560 cgtggccggt gggcaggatg tcgctcagca ggacggcgat ttcgtcgtca attgtctggg 7620 ggatcttgta gaggctgttg tcggcatgcg ggatgcggac gtattcggcc tgcacgccat 7680 cgatcatgta acccaggatc cacccgccgt cgcggcaatg ggagtaaagc tgcttcttgc 7740 agtagtcgca cgagccgcaa gaagtgacgc aggaaatcag gaccttgtcg cctttcttga 7800 actgcgtgac actctcgccc acttcctcga tgacgcctac cccttcatgg cccaggatgc 7860 gcccgtcggc gacctctgga ttcttgcctt tgtagatgcc gagatccgtg ccgcagatcg 7920 tggtcttcaa aacccgtact actacatccg tgggcttttg aagggtgggc ttgggcttgt 7980 cttcaagcga gatcttgtgg tcaccgtgat aaaccagagc tttcatcctc agctattgta 8040 atatgtgtgt ttgtttggat tattaagaag aataattaca aaaaaaatta caaaggaagg 8100 taattacaac agaattaaga aaggacaaga aggaggaaga gaatcagttc attatttctt 8160 ctttgttata taacaaaccc aagtagcgat ttggccatac attaaaagtt gagaaccacc 8220 ctccctggca acagccacaa ctcgttacca ttgttcatca cgatcatgaa actcgctgtc 8280 agctgaaatt tcacctcagt ggatctctct ttttattctt catcgttcca ctaacctttt 8340 tccatcagct ggcagggaac ggaaagtgga atcccattta gcgagcttcc tcttttcttc 8400 aagaaaagac gaagcttgtg tgtgggtgcg cgcgctagta tctttccaca ttaagaaata 8460 taccataaag gttacttaga catcactatg gctatatata tatatatata tatatatgta 8520 acttagcacc atcgcgcgtg catcactgca tgtgttaacc gaaaagtttg gcgaacactt 8580 caccgacacg gtcatttaga tctgtcgtct gcattgcacg tcccttagcc ttaaatccta 8640 ggcgggagca ttctcgtgta attgtgcagc ctgcgtagca actcaacata gcgtagtcta 8700 cccagttttt caagggttta tcgttagaag attctccctt ttcttcctgc tcacaaatct 8760 taaagtcata cattgcacga ctaaatgcaa gcatgcggat cccccgggct gcaggaattc 8820 gatatcaagc ttatcgatac cgtcgactgg ccattaatct ttcccatatt agatttcgcc 8880 aagccatgaa agttcaagaa aggtctttag acgaattacc cttcatttct caaactggcg 8940 tcaagggatc ctggtatggt tttatcgttt tatttctggt tcttatagca tcgttttgga 9000 cttctctgtt cccattaggc ggttcaggag ccagcgcaga atcattcttt gaaggatact 9060 tatcctttcc aattttgatt gtctgttacg ttggacataa actgtatact agaaattgga 9120 ctttgatggt gaaactagaa gatatggatc ttgataccgg cagaaaacaa gtagatttga 9180 ctcttcgtag ggaagaaatg aggattgagc gagaaacatt agcaaaaaga tccttcgtaa 9240 caagattttt acatttctgg tgttgaaggg aaagatatga gctatacagc ggaatttcca 9300 tatcactcag attttgttat ctaatttttt ccttcccacg tccgcgggaa tctgtgtata 9360 ttactgcatc tagatatatg ttatcttatc ttggcgcgta catttaattt tcaacgtatt 9420 ctataagaaa ttgcgggagt ttttttcatg tagatgatac tgactgcacg caaatatagg 9480 catgatttat aggcatgatt tgatggctgt accgatagga acgctaagag taacttcaga 9540 atcgttatcc tggcggaaaa aattcatttg taaactttaa aaaaaaaagc caatatcccc 9600 aaaattatta agagcgcctc cattattaac taaaatttca ctcagcatcc acaatgtatc 9660 aggtatctac tacagatatt acatgtggcg aaaaagacaa gaacaatgca atagcgcatc 9720 aagaaaaaac acaaagcttt caatcaatga atcgaaaatg tcattaaaat agtatataaa 9780 ttgaaactaa gtcataaagc tataaaaaga aaatttattt aaatgcaaga tttaaagtaa 9840 attcacggcc ctgcaggcct cagctcttgt tttgttctgc aaataactta cccatctttt 9900 tcaaaacttt aggtgcaccc tcctttgcta gaataagttc tatccaatac atcctatttg 9960 gatctgcttg agcttctttc atcacggata cgaattcatt ttctgttctc acaattttgg 10020 acacaactct gtcttccgtt gccccgaaac tttctggcag ttttgagtaa ttccacatag 10080 gaatgtcatt ataactctgg ttcggaccat gaatttccct ctcaaccgtg taaccatcgt 10140 tattaatgat aaagcagatt gggtttatct tctctctaat ggctagtcct aattcttgga 10200 cagtcagttg caatgatcca tctccgataa acaataaatg tctagattct ttatctgcaa 10260 tttggctgcc tagagctgcg gggaaagtgt atcctataga tccccacaag ggttgaccaa 10320 taaaatgtga tttcgatttc agaaatatag atgaggcacc gaagaaagaa gtgccttgtt 10380 cagccacgat cgtctcatta ctttgggtca aattttcgac agcttgccac agtctatctt 10440 gtgacaacag cgcgttagaa ggtacaaaat cttcttgctt tttatctatg tacttgcctt 10500 tatattcaat ttcggacaag tcaagaagag atgatatcag ggattcgaag tcgaaatttt 10560 ggattctttc gttgaaaatt ttaccttcat cgatattcaa ggaaatcatt ttattttcat 10620 taagatggtg agtaaatgca cccgtactag aatcggtaag ctttacaccc aacataagaa 10680 taaaatcagc agattccaca aattccttca agtttggctc tgacagagta ccgttgtaaa 10740 tccccaaaaa tgagggcaat gcttcatcaa cagatgattt accaaagttc aaagtagtaa 10800 taggtaactt agtctttgaa ataaactgag taacagtctt ctctaggccg aacgatataa 10860 tttcatggcc tgtgattaca attggtttct tggcattctt cagactttcc tgtattttgt 10920 tcagaatctc ttgatcagat gtattcgacg tggaattttc cttcttaaga ggcaaggatg 10980 gtttttcagc cttagcggca gctacatcta caggtaaatt gatgtaaacc ggctttcttt 11040 cctttagtaa ggcagacaac actctatcaa tttcaacagt tgcattctcg gctgtcaata 11100 aagtcctggc agcagtaacc ggttcgtgca tcttcataaa gtgcttgaaa tcaccatcag 11160 ccaacgtatg gtgaacaaac ttaccttcgt tctgcacttt cgaggtagga gatcccacga 11220 tctcaacaac aggcaggttc tcagcatagg agcccgctaa gccattaact gcggataatt 11280 cgccaacacc aaatgtagtc aagaatgccg cagccttttt cgttcttgcg tacccgtcgg 11340 ccatatagga ggcatttaac tcattagcat ttcccaccca tttcatatct ttgtgtgaaa 11400 taatttgatc tagaaattgc aaattgtagt cacctggtac tccgaatatt tcttctatac 11460 ctaattcgtg taatctgtcc aacagatagt cacctactgt atacattttg tttactagtt 11520 tatgtgtgtt tattcgaaac taagttcttg gtgttttaaa actaaaaaaa agactaacta 11580 taaaagtaga atttaagaag tttaagaaat agatttacag aattacaatc aatacctacc 11640 gtctttatat acttattagt caagtagggg aataatttca gggaactggt ttcaaccttt 11700 tttttcagct ttttccaaat cagagagagc agaaggtaat agaaggtgta agaaaatgag 11760 atagatacat gcgtgggtca attgccttgt gtcatcattt actccaggca ggttgcatca 11820 ctccattgag gttgtgcccg ttttttgcct gtttgtgccc ctgttctctg tagttgcgct 11880 aagagaatgg acctatgaac tgatggttgg tgaagaaaac aatattttgg tgctgggatt 11940 cttttttttt ctggatgcca gcttaaaaag cgggctccat tatatttagt ggatgccagg 12000 aataaactgt tcacccagac acctacgatg ttatatattc tgtgtaaccc gccccctatt 12060 ttgggcatgt acgggttaca gcagaattaa aaggctaatt ttttgactaa ataaagttag 12120 gaaaatcact actattaatt atttacgtat tctttgaaat ggcagtattg ataatgataa 12180 actcgaactg aaaaagcgtg ttttttattc aaaatgattc taactccctt acgtaatcaa 12240 ggaatctttt tgccttggcc tccgcgtcat taaacttctt gttgttgacg ctaacattca 12300 acgctagtat atattcgttt ttttcaggta agttcttttc aacgggtctt actgatgagg 12360 cagtcgcgtc tgaacctgtt aagaggtcaa atatgtcttc ttgaccgtac gtgtcttgca 12420 tgttattagc tttgggaatt tgcatcaagt cataggaaaa tttaaatctt ggctctcttg 12480 ggctcaaggt gacaaggtcc tcgaaaatag ggcgcgcccc accgcggtgg agctccagct 12540 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc 12600 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 12660 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 12720 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 12780 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 12840 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 12900 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 12960 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 13020 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 13080 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 13140 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 13200 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 13260 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 13320 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 13380 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 13440 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 13500 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 13560 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 13620 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 13680 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 13740 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 13800 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 13860 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 13920 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 13980 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 14040 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 14100 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 14160 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 14220 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 14280 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 14340 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 14400 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 14460 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 14520 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 14580 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 14640 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 14700 taggggttcc gcgcacattt ccccgaaaag tgccacctga acgaagcatc tgtgcttcat 14760 tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 14820 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct 14880 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga 14940 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct 15000 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat 15060 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg 15120 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa 15180 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt 15240 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg 15300 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc 15360 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc 15420 actctatgaa tagttcttac tacaattttt ttgtct 15456 <210> 68 <211> 1559 <212> DNA <213> Artificial sequence <220> <223> Template <400> 68 gcattgcgga ttacgtattc taatgttcag taccgttcgt ataatgtatg ctatacgaag 60 ttatgcagat tgtactgaga gtgcaccata ccaccttttc aattcatcat ttttttttta 120 ttcttttttt tgatttcggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa 180 ggaagaacga aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga 240 agaaacatga aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa 300 acgaagataa atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc 360 tgttgctgcc aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt 420 ggatgttcgt accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg 480 tttactaaaa acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc 540 gctaaaggca ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga 600 cattggtaat acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc 660 agacattacg aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc 720 ggcagaagaa gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa 780 gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa 840 agattttgtt atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga 900 ttggttgatt atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca 960 gtatagaacc gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg 1020 actatttgca aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg 1080 ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc 1140 atgtatacta aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc 1200 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt 1260 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1320 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1380 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1440 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagataact 1500 tcgtataatg tatgctatac gaacggtacc agtgatgata caacgagtta gccaaggtg 1559 <210> 69 <211> 1047 <212> DNA <213> Achromobacter xylosoxidans <400> 69 atgaaagctc tggtttatca cggtgaccac aagatctcgc ttgaagacaa gcccaagccc 60 acccttcaaa agcccacgga tgtagtagta cgggttttga agaccacgat ctgcggcacg 120 gatctcggca tctacaaagg caagaatcca gaggtcgccg acgggcgcat cctgggccat 180 gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca cgcagttcaa gaaaggcgac 240 aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg actactgcaa gaagcagctt 300 tactcccatt gccgcgacgg cgggtggatc ctgggttaca tgatcgatgg cgtgcaggcc 360 gaatacgtcc gcatcccgca tgccgacaac agcctctaca agatccccca gacaattgac 420 gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg gccacgaaat cggcgtccag 480 tatgggaatg tccagccggg cgatgcggtg gctattgtcg gcgcgggccc cgtcggcatg 540 tccgtactgt tgaccgccca gttctactcc ccctcgacca tcatcgtgat cgacatggac 600 gagaatcgcc tccagctcgc caaggagctc ggggcaacgc acaccatcaa ctccggcacg 660 gagaacgttg tcgaagccgt gcataggatt gcggcagagg gagtcgatgt tgcgatcgag 720 gcggtgggca taccggcgac ttgggacatc tgccaggaga tcgtcaagcc cggcgcgcac 780 atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg agattcagaa gctctggatc 840 aagaacctga cgatcaccac gggactggtg aacacgaaca cgacgcccat gctgatgaag 900 gtcgcctcga ccgacaagct tccgttgaag aagatgatta cccatcgctt cgagctggcc 960 gagatcgagc acgcctatca ggtattcctc aatggcgcca aggagaaggc gatgaagatc 1020 atcctctcga acgcaggcgc tgcctga 1047 <210> 70 <211> 348 <212> PRT <213> Achromobacter xylosoxidans <400> 70 Met Lys Ala Leu Val Tyr His Gly Asp His Lys Ile Ser Leu Glu Asp 1 5 10 15 Lys Pro Lys Pro Thr Leu Gln Lys Pro Thr Asp Val Val Val Arg Val 20 25 30 Leu Lys Thr Thr Ile Cys Gly Thr Asp Leu Gly Ile Tyr Lys Gly Lys 35 40 45 Asn Pro Glu Val Ala Asp Gly Arg Ile Leu Gly His Glu Gly Val Gly 50 55 60 Val Ile Glu Glu Val Gly Glu Ser Val Thr Gln Phe Lys Lys Gly Asp 65 70 75 80 Lys Val Leu Ile Ser Cys Val Thr Ser Cys Gly Ser Cys Asp Tyr Cys 85 90 95 Lys Lys Gln Leu Tyr Ser His Cys Arg Asp Gly Gly Trp Ile Leu Gly 100 105 110 Tyr Met Ile Asp Gly Val Gln Ala Glu Tyr Val Arg Ile Pro His Ala 115 120 125 Asp Asn Ser Leu Tyr Lys Ile Pro Gln Thr Ile Asp Asp Glu Ile Ala 130 135 140 Val Leu Leu Ser Asp Ile Leu Pro Thr Gly His Glu Ile Gly Val Gln 145 150 155 160 Tyr Gly Asn Val Gln Pro Gly Asp Ala Val Ala Ile Val Gly Ala Gly 165 170 175 Pro Val Gly Met Ser Val Leu Leu Thr Ala Gln Phe Tyr Ser Pro Ser 180 185 190 Thr Ile Ile Val Ile Asp Met Asp Glu Asn Arg Leu Gln Leu Ala Lys 195 200 205 Glu Leu Gly Ala Thr His Thr Ile Asn Ser Gly Thr Glu Asn Val Val 210 215 220 Glu Ala Val His Arg Ile Ala Ala Glu Gly Val Asp Val Ala Ile Glu 225 230 235 240 Ala Val Gly Ile Pro Ala Thr Trp Asp Ile Cys Gln Glu Ile Val Lys 245 250 255 Pro Gly Ala His Ile Ala Asn Val Gly Val His Gly Val Lys Val Asp 260 265 270 Phe Glu Ile Gln Lys Leu Trp Ile Lys Asn Leu Thr Ile Thr Thr Gly 275 280 285 Leu Val Asn Thr Asn Thr Thr Pro Met Leu Met Lys Val Ala Ser Thr 290 295 300 Asp Lys Leu Pro Leu Lys Lys Met Ile Thr His Arg Phe Glu Leu Ala 305 310 315 320 Glu Ile Glu His Ala Tyr Gln Val Phe Leu Asn Gly Ala Lys Glu Lys 325 330 335 Ala Met Lys Ile Ile Leu Ser Asn Ala Gly Ala Ala 340 345 <210> 71 <211> 1644 <212> DNA <213> artificial sequence <220> <223> codon optimized L. lactis kivD coding region for S. cerevisiae expression <400> 71 atgtatacag taggtgacta tctgttggac agattacacg aattaggtat agaagaaata 60 ttcggagtac caggtgacta caatttgcaa tttctagatc aaattatttc acacaaagat 120 atgaaatggg tgggaaatgc taatgagtta aatgcctcct atatggccga cgggtacgca 180 agaacgaaaa aggctgcggc attcttgact acatttggtg ttggcgaatt atccgcagtt 240 aatggcttag cgggctccta tgctgagaac ctgcctgttg ttgagatcgt gggatctcct 300 acctcgaaag tgcagaacga aggtaagttt gttcaccata cgttggctga tggtgatttc 360 aagcacttta tgaagatgca cgaaccggtt actgctgcca ggactttatt gacagccgag 420 aatgcaactg ttgaaattga tagagtgttg tctgccttac taaaggaaag aaagccggtt 480 tacatcaatt tacctgtaga tgtagctgcc gctaaggctg aaaaaccatc cttgcctctt 540 aagaaggaaa attccacgtc gaatacatct gatcaagaga ttctgaacaa aatacaggaa 600 agtctgaaga atgccaagaa accaattgta atcacaggcc atgaaattat atcgttcggc 660 ctagagaaga ctgttactca gtttatttca aagactaagt tacctattac tactttgaac 720 tttggtaaat catctgttga tgaagcattg ccctcatttt tggggattta caacggtact 780 ctgtcagagc caaacttgaa ggaatttgtg gaatctgctg attttattct tatgttgggt 840 gtaaagctta ccgattctag tacgggtgca tttactcacc atcttaatga aaataaaatg 900 atttccttga atatcgatga aggtaaaatt ttcaacgaaa gaatccaaaa tttcgacttc 960 gaatccctga tatcatctct tcttgacttg tccgaaattg aatataaagg caagtacata 1020 gataaaaagc aagaagattt tgtaccttct aacgcgctgt tgtcacaaga tagactgtgg 1080 caagctgtcg aaaatttgac ccaaagtaat gagacgatcg tggctgaaca aggcacttct 1140 ttcttcggtg cctcatctat atttctgaaa tcgaaatcac attttattgg tcaacccttg 1200 tggggatcta taggatacac tttccccgca gctctaggca gccaaattgc agataaagaa 1260 tctagacatt tattgtttat cggagatgga tcattgcaac tgactgtcca agaattagga 1320 ctagccatta gagagaagat aaacccaatc tgctttatca ttaataacga tggttacacg 1380 gttgagaggg aaattcatgg tccgaaccag agttataatg acattcctat gtggaattac 1440 tcaaaactgc cagaaagttt cggggcaacg gaagacagag ttgtgtccaa aattgtgaga 1500 acagaaaatg aattcgtatc cgtgatgaaa gaagctcaag cagatccaaa taggatgtat 1560 tggatagaac ttattctagc aaaggagggt gcacctaaag ttttgaaaaa gatgggtaag 1620 ttatttgcag aacaaaacaa gagc 1644 <210> 72 <211> 753 <212> DNA <213> Saccharomyces cerevisiae <400> 72 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300 gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360 cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420 ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480 agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540 aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600 ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660 gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720 ttaataatcc aaacaaacac acatattaca ata 753 <210> 73 <211> 316 <212> DNA <213> Saccharomyces cerevisiae <400> 73 gagtaagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata 60 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 120 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 180 cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cacccaattg 240 tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta tgtcctcaga 300 ggacaacacc tgtggt 316 <210> 74 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 74 ggaattcaca catgaaagct ctggtttatc 30 <210> 75 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 75 gcgtccaggg cgtcaaagat caggcagc 28 <210> 76 <211> 55 <212> DNA <213> artificial sequence <220> <223> primer <400> 76 aaacaaacac acatattaca atagctgagg atgaaagctc tggtttatca cggtg 55 <210> 77 <211> 55 <212> DNA <213> artificial sequence <220> <223> primer <400> 77 atcataagaa attcgcttac tcttaattaa tcaggcagcg cctgcgttcg agagg 55 <210> 78 <211> 8994 <212> DNA <213> artificial sequence <220> <223> constructed plasmid <400> 78 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg 60 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 120 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa 180 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 240 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 300 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 360 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 420 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 480 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 540 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 600 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 660 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 720 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 780 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 840 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 900 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 960 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 1020 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 1080 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 1140 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1200 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 1260 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 1320 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 1380 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 1440 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 1500 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 1560 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 1620 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 1680 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 1740 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 1800 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 1860 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 1920 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 1980 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 2040 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 2100 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 2160 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 2220 ccccgaaaag tgccacctga acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa 2280 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 2340 caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa 2400 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag 2460 aaatgcaacg cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac 2520 aaaaatgcat cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc 2580 ctttgtgcgc tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt 2640 tagaagaagg ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc 2700 ccgcgtttac tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc 2760 gattatattc tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg 2820 atgattcttc attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta 2880 cgtataggaa atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac 2940 tacaattttt ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag 3000 tttagatgca agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca 3060 gagatatata gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt 3120 ttagtagctc gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc 3180 ttttggtttt caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa 3240 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca 3300 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca 3360 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat 3420 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg 3480 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt 3540 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcatactaag aaaccattat 3600 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt 3660 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 3720 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 3780 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatcga 3840 ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc accattatgg 3900 gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca ttgagtgttt 3960 tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat taggaatcgt 4020 agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc ttgtcaatat 4080 taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc aatttgctta 4140 cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt agattgcgta 4200 tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg tttctattat 4260 gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct ttttaagcaa 4320 ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg ttggaaccac 4380 ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct tcaatggcct 4440 taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac aagatagtgg 4500 cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat ggttcgtaca 4560 aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc aacaaaccca 4620 aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg ttgctggtga 4680 ttataatacc atttaggtgg gttgggttct taactaggat catggcggca gaatcaatca 4740 attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc acagtttttc 4800 tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata ggcaatggtg 4860 gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact tctggaacgg 4920 tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc ttaccaaagt 4980 aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca aattgtggct 5040 tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt aagttggcgt 5100 acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca ctaccggtac 5160 cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg gaggcttcca 5220 gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca attaaatgat 5280 tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga accttaatgg 5340 cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc ttcttagggg 5400 cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata tattgctgaa 5460 atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac 5520 aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga 5580 acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct ttttctccca 5640 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc 5700 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa 5760 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac 5820 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac 5880 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac 5940 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt 6000 tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta gaagttctcc 6060 tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 6120 aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 6180 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 6240 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 6300 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 6360 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 6420 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 6480 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 6540 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 6600 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 6660 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 6720 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 6780 ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc ctgcagcccg 6840 ggggatccgc atgcttgcat ttagtcgtgc aatgtatgac tttaagattt gtgagcagga 6900 agaaaaggga gaatcttcta acgataaacc cttgaaaaac tgggtagact acgctatgtt 6960 gagttgctac gcaggctgca caattacacg agaatgctcc cgcctaggat ttaaggctaa 7020 gggacgtgca atgcagacga cagatctaaa tgaccgtgtc ggtgaagtgt tcgccaaact 7080 tttcggttaa cacatgcagt gatgcacgcg cgatggtgct aagttacata tatatatata 7140 tatatatata tagccatagt gatgtctaag taacctttat ggtatatttc ttaatgtgga 7200 aagatactag cgcgcgcacc cacacacaag cttcgtcttt tcttgaagaa aagaggaagc 7260 tcgctaaatg ggattccact ttccgttccc tgccagctga tggaaaaagg ttagtggaac 7320 gatgaagaat aaaaagagag atccactgag gtgaaatttc agctgacagc gagtttcatg 7380 atcgtgatga acaatggtaa cgagttgtgg ctgttgccag ggagggtggt tctcaacttt 7440 taatgtatgg ccaaatcgct acttgggttt gttatataac aaagaagaaa taatgaactg 7500 attctcttcc tccttcttgt cctttcttaa ttctgttgta attaccttcc tttgtaattt 7560 tttttgtaat tattcttctt aataatccaa acaaacacac atattacaat agctagctga 7620 ggatgaaggc attagtttat catggggatc acaaaatttc gttagaagac aaaccaaaac 7680 ccactctgca gaaaccaaca gacgttgtgg ttagggtgtt gaaaacaaca atttgcggta 7740 ctgacttggg aatatacaaa ggtaagaatc ctgaagtggc agatggcaga atcctgggtc 7800 atgagggcgt tggcgtcatt gaagaagtgg gcgaatccgt gacacaattc aaaaaggggg 7860 ataaagtttt aatctcctgc gttactagct gtggatcgtg tgattattgc aagaagcaac 7920 tgtattcaca ctgtagagac ggtggctgga ttttaggtta catgatcgac ggtgtccaag 7980 ccgaatacgt cagaatacca catgctgaca attcattgta taagatcccg caaactatcg 8040 atgatgaaat tgcagtacta ctgtccgata ttttacctac tggacatgaa attggtgttc 8100 aatatggtaa cgttcaacca ggcgatgctg tagcaattgt aggagcaggt cctgttggaa 8160 tgtcagtttt gttaactgct caattttact cgcctagtac cattattgtt atcgacatgg 8220 acgaaaaccg tttacaatta gcgaaggagc ttggggccac acacactatt aactccggta 8280 ctgaaaatgt tgtcgaagct gtgcatcgta tagcagccga aggagtggat gtagcaatag 8340 aagctgttgg tatacccgca acctgggaca tctgtcagga aattgtaaaa cccggcgctc 8400 atattgccaa cgtgggagtt catggtgtta aggtggactt tgaaattcaa aagttgtgga 8460 ttaagaatct aaccatcacc actggtttgg ttaacactaa tactacccca atgttgatga 8520 aggtagcctc tactgataaa ttgcctttaa agaaaatgat tactcacagg tttgagttag 8580 ctgaaatcga acacgcatat caggttttct tgaatggcgc taaagaaaaa gctatgaaga 8640 ttattctatc taatgcaggt gccgcctaat taattaagag taagcgaatt tcttatgatt 8700 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga 8760 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt 8820 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccgag 8880 caaatgcctg caaatcgctc cccatttcac ccaattgtag atatgctaac tccagcaatg 8940 agttgatgaa tctcggtgtg tattttatgt cctcagagga caacacctgt ggta 8994 <210> 79 <211> 2145 <212> DNA <213> artificial sequence <220> <223> constructed chimeric gene <400> 79 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatatatata 300 tatagccata gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact 360 agcgcgcgca cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa 420 tgggattcca ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga 480 ataaaaagag agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat 540 gaacaatggt aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat 600 ggccaaatcg ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt 660 cctccttctt gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta 720 attattcttc ttaataatcc aaacaaacac acatattaca atagctagct gaggatgaag 780 gcattagttt atcatgggga tcacaaaatt tcgttagaag acaaaccaaa acccactctg 840 cagaaaccaa cagacgttgt ggttagggtg ttgaaaacaa caatttgcgg tactgacttg 900 ggaatataca aaggtaagaa tcctgaagtg gcagatggca gaatcctggg tcatgagggc 960 gttggcgtca ttgaagaagt gggcgaatcc gtgacacaat tcaaaaaggg ggataaagtt 1020 ttaatctcct gcgttactag ctgtggatcg tgtgattatt gcaagaagca actgtattca 1080 cactgtagag acggtggctg gattttaggt tacatgatcg acggtgtcca agccgaatac 1140 gtcagaatac cacatgctga caattcattg tataagatcc cgcaaactat cgatgatgaa 1200 attgcagtac tactgtccga tattttacct actggacatg aaattggtgt tcaatatggt 1260 aacgttcaac caggcgatgc tgtagcaatt gtaggagcag gtcctgttgg aatgtcagtt 1320 ttgttaactg ctcaatttta ctcgcctagt accattattg ttatcgacat ggacgaaaac 1380 cgtttacaat tagcgaagga gcttggggcc acacacacta ttaactccgg tactgaaaat 1440 gttgtcgaag ctgtgcatcg tatagcagcc gaaggagtgg atgtagcaat agaagctgtt 1500 ggtatacccg caacctggga catctgtcag gaaattgtaa aacccggcgc tcatattgcc 1560 aacgtgggag ttcatggtgt taaggtggac tttgaaattc aaaagttgtg gattaagaat 1620 ctaaccatca ccactggttt ggttaacact aatactaccc caatgttgat gaaggtagcc 1680 tctactgata aattgccttt aaagaaaatg attactcaca ggtttgagtt agctgaaatc 1740 gaacacgcat atcaggtttt cttgaatggc gctaaagaaa aagctatgaa gattattcta 1800 tctaatgcag gtgccgccta attaattaag agtaagcgaa tttcttatga tttatgattt 1860 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg 1920 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc 1980 aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg agcaaatgcc 2040 tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa tgagttgatg 2100 aatctcggtg tgtattttat gtcctcagag gacaacacct gtggt 2145 <210> 80 <211> 4280 <212> DNA <213> artificial sequence <220> <223> vector <400> 80 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg 60 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 120 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 180 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 240 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 300 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 360 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 420 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 480 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 540 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 600 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 660 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 720 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 780 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 840 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 900 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 960 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 1020 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 1080 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 1140 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 1200 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 1260 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 1320 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1380 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1440 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1500 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1560 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1620 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1680 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1740 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1800 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1860 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1920 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1980 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 2040 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 2100 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 2160 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 2220 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 2280 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 2340 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 2400 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 2460 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca 2520 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 2580 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 2640 tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccccgg ctctgagaca 2700 gtagtaggtt agtcatcgct ctaccgacgc gcaggaaaag aaagaagcat tgcggattac 2760 gtattctaat gttcagcccg cggaacgcca gcaaatcacc acccatgcgc atgatactga 2820 gtcttgtaca cgctgggctt ccagtgtact gagagtgcac cataccacag cttttcaatt 2880 caattcatca tttttttttt attctttttt ttgatttcgg tttctttgaa atttttttga 2940 ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata 3000 tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca 3060 cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg 3120 tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca 3180 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga 3240 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc 3300 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt 3360 cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt 3420 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat 3480 tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat 3540 gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt 3600 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg 3660 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa 3720 gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta caggatctga 3780 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga 3840 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa 3900 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt 3960 atatcagtta ttaccctatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 4020 gcatcaggaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 4080 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ttcagcccgc ggaacgccag 4140 caaatcacca cccatgcgca tgatactgag tcttgtacac gctgggcttc cagtgatgat 4200 acaacgagtt agccaaggtg agcacggatg tctaaattag aattacgttt taatatcttt 4260 ttttccatat ctagggctag 4280 <210> 81 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 81 gcatgcttgc atttagtcgt gcaatgtatg 30 <210> 82 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 82 gaacattaga atacgtaatc cgcaatgcac tagtaccaca ggtgttgtcc tctg 54 <210> 83 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 83 cagaggacaa cacctgtggt actagtgcat tgcggattac gtattctaat gttc 54 <210> 84 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 84 caccttggct aactcgttgt atcatcac 28 <210> 85 <211> 100 <212> DNA <213> artificial sequence <220> <223> primer <400> 85 ttttaagccg aatgagtgac agaaaaagcc cacaacttat caagtgatat tgaacaaagg 60 gcgaaacttc gcatgcttgc atttagtcgt gcaatgtatg 100 <210> 86 <211> 98 <212> DNA <213> artificial sequence <220> <223> primer <400> 86 cccaattggt aaatattcaa caagagacgc gcagtacgta acatgcgaat tgcgtaattc 60 acggcgataa caccttggct aactcgttgt atcatcac 98 <210> 87 <211> 29 <212> DNA <213> artificial sequence <220> <223> primer <400> 87 caaaagccca tgtcccacac caaaggatg 29 <210> 88 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 88 caccatcgcg cgtgcatcac tgcatg 26 <210> 89 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 89 tcggtttttg caatatgacc tgtgggcc 28 <210> 90 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 90 gagaagatgc ggccagcaaa ac 22 <210> 91 <211> 2745 <212> DNA <213> artificial sequence <220> <223> constructed coding region-terminator segment <400> 91 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaatgttgtc ctggttgctg tggttaagcg 1740 gccgcgttaa ttcaaattaa ttgatatagt tttttaatga gtattgaatc tgtttagaaa 1800 taatggaata ttatttttat ttatttattt atattattgg tcggctcttt tcttctgaag 1860 gtcaatgaca aaatgatatg aaggaaataa tgatttctaa aattttacaa cgtaagatat 1920 ttttacaaaa gcctagctca tcttttgtca tgcactattt tactcacgct tgaaattaac 1980 ggccagtcca ctgcggagtc atttcaaagt catcctaatc gatctatcgt ttttgatagc 2040 tcattttgga gttcgcgatt gtcttctgtt attcacaact gttttaattt ttatttcatt 2100 ctggaactct tcgagttctt tgtaaagtct ttcatagtag cttactttat cctccaacat 2160 atttaacttc atgtcaattt cggctcttaa attttccaca tcatcaagtt caacatcatc 2220 ttttaacttg aatttattct ctagctcttc caaccaagcc tcattgctcc ttgatttact 2280 ggtgaaaagt gatacacttt gcgcgcaatc caggtcaaaa ctttcctgca aagaattcac 2340 caatttctcg acatcatagt acaatttgtt ttgttctccc atcacaattt aatatacctg 2400 atggattctt atgaagcgct gggtaatgga cgtgtcactc tacttcgcct ttttccctac 2460 tccttttagt acggaagaca atgctaataa ataagagggt aataataata ttattaatcg 2520 gcaaaaaaga ttaaacgcca agcgtttaat tatcagaaag caaacgtcgt accaatcctt 2580 gaatgcttcc caattgtata ttaagagtca tcacagcaac atattcttgt tattaaatta 2640 attattattg atttttgata ttgtataaaa aaaccaaata tgtataaaaa aagtgaataa 2700 aaaataccaa gtatggagaa atatattaga agtctatacg ttaaa 2745 <210> 92 <211> 27 <212> DNA <213> artificial sequence <220> <223> primer <400> 92 gacttttgga agcctgaaga aactggc 27 <210> 93 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 93 cttggcagca acaggactag 20 <210> 94 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 94 ccaggccaat tcaacagact gtcggc 26 <210> 95 <211> 2347 <212> DNA <213> artificial sequence <220> <223> constructed URA3 marker with flanking homologous repeat sequences for HIS gene replacement and marker excision <400> 95 gcattgcgga ttacgtattc taatgttcag gtgctggaag aagagctgct taaccgccgc 60 gcccagggtg aagatccacg ctactttacc ctgcgtcgtc tggatttcgg cggctgtcgt 120 ctttcgctgg caacgccggt tgatgaagcc tgggacggtc cgctctcctt aaacggtaaa 180 cgtatcgcca cctcttatcc tcacctgctc aagcgttatc tcgaccagaa aggcatctct 240 tttaaatcct gcttactgaa cggttctgtt gaagtcgccc cgcgtgccgg actggcggat 300 gcgatttgcg atctggtttc caccggtgcc acgctggaag ctaacggcct gcgcgaagtc 360 gaagttatct atcgctcgaa agcctgcctg attcaacgcg atggcgaaat ggaagaatcc 420 aaacagcaac tgatcgacaa actgctgacc cgtattcagg gtgtgatcca ggcgcgcgaa 480 tcaaaataca tcatgatgca cgcaccgacc gaacgtctgg atgaagtcat ggtacctact 540 gagagtgcac cataccacag cttttcaatt caattcatca tttttttttt attctttttt 600 ttgatttcgg tttctttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg 660 aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg 720 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata 780 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc 840 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg 900 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa 960 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc 1020 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa 1080 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac 1140 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga 1200 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct 1260 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt 1320 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat 1380 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac 1440 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc 1500 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata 1560 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact 1620 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa 1680 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg ttaatatttt 1740 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 1800 cggcaaaatc tctagagtgc tggaagaaga gctgcttaac cgccgcgccc agggtgaaga 1860 tccacgctac tttaccctgc gtcgtctgga tttcggcggc tgtcgtcttt cgctggcaac 1920 gccggttgat gaagcctggg acggtccgct ctccttaaac ggtaaacgta tcgccacctc 1980 ttatcctcac ctgctcaagc gttatctcga ccagaaaggc atctctttta aatcctgctt 2040 actgaacggt tctgttgaag tcgccccgcg tgccggactg gcggatgcga tttgcgatct 2100 ggtttccacc ggtgccacgc tggaagctaa cggcctgcgc gaagtcgaag ttatctatcg 2160 ctcgaaagcc tgcctgattc aacgcgatgg cgaaatggaa gaatccaaac agcaactgat 2220 cgacaaactg ctgacccgta ttcagggtgt gatccaggcg cgcgaatcaa aatacatcat 2280 gatgcacgca ccgaccgaac gtctggatga agtcatccag tgatgataca acgagttagc 2340 caaggtg 2347 <210> 96 <211> 80 <212> DNA <213> artificial sequence <220> <223> primer <400> 96 cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 97 <211> 81 <212> DNA <213> artificial sequence <220> <223> primer <400> 97 tatacacatg tatatatatc gtatgctgca gctttaaata atcggtgtca caccttggct 60 aactcgttgt atcatcactg g 81 <210> 98 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 98 gacttgaata atgcagcggc gcttgc 26 <210> 99 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 99 ccaccctctt caattagcta agatcatagc 30 <210> 100 <211> 25 <212> DNA <213> artificial sequence <220> <223> primer <400> 100 aaaaattgat tctcatcgta aatgc 25 <210> 101 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 101 ctgcagcgag gagccgtaat 20 <210> 102 <211> 90 <212> DNA <213> artificial sequence <220> <223> primer <400> 102 atggttcatt taggtccaaa aaaaccacaa gccagaaagg gttccatggc cgatgtgcca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 103 <211> 91 <212> DNA <213> artificial sequence <220> <223> primer <400> 103 ttaagcaccg atgataccaa cggacttacc ttcagcaatt cttttttggg ccaaagcagc 60 caccttggct aactcgttgt atcatcactg g 91 <210> 104 <211> 24 <212> DNA <213> artificial sequence <220> <223> primer <400> 104 ctaggatgag tagcagcacg ttcc 24 <210> 105 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 105 ccaattccgt gatgtctctt tgttgc 26 <210> 106 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 106 gtgaacgagt tcacaaccgc 20 <210> 107 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 107 gttcgttcca gaattatcac gc 22 <210> 108 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 108 ggatccgcat gcttgcattt agtcgtgc 28 <210> 109 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 109 gggatgcgga cgtattcggc 20 <210> 110 <211> 4506 <212> DNA <213> Saccharomyces cerevisiae <400> 110 atgtcgaata ccccttataa ttcatctgtg ccttccattg catccatgac ccagtcttcg 60 gtctcaagaa gtcctaacat gcatacagca actacgcccg gtgccaacac cagctctaac 120 tctccaccct tgcacatgtc ttcagattcg tccaagatca agaggaagcg taacagaatt 180 ccgctcagtt gcaccatttg tcggaaaagg aaagtcaaat gtgacaaact cagaccacac 240 tgccagcagt gcactaaaac tggggtagcc catctctgcc actacatgga acagacctgg 300 gcagaagagg cagagaaaga attgctgaag gacaacgaat taaagaagct tagggagcgc 360 gtaaaatctt tagaaaagac tctttctaag gtgcactctt ctccttcgtc taactccttg 420 aaaagttaca acactcccga gagcagcaac ctgtttatgg gtagcgatga acacaccacc 480 cttgttaatg caaatacagg ctctgcttcc tctgcctcgc atatgcatca gcaacaacag 540 caacagcagc aacaggaaca acaacaagac ttttccagaa gtgcgaacgc caacgcgaat 600 tcctcgtccc tttctatctc aaataaatat gacaacgatg agctggactt aactaaggac 660 tttgatcttt tgcatatcaa aagtaacgga accatccact taggtgccac ccactggttg 720 tctatcatga aaggtgaccc gtacctaaaa cttttgtggg gtcatatctt cgctatgagg 780 gaaaagttaa atgaatggta ctaccaaaaa aattcgtact ctaagctgaa gtcaagcaaa 840 tgtcccatca atcacgcgca agcgccgcct tctgccgctg ccgccgctac cagaaaatgt 900 cctgttgatc actccgcgtt ttcgtctggc atggtggccc caaaggagga gactcctctt 960 cctaggaaat gtccagttga ccacaccatg ttctcttcgg gaatgattcc tcccagagag 1020 gacacttcgt cccagaagag gtgtcccgtt gaccacacca tgtattccgc aggaatgatg 1080 ccgcccaagg acgagacacc ttccccattt tccactaaag ctatgataga ccataacaag 1140 catacaatga atccgcctca gtcaaaatgt cctgtggacc atagaaacta tatgaaggat 1200 tatccctctg acatggcaaa ttcttcttcg aacccggcaa gtcgttgccc cattgaccat 1260 tcaagcatga aaaatacagc ggccttacca gcttcaacgc acaataccat cccacaccac 1320 caaccacagt ccggatctca tgctcgttcg catcccgcac aaagcaggaa acatgattcc 1380 tacatgacag aatctgaagt cctcgcaaca ctttgtgaga tgttgccacc aaagcgcgtc 1440 atcgcattat tcatcgagaa attcttcaaa catttatacc ctgccattcc aatcttagat 1500 gaacagaatt tcaaaaatca cgtgaatcaa atgctttcgt tgtcttcgat gaatcccaca 1560 gttaacaact ttggtatgag catgccatct tcatctacac tagagaacca acccataaca 1620 caaatcaatc ttccaaaact ttccgattct tgtaacttag gtattctgat aataatcttg 1680 agattgacat ggctatccat accttctaat tcctgcgaag tcgacctggg agaagaaagt 1740 ggctcatttt tagtgcccaa cgaatctagc aatatgtctg catctgcatt gacctcgatg 1800 gctaaagaag aatcacttct gctaaagcat gagacaccgg tcgaggcact ggagctatgt 1860 caaaaatact tgattaaatt cgatgaactt tctagtattt ccaataacaa cgttaattta 1920 accacggtgc agtttgccat tttttacaac ttctatatga aaagtgcctc taatgatttg 1980 actaccttga caaataccaa caacactggc atggccaatc ctggtcacga ttccgagtct 2040 caccagatcc tattgtccaa tattactcaa atggccttta gttgtgggtt acacagagac 2100 cctgataatt ttcctcaatt aaacgctacc attccagcaa ccagccagga cgtgtctaac 2160 aacgggagca aaaaggcaaa ccctagcacc aatccaactt tgaataacaa catgtctgct 2220 gccactacca acagcagtag cagatctggc agtgctgatt caagaagtgg ttctaaccct 2280 gtgaacaaga aggaaaatca ggttagtatc gaaagattta aacacacttg gaggaaaatt 2340 tggtattaca ttgttagcat ggatgttaac caatctcttt ccctggggag ccctcgacta 2400 ctaagaaatc tgagggattt cagcgataca aagctaccaa gtgcgtcaag gattgattat 2460 gttcgcgata tcaaagagtt aatcattgtg aagaatttta ctcttttttt ccaaattgat 2520 ttgtgtatta ttgctgtatt aaatcacatt ttgaatgttt ctttagcaag aagcgtgaga 2580 aaatttgaac tggattcatt gattaattta ttgaaaaatc tgacctatgg tactgagaat 2640 gtcaatgatg tagtgagctc cttgatcaac aaagggttat taccaacttc ggaaggtggt 2700 tctgtagatt caaataatga tgaaatttac ggtctaccga aactacccga tattctaaac 2760 catggtcaac ataaccaaaa cttgtatgct gatggaagaa atacttctag tagtgatata 2820 gataagaaat tggaccttcc tcacgaatct acaacgagag ctctattctt ttccaagcat 2880 atgacaatta gaatgttgct gtacttattg aactacattt tgtttactca ttatgaacca 2940 atgggcagtg aagatcctgg tactaatatc ctagctaagg agtacgctca agaggcatta 3000 aattttgcca tggatggcta cagaaactgc atgattttct tcaacaatat cagaaacacc 3060 aattcactat tcgattacat gaatgttatc ttgtcttacc cttgtttgga cattggacat 3120 cgttctttac aatttatcgt ttgtttgatc ctgagagcta aatgtggccc attgactggt 3180 atgcgtgaat catcgatcat tactaatggt acatcaagtg gatttaatag ttcggtagaa 3240 gatgaggacg tcaaagttaa acaagaatct tctgatgaat tgaaaaaaga cgatttcatg 3300 aaagatgtaa atttggattc aggcgattca ttagcagaga ttctaatgtc aagaatgctg 3360 ctatttcaaa aactaacaaa acaactatca aagaagtaca actacgctat tcgtatgaac 3420 aaatccactg gattctttgt ctctttacta gatacacctt caaagaaatc agactcgaaa 3480 tcgggtggta gttcattcat gttgggtaat tggaaacatc caaaggtttc aaacatgagc 3540 ggatttcttg ctggtgacaa agaccaatta cagaaatgcc ccgtgtacca agatgcgctg 3600 gggtttgtta gtccaaccgg tgctaatgaa ggttctgctc cgatgcaagg catgtcctta 3660 cagggctcta ctgctaggat gggagggacc cagttgccac caattagatc atacaaacct 3720 atcacgtaca caagtagtaa tctacgtcgt atgaatgaaa cgggtgaggc agaagctaag 3780 agaagaagat ttaatgatgg ctatattgat aataatagta acaacgatat acctagagga 3840 atcagcccaa aaccttcaaa tgggctatca tcggtgcagc cactattatc gtcattttcc 3900 atgaaccagc taaacggggg taccattcca acggttccat cgttaaccaa cattacttca 3960 caaatgggag ctttaccatc tttagatagg atcaccacta atcaaataaa tttgccagac 4020 ccatctagag atgaagcatt tgacaactcc atcaagcaaa tgacgcctat gacaagtgca 4080 ttcatgaatg ctaatactac aattccaagt tcaactttaa acgggaatat gaacatgaat 4140 ggagctggaa ctgcgaatac agatacaagt gccaacggca gtgctttatc gacactgaca 4200 agcccacaag gctcagactt agcatccaat tctgctacac agtataaacc tgacttagaa 4260 gactttttga tgcaaaattc taactttaat gggctaatga taaatccttc cagtctggta 4320 gaagttgttg gtggatacaa cgatcctaat aaccttggaa gaaatgacgc ggttgatttt 4380 ctacccgttg ataatgttga aattgatggt gttggaataa aaatcaacta tcatctacta 4440 actagtattt acgttactag tatattatca tatacggtgt tagaagatga cgcaaatgat 4500 gagaaa 4506 <210> 111 <211> 99 <212> DNA <213> artificial sequence <220> <223> primer <400> 111 tcctttctca attattattt tctactcata acctcacgca aaataacaca gtcaaatcaa 60 tcaaagtatg actgacaaaa aaactcttaa agacttaag 99 <210> 112 <211> 77 <212> DNA <213> artificial sequence <220> <223> primer <400> 112 gaacattaga atacgtaatc cgcaatgctt ctttcttttc cgtttaacgt atagacttct 60 aatatatttc tccatac 77 <210> 113 <211> 45 <212> DNA <213> artificial sequence <220> <223> primer <400> 113 aaacggaaaa gaaagaagca ttgcggatta cgtattctaa tgttc 45 <210> 114 <211> 88 <212> DNA <213> artificial sequence <220> <223> primer <400> 114 tatttttcgt tacataaaaa tgcttataaa actttaacta ataattagag attaaatcgc 60 caccttggct aactcgttgt atcatcac 88 <210> 115 <211> 1713 <212> DNA <213> Streptococcus mutans <400> 115 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaa 1713 <210> 116 <211> 571 <212> PRT <213> Streptococcus mutans <400> 116 Met Thr Asp Lys Lys Thr Leu Lys Asp Leu Arg Asn Arg Ser Ser Val 1 5 10 15 Tyr Asp Ser Met Val Lys Ser Pro Asn Arg Ala Met Leu Arg Ala Thr 20 25 30 Gly Met Gln Asp Glu Asp Phe Glu Lys Pro Ile Val Gly Val Ile Ser 35 40 45 Thr Trp Ala Glu Asn Thr Pro Cys Asn Ile His Leu His Asp Phe Gly 50 55 60 Lys Leu Ala Lys Val Gly Val Lys Glu Ala Gly Ala Trp Pro Val Gln 65 70 75 80 Phe Gly Thr Ile Thr Val Ser Asp Gly Ile Ala Met Gly Thr Gln Gly 85 90 95 Met Arg Phe Ser Leu Thr Ser Arg Asp Ile Ile Ala Asp Ser Ile Glu 100 105 110 Ala Ala Met Gly Gly His Asn Ala Asp Ala Phe Val Ala Ile Gly Gly 115 120 125 Cys Asp Lys Asn Met Pro Gly Ser Val Ile Ala Met Ala Asn Met Asp 130 135 140 Ile Pro Ala Ile Phe Ala Tyr Gly Gly Thr Ile Ala Pro Gly Asn Leu 145 150 155 160 Asp Gly Lys Asp Ile Asp Leu Val Ser Val Phe Glu Gly Val Gly His 165 170 175 Trp Asn His Gly Asp Met Thr Lys Glu Glu Val Lys Ala Leu Glu Cys 180 185 190 Asn Ala Cys Pro Gly Pro Gly Gly Cys Gly Gly Met Tyr Thr Ala Asn 195 200 205 Thr Met Ala Thr Ala Ile Glu Val Leu Gly Leu Ser Leu Pro Gly Ser 210 215 220 Ser Ser His Pro Ala Glu Ser Ala Glu Lys Lys Ala Asp Ile Glu Glu 225 230 235 240 Ala Gly Arg Ala Val Val Lys Met Leu Glu Met Gly Leu Lys Pro Ser 245 250 255 Asp Ile Leu Thr Arg Glu Ala Phe Glu Asp Ala Ile Thr Val Thr Met 260 265 270 Ala Leu Gly Gly Ser Thr Asn Ser Thr Leu His Leu Leu Ala Ile Ala 275 280 285 His Ala Ala Asn Val Glu Leu Thr Leu Asp Asp Phe Asn Thr Phe Gln 290 295 300 Glu Lys Val Pro His Leu Ala Asp Leu Lys Pro Ser Gly Gln Tyr Val 305 310 315 320 Phe Gln Asp Leu Tyr Lys Val Gly Gly Val Pro Ala Val Met Lys Tyr 325 330 335 Leu Leu Lys Asn Gly Phe Leu His Gly Asp Arg Ile Thr Cys Thr Gly 340 345 350 Lys Thr Val Ala Glu Asn Leu Lys Ala Phe Asp Asp Leu Thr Pro Gly 355 360 365 Gln Lys Val Ile Met Pro Leu Glu Asn Pro Lys Arg Glu Asp Gly Pro 370 375 380 Leu Ile Ile Leu His Gly Asn Leu Ala Pro Asp Gly Ala Val Ala Lys 385 390 395 400 Val Ser Gly Val Lys Val Arg Arg His Val Gly Pro Ala Lys Val Phe 405 410 415 Asn Ser Glu Glu Glu Ala Ile Glu Ala Val Leu Asn Asp Asp Ile Val 420 425 430 Asp Gly Asp Val Val Val Val Arg Phe Val Gly Pro Lys Gly Gly Pro 435 440 445 Gly Met Pro Glu Met Leu Ser Leu Ser Ser Met Ile Val Gly Lys Gly 450 455 460 Gln Gly Glu Lys Val Ala Leu Leu Thr Asp Gly Arg Phe Ser Gly Gly 465 470 475 480 Thr Tyr Gly Leu Val Val Gly His Ile Ala Pro Glu Ala Gln Asp Gly 485 490 495 Gly Pro Ile Ala Tyr Leu Gln Thr Gly Asp Ile Val Thr Ile Asp Gln 500 505 510 Asp Thr Lys Glu Leu His Phe Asp Ile Ser Asp Glu Glu Leu Lys His 515 520 525 Arg Gln Glu Thr Ile Glu Leu Pro Pro Leu Tyr Ser Arg Gly Ile Leu 530 535 540 Gly Lys Tyr Ala His Ile Val Ser Ser Ala Ser Arg Gly Ala Val Thr 545 550 555 560 Asp Phe Trp Lys Pro Glu Glu Thr Gly Lys Lys 565 570 <210> 117 <211> 548 <212> PRT <213> Lactococcus lactis <400> 117 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys Ser 545 <210> 118 <211> 1125 <212> DNA <213> artificial sequence <220> <223> horse ADH coding region codon optimized for S. cerevisiae expression <400> 118 atgtcaacag ccggtaaagt tattaagtgt aaagcggcag ttttgtggga agagaaaaag 60 ccgtttagca tagaagaagt agaagtagcg ccaccaaaag cacacgaggt tagaatcaag 120 atggttgcca ccggaatctg tagatccgac gaccatgtgg tgagtggcac tctagttact 180 cctttgccag taatcgcggg acacgaggct gccggaatcg ttgaatccat aggtgaaggt 240 gttaccactg ttcgtcctgg tgataaagtg atcccactgt tcactcctca atgtggtaag 300 tgtagagtct gcaaacatcc tgagggtaat ttctgcctta aaaatgattt gtctatgcct 360 agaggtacta tgcaggatgg tacaagcaga tttacatgca gagggaaacc tatacaccat 420 ttccttggta cttctacatt ttcccaatac acagtggtgg acgagatatc tgtcgctaaa 480 atcgatgcag cttcaccact ggaaaaagtt tgcttgatag ggtgcggatt ttccaccggt 540 tacggttccg cagttaaagt tgcaaaggtt acacagggtt cgacttgtgc agtattcggt 600 ttaggaggag taggactaag cgttattatg gggtgtaaag ctgcaggcgc agcgaggatt 660 ataggtgtag acatcaataa ggacaaattt gcaaaagcta aggaggtcgg ggctactgaa 720 tgtgttaacc ctcaagatta taagaaacca atacaagaag tccttactga aatgtcaaac 780 ggtggagttg atttctcttt tgaagttata ggccgtcttg atactatggt aactgcgttg 840 tcctgctgtc aagaggcata tggagtcagt gtgatcgtag gtgttcctcc tgattcacaa 900 aatttgtcga tgaatcctat gctgttgcta agcggtcgta catggaaggg agctatattt 960 ggcggtttta agagcaagga tagtgttcca aaacttgttg ccgactttat ggcgaagaag 1020 tttgctcttg atcctttaat tacacatgta ttgccattcg agaaaatcaa tgaagggttt 1080 gatttgttaa gaagtggtga atctattcgt acaattttaa ctttt 1125 <210> 119 <211> 375 <212> PRT <213> Equus caballus <400> 119 Met Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro 20 25 30 Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg 35 40 45 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val 50 55 60 Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly 65 70 75 80 Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro 85 90 95 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys 100 105 110 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr 115 120 125 Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr 130 135 140 Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys 145 150 155 160 Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly 165 170 175 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln 180 185 190 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val 195 200 205 Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp 210 215 220 Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 240 Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr 245 250 255 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg 260 265 270 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly 275 280 285 Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met 290 295 300 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe 305 310 315 320 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe 325 330 335 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro 340 345 350 Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser 355 360 365 Ile Arg Thr Ile Leu Thr Phe 370 375 <210> 120 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 120 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 121 <211> 9089 <212> DNA <213> artificial sequence <220> <223> constructed plasmid <400> 121 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac tggccattaa 2040 tctttcccat attagatttc gccaagccat gaaagttcaa gaaaggtctt tagacgaatt 2100 acccttcatt tctcaaactg gcgtcaaggg atcctggtat ggttttatcg ttttatttct 2160 ggttcttata gcatcgtttt ggacttctct gttcccatta ggcggttcag gagccagcgc 2220 agaatcattc tttgaaggat acttatcctt tccaattttg attgtctgtt acgttggaca 2280 taaactgtat actagaaatt ggactttgat ggtgaaacta gaagatatgg atcttgatac 2340 cggcagaaaa caagtagatt tgactcttcg tagggaagaa atgaggattg agcgagaaac 2400 attagcaaaa agatccttcg taacaagatt tttacatttc tggtgttgaa gggaaagata 2460 tgagctatac agcggaattt ccatatcact cagattttgt tatctaattt tttccttccc 2520 acgtccgcgg gaatctgtgt atattactgc atctagatat atgttatctt atcttggcgc 2580 gtacatttaa ttttcaacgt attctataag aaattgcggg agtttttttc atgtagatga 2640 tactgactgc acgcaaatat aggcatgatt tataggcatg atttgatggc tgtaccgata 2700 ggaacgctaa gagtaacttc agaatcgtta tcctggcgga aaaaattcat ttgtaaactt 2760 taaaaaaaaa agccaatatc cccaaaatta ttaagagcgc ctccattatt aactaaaatt 2820 tcactcagca tccacaatgt atcaggtatc tactacagat attacatgtg gcgaaaaaga 2880 caagaacaat gcaatagcgc atcaagaaaa aacacaaagc tttcaatcaa tgaatcgaaa 2940 atgtcattaa aatagtatat aaattgaaac taagtcataa agctataaaa agaaaattta 3000 tttaaatgca agatttaaag taaattcacg gccctgcagg ccctaacctg ctaggacaca 3060 acgtctttgc ctggtaaagt ttctagctga cgtgattcct tcacctgtgg atccggcaat 3120 tgtaaaggtt gtgaaaccct cagcttcata accgacacct gcaaatgact ttgcattctt 3180 aacaaagata gttgtatcaa tttcacgttc gaatctatta aggttatcga tgttcttaga 3240 ataaatgtag gcggaatgtt ttctattctg ctcagctatc ttggcgtatt taatggcttc 3300 atcaatgtcc ttcactctaa ctataggcaa aattggcatc atcaactccg tcataacgaa 3360 cggatggttt gcgttgactt cacaaataat acactttaca ttacttggtg actctacatc 3420 tatttcatcc aaaaacagtt tagcgtcctt accaacccac ttcttattaa tgaaatattc 3480 ttgagtttca ttgttctttt gaagaacaag gtctatcagc ttggatactt ggtcttcatt 3540 gataatgacg gcgttgtttt tcaacatgtt agagatcaga tcatctgcaa cgttttcaaa 3600 cacgaacact tctttttccg cgatacaagg aagattgttg tcaaacgaac aaccttcaat 3660 aatgcttctg ccggccttct cgatatctgc tgtatcgtct acaataaccg gaggattacc 3720 cgcgccagct ccgatggcct ttttaccaga attaagaagg gtttttacca tacccgggcc 3780 acccgtaccg cacaacaatt ttatggatgg atgtttgata atagcgtcta aactttccat 3840 agttgggttc tttatagtag tgacaaggtt ttcaggtcca ccacagctaa ttatggcttt 3900 gtttatcatt tctactgcga aagcgacaca ctttttggcg catgggtgac cattaaatac 3960 aactgcattc cccgcagcta tcatacctat agaattgcag ataacggttt ctgttggatt 4020 cgtgcttgga gttatagcgc cgataactcc gtatggactc atttcaacca ctgttagtcc 4080 attatcgccg gaccatgctg ttgttgtcag atcttcagtg cctggggtat acttggccac 4140 taattcatgt ttcaagattt tatcctcata ccttcccatg tgggtttcct ccaggatcat 4200 tgtggctaag acctctttat tctgtaatgc ggcttttctt atttcggtga ttattttctc 4260 tctttgttcc tttgtgtagt gtagggaaag aatcttttgt gcatgtactg cagaagaaat 4320 ggcattctca acattttcaa atactccaaa acatgaagag ttatctttgt aattctttaa 4380 gttgatgttt tcaccattag tcttcacttt caagtctttg gtggttggga ttaaggtatc 4440 tttatccatg gtgtttgttt atgtgtgttt attcgaaact aagttcttgg tgttttaaaa 4500 ctaaaaaaaa gactaactat aaaagtagaa tttaagaagt ttaagaaata gatttacaga 4560 attacaatca atacctaccg tctttatata cttattagtc aagtagggga ataatttcag 4620 ggaactggtt tcaacctttt ttttcagctt tttccaaatc agagagagca gaaggtaata 4680 gaaggtgtaa gaaaatgaga tagatacatg cgtgggtcaa ttgccttgtg tcatcattta 4740 ctccaggcag gttgcatcac tccattgagg ttgtgcccgt tttttgcctg tttgtgcccc 4800 tgttctctgt agttgcgcta agagaatgga cctatgaact gatggttggt gaagaaaaca 4860 atattttggt gctgggattc tttttttttc tggatgccag cttaaaaagc gggctccatt 4920 atatttagtg gatgccagga ataaactgtt cacccagaca cctacgatgt tatatattct 4980 gtgtaacccg ccccctattt tgggcatgta cgggttacag cagaattaaa aggctaattt 5040 tttgactaaa taaagttagg aaaatcacta ctattaatta tttacgtatt ctttgaaatg 5100 gcagtattga taatgataaa ctcgaactga aaaagcgtgt tttttattca aaatgattct 5160 aactccctta cgtaatcaag gaatcttttt gccttggcct ccgcgtcatt aaacttcttg 5220 ttgttgacgc taacattcaa cgctagtata tattcgtttt tttcaggtaa gttcttttca 5280 acgggtctta ctgatgaggc agtcgcgtct gaacctgtta agaggtcaaa tatgtcttct 5340 tgaccgtacg tgtcttgcat gttattagct ttgggaattt gcatcaagtc ataggaaaat 5400 ttaaatcttg gctctcttgg gctcaaggtg acaaggtcct cgaaaatagg gcgcgcccca 5460 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 5520 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5580 ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta 5640 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5700 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5760 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5820 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5880 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5940 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6000 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6060 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6120 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6180 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6240 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6300 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6360 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6420 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6480 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6540 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6600 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6660 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6720 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6780 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6840 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6900 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6960 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7020 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7080 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7140 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7200 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7260 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7320 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7380 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7440 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7500 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7560 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7620 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa 7680 cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca 7740 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta 7800 ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt 7860 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta 7920 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc 7980 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca 8040 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg 8100 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc 8160 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt 8220 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa 8280 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt 8340 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga 8400 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag 8460 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata 8520 cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg 8580 gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc 8640 tgaagttcct atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc 8700 gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt 8760 cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg 8820 tgtttatgct taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag 8880 tacctcctgt gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc 8940 ctttagctgt tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat 9000 catttccttt gatattggat catactaaga aaccattatt atcatgacat taacctataa 9060 aaataggcgt atcacgaggc cctttcgtc 9089 <210> 122 <211> 1023 <212> DNA <213> Saccharomyces cerevisiae <400> 122 caccgcggtg gggcgcgccc tattttcgag gaccttgtca ccttgagccc aagagagcca 60 agatttaaat tttcctatga cttgatgcaa attcccaaag ctaataacat gcaagacacg 120 tacggtcaag aagacatatt tgacctctta acaggttcag acgcgactgc ctcatcagta 180 agacccgttg aaaagaactt acctgaaaaa aacgaatata tactagcgtt gaatgttagc 240 gtcaacaaca agaagtttaa tgacgcggag gccaaggcaa aaagattcct tgattacgta 300 agggagttag aatcattttg aataaaaaac acgctttttc agttcgagtt tatcattatc 360 aatactgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc taactttatt 420 tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa atagggggcg 480 ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca 540 ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga atcccagcac 600 caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac 660 agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct 720 gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt 780 acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa 840 ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg gtaggtattg 900 attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt 960 ttttttagtt ttaaaacacc aagaacttag tttcgaataa acacacataa actagtaaac 1020 aaa 1023 <210> 123 <211> 21 <212> DNA <213> artificial sequence <220> <223> primer <400> 123 caaaagctga gctccaccgc g 21 <210> 124 <211> 44 <212> DNA <213> artificial sequence <220> <223> primer <400> 124 gtttactagt ttatgtgtgt ttattcgaaa ctaagttctt ggtg 44 <210> 125 <400> 125 000 <210> 126 <211> 39 <212> DNA <213> artificial sequence <220> <223> primer <400> 126 cacacatatt acaatagcta gctgaggatg aaagctctg 39 <210> 127 <211> 39 <212> DNA <213> artificial sequence <220> <223> primer <400> 127 cagagctttc atcctcagct agctattgta atatgtgtg 39 <210> 128 <211> 9491 <212> DNA <213> artificial sequence <220> <223> constructed plasmid <400> 128 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt 720 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca 780 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag 840 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag 900 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag 960 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta 1020 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca 1080 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct 1140 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat 1200 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat 1260 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt 1320 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt 1380 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata 1440 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 1500 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 1560 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 1620 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 1680 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 1740 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 1800 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 1860 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 1980 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2040 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcg 2100 acggcgcgcc actggtagag agcgactttg tatgccccaa ttgcgaaacc cgcgatatcc 2160 ttctcgattc tttagtaccc gaccaggaca aggaaaagga ggtcgaaacg tttttgaaga 2220 aacaagagga actacacgga agctctaaag atggcaacca gccagaaact aagaaaatga 2280 agttgatgga tccaactggc accgctggct tgaacaacaa taccagcctt ccaacttctg 2340 taaataacgg cggtacgcca gtgccaccag taccgttacc tttcggtata cctcctttcc 2400 ccatgtttcc aatgcccttc atgcctccaa cggctactat cacaaatcct catcaagctg 2460 acgcaagccc taagaaatga ataacaatac tgacagtact aaataattgc ctacttggct 2520 tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg attatcgtaa 2580 tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg 2640 ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt ggcatcctct 2700 ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat atctaacaac 2760 tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag tattggatgg 2820 ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa aatctacaat 2880 caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt cgcccacgtt 2940 aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat aaaaagacaa 3000 agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat tcttctgttc 3060 ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa ctagtatgac 3120 tgacaaaaaa actcttaaag acttaagaaa tcgtagttct gtttacgatt caatggttaa 3180 atcacctaat cgtgctatgt tgcgtgcaac tggtatgcaa gatgaagact ttgaaaaacc 3240 tatcgtcggt gtcatttcaa cttgggctga aaacacacct tgtaatatcc acttacatga 3300 ctttggtaaa ctagccaaag tcggtgttaa ggaagctggt gcttggccag ttcagttcgg 3360 aacaatcacg gtttctgatg gaatcgccat gggaacccaa ggaatgcgtt tctccttgac 3420 atctcgtgat attattgcag attctattga agcagccatg ggaggtcata atgcggatgc 3480 ttttgtagcc attggcggtt gtgataaaaa catgcccggt tctgttatcg ctatggctaa 3540 catggatatc ccagccattt ttgcttacgg cggaacaatt gcacctggta atttagacgg 3600 caaagatatc gatttagtct ctgtctttga aggtgtcggc cattggaacc acggcgatat 3660 gaccaaagaa gaagttaaag ctttggaatg taatgcttgt cccggtcctg gaggctgcgg 3720 tggtatgtat actgctaaca caatggcgac agctattgaa gttttgggac ttagccttcc 3780 gggttcatct tctcacccgg ctgaatccgc agaaaagaaa gcagatattg aagaagctgg 3840 tcgcgctgtt gtcaaaatgc tcgaaatggg cttaaaacct tctgacattt taacgcgtga 3900 agcttttgaa gatgctatta ctgtaactat ggctctggga ggttcaacca actcaaccct 3960 tcacctctta gctattgccc atgctgctaa tgtggaattg acacttgatg atttcaatac 4020 tttccaagaa aaagttcctc atttggctga tttgaaacct tctggtcaat atgtattcca 4080 agacctttac aaggtcggag gggtaccagc agttatgaaa tatctcctta aaaatggctt 4140 ccttcatggt gaccgtatca cttgtactgg caaaacagtc gctgaaaatt tgaaggcttt 4200 tgatgattta acacctggtc aaaaggttat tatgccgctt gaaaatccta aacgtgaaga 4260 tggtccgctc attattctcc atggtaactt ggctccagac ggtgccgttg ccaaagtttc 4320 tggtgtaaaa gtgcgtcgtc atgtcggtcc tgctaaggtc tttaattctg aagaagaagc 4380 cattgaagct gtcttgaatg atgatattgt tgatggtgat gttgttgtcg tacgttttgt 4440 aggaccaaag ggcggtcctg gtatgcctga aatgctttcc ctttcatcaa tgattgttgg 4500 taaagggcaa ggtgaaaaag ttgcccttct gacagatggc cgcttctcag gtggtactta 4560 tggtcttgtc gtgggtcata tcgctcctga agcacaagat ggcggtccaa tcgcctacct 4620 gcaaacagga gacatagtca ctattgacca agacactaag gaattacact ttgatatctc 4680 cgatgaagag ttaaaacatc gtcaagagac cattgaattg ccaccgctct attcacgcgg 4740 tatccttggt aaatatgctc acatcgtttc gtctgcttct aggggagccg taacagactt 4800 ttggaagcct gaagaaactg gcaaaaaatg ttgtcctggt tgctgtggtt aagcggccgc 4860 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 4920 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 4980 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 5040 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 5100 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 5160 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 5220 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 5280 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 5340 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 5400 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 5460 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 5520 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 5580 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 5640 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 5700 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 5760 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 5820 accaagtatg gagaaatata ttagaagtct atacgttaaa ccaccgcggt ggagctccag 5880 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt 5940 tcctgtgtga aattgttatc cgctcacaat tccacacaac ataggagccg gaagcataaa 6000 gtgtaaagcc tggggtgcct aatgagtgag gtaactcaca ttaattgcgt tgcgctcact 6060 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 6120 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 6180 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 6240 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 6300 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 6360 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 6420 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 6480 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 6540 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 6600 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 6660 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 6720 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 6780 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6840 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6900 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6960 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 7020 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 7080 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 7140 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 7200 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 7260 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 7320 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 7380 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 7440 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 7500 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 7560 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 7620 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 7680 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7740 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7800 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7860 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7920 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7980 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 8040 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc 8100 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct 8160 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg 8220 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 8280 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat 8340 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc 8400 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt 8460 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat 8520 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt 8580 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg 8640 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct 8700 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat 8760 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa 8820 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta 8880 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg 8940 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt 9000 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt 9060 ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg 9120 aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt 9180 gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc 9240 gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta 9300 tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat 9360 gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg 9420 gatcatctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 9480 gccctttcgt c 9491 <210> 129 <211> 1000 <212> DNA <213> Saccharymoces cerevisiae <400> 129 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 420 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 480 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 540 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 600 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 660 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 720 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 780 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 840 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 900 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 960 accaagtatg gagaaatata ttagaagtct atacgttaaa 1000 <210> 130 <211> 16387 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 130 gatcctctag tttctcggta ctatgcatat gatccaatat caaaggaaat gatagcattg 60 aaggatgaga ctaatccaat tgaggagtgg cagcatatag aacagctaaa gggtagtgct 120 gaaggaagca tacgataccc cgcatggaat gggataatat cacaggaggt actagactac 180 ctttcatcct acataaatag acgcatataa gtacgcattt aagcataaac acgcactatg 240 ccgttcttct catgtatata tatatacagg caacacgcag atataggtgc gacgtgaaca 300 gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct 360 ttgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc agagcgcttt 420 tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac caaaaacgca ccggactgta 480 acgagctact aaaatattgc gaataccgct tccacaaaca ttgctcaaaa gtatctcttt 540 gctatatatc tctgtgctat atccctatat aacctaccca tccacctttc gctccttgaa 600 cttgcatcta aactcgacct ctacattttt tatgtttatc tctagtatta ctctttagac 660 aaaaaaattg tagtaagaac tattcataga gtgaatcgaa aacaatacga aaatgtaaac 720 atttcctata cgtagtatat agagacaaaa tagaagaaac cgttcataat tttctgacca 780 atgaagaatc atcaacgcta tcactttctg ttcacaaagt atgcgcaatc cacatcggta 840 tagaatataa tcggggatgc ctttatcttg aaaaaatgca cccgcagctt cgctagtaat 900 cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa gagaaaatag acaccaaagt 960 agccttcttc taaccttaac ggacctacag tgcaaaaagt tatcaagaga ctgcattata 1020 gagcgcacaa aggagaaaaa aagtaatcta agatgctttg ttagaaaaat agcgctctcg 1080 ggatgcattt ttgtagaaca aaaaagaagt atagattctt tgttggtaaa atagcgctct 1140 cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc 1200 tctcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc 1260 gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 1320 agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc gttaacaaag 1380 atatgctatt gaagtgcaag atggaaacgc agaaaatgaa ccggggatgc gacgtgcaag 1440 attacctatg caatagatgc aatagtttct ccaggaaccg aaatacatac attgtcttcc 1500 gtaaagcgct agactatata ttattataca ggttcaaata tactatctgt ttcagggaaa 1560 actcccaggt tcggatgttc aaaattcaat gatgggtaac aagtacgatc gtaaatctgt 1620 aaaacagttt gtcggatatt aggctgtatc tcctcaaagc gtattcgaat atcattgaga 1680 agctgcattt tttttttttt tttttttttt tttttttata tatatttcaa ggatatacca 1740 ttgtaatgtc tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa 1800 tcacagccga agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt 1860 tcgatttcga aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc 1920 cagatgaggc gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg 1980 gtcctaaatg gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag 2040 aacttcaatt gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact 2100 tatctccaat caagccacaa tttgctaaag gtactgactt cgttgttgtt agagaattag 2160 tgggaggtat ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata 2220 gtgaacaata caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc 2280 tacaacatga gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt 2340 caagattatg gagaaaaact gtggaggaaa ccatcaagaa cgaattccct acattgaaag 2400 ttcaacatca attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa 2460 atggtattat aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta 2520 tcccaggctc cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca 2580 ccgcatttgg tttgtacgaa ccatgccatg gttccgctcc agatttgcca aagaataagg 2640 tcaaccctat cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc 2700 ctgaagaagg taaagccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa 2760 ctggtgattt aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag 2820 ttaagaaaat ccttgcttaa aaagattctc tttttttatg atatttgtac aaaaaaaaaa 2880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aatgcagcgt cacatcggat 2940 aataatgatg gcagccattg tagaagtgcc ttttgcattt ctagtctctt tctcggtcta 3000 gctagtttta ctacatcgcg aagatagaat cttagatcac actgcctttg ctgagctgga 3060 tcaatagagt aacaaaagag tggtaaggcc tcgttaaagg acaaggacct gagcggaagt 3120 gtatcgtaca gtagacggag tatactagag tcgacctgca ggcatgcaag cttttcaatt 3180 catcattttt tttttattct tttttttgat ttcggtttcc ttgaaatttt tttgattcgg 3240 taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 3300 gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 3360 caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 3420 ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 3480 acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 3540 taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 3600 agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag 3660 acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 3720 gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 3780 gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 3840 cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 3900 ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 3960 gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 4020 acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta 4080 ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 4140 acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 4200 tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 4260 agttattacc cgggaatctc ggtcgtaatg atttttataa tgacgaaaaa aaaaaaattg 4320 gaaagaaaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 4380 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 4440 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 4500 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 4560 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 4620 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 4680 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 4740 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 4800 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 4860 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 4920 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 4980 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 5040 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 5100 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 5160 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 5220 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 5280 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 5340 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 5400 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 5460 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 5520 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 5580 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 5640 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 5700 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 5760 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 5820 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 5880 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 5940 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 6000 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 6060 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 6120 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 6180 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 6240 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 6300 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 6360 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 6420 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 6480 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 6540 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 6600 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 6660 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 6720 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta 6780 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac cgaaatcggc 6840 aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt tccagtttgg 6900 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 6960 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc 7020 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 7080 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc taaggcgctg 7140 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta 7200 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa 7260 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 7320 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 7380 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 7440 aattcgagct ccaccgcgga tagatctgaa atgaataaca atactgacag tactaaataa 7500 ttgcctactt ggcttcacat acgttgcata cgtcgatata gataataatg ataatgacag 7560 caggattatc gtaatacgta atagttgaaa atctcaaaaa tgtgtgggtc attacgtaaa 7620 taatgatagg aatgggattc ttctattttt cctttttcca ttctagcagc cgtcgggaaa 7680 acgtggcatc ctctctttcg ggctcaattg gagtcacgct gccgtgagca tcctctcttt 7740 ccatatctaa caactgagca cgtaaccaat ggaaaagcat gagcttagcg ttgctccaaa 7800 aaagtattgg atggttaata ccatttgtct gttctcttct gactttgact cctcaaaaaa 7860 aaaaaatcta caatcaacag atcgcttcaa ttacgccctc acaaaaactt ttttccttct 7920 tcttcgccca cgttaaattt tatccctcat gttgtctaac ggatttctgc acttgattta 7980 ttataaaaag acaaagacat aatacttctc tatcaatttc agttattgtt cttccttgcg 8040 ttattcttct gttcttcttt ttcttttgtc atatataacc ataaccaagt aatacatatt 8100 caaatctaga gctgaggatg ttgaagcaaa tcaacttcgg tggtactgtt gaaaccgtct 8160 acgaaagagc tgactggcca agagaaaagt tgttggacta cttcaagaac gacacttttg 8220 ctttgatcgg ttacggttcc caaggttacg gtcaaggttt gaacttgaga gacaacggtt 8280 tgaacgttat cattggtgtc cgtaaagatg gtgcttcttg gaaggctgcc atcgaagacg 8340 gttgggttcc aggcaagaac ttgttcactg ttgaagatgc tatcaagaga ggtagttacg 8400 ttatgaactt gttgtccgat gccgctcaat cagaaacctg gcctgctatc aagccattgt 8460 tgaccaaggg taagactttg tacttctccc acggtttctc cccagtcttc aaggacttga 8520 ctcacgttga accaccaaag gacttagatg ttatcttggt tgctccaaag ggttccggta 8580 gaactgtcag atctttgttc aaggaaggtc gtggtattaa ctcttcttac gccgtctgga 8640 acgatgtcac cggtaaggct cacgaaaagg cccaagcttt ggccgttgcc attggttccg 8700 gttacgttta ccaaaccact ttcgaaagag aagtcaactc tgacttgtac ggtgaaagag 8760 gttgtttaat gggtggtatc cacggtatgt tcttggctca atacgacgtc ttgagagaaa 8820 acggtcactc cccatctgaa gctttcaacg aaaccgtcga agaagctacc caatctctat 8880 acccattgat cggtaagtac ggtatggatt acatgtacga tgcttgttcc accaccgcca 8940 gaagaggtgc tttggactgg tacccaatct tcaagaatgc tttgaagcct gttttccaag 9000 acttgtacga atctaccaag aacggtaccg aaaccaagag atctttggaa ttcaactctc 9060 aacctgacta cagagaaaag ctagaaaagg aattagacac catcagaaac atggaaatct 9120 ggaaggttgg taaggaagtc agaaagttga gaccagaaaa ccaataatta attaatcatg 9180 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag 9240 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 9300 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 9360 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 9420 tgcgggcggc cgctctagag agttgttagc aaccttttgt ttcttttgag ctggttcaga 9480 cattatgtac acgtatatgt gacgagttcg agaagtattt tactatcgta ctaaatttta 9540 cctgaaaaat tatatactcg agaaagagga agccaagaat tgagaaaaaa gaaaaacccg 9600 cgagtaagga aattaaatac aggtgtacac atacacgcac acatatatat atatatatat 9660 atgtatatgt gtatatagga agcgcgcgca tgttagtata tacgattcgt tggaaagggg 9720 ccgtccacca aacgtgactt gacgagttga caaattgacc tcaatatggc tcagtcagta 9780 atttttagtt ccgctttatt cccgccatct ttcaggccac gagggtagct cataacgccg 9840 cgctaatgcc gctgcgtcac agcaaccagt agctcagcca aaaccgaaag agaaatcgta 9900 gctgtcccga tgaggactta tacacttgtc accatctaaa taaattattt attcgcgttt 9960 cggttcttgt tttcgattta attagattgt tcattgaatc ataataaata tgtaaaaaat 10020 atatatattt gaagctgctt cagaaaaaca gggcttccta gtgtacagat gtatgtcgga 10080 tgaaaaaaaa aaaatcttaa atgtgaaatt gggtcaattc aattgactat gacttgatgt 10140 tgcaaaaatt ccaagagaaa aagtttccag cacttgatat tattttcctc tttaattttt 10200 cgccttgtct acgatcttat tagcaccgat ccagggcatc atagacctta actgttcacc 10260 aataatttcg ataccatgtg ctgcattgtt tcttctttta gcagtcatac tcgggtaacc 10320 cgtagcgcct tcacttatga acatcttagc gtattcaccg tcctggatac gtttcaaggc 10380 atttctcatg gcttgtcttg attctgcgtt aatgacttca ggtccggtga catactcacc 10440 atattctgca ttatttgaaa tggaatagtt catattagct ataccacctt catacattaa 10500 gtctactatc aacttcaatt catgtagaca ttcgaagtat gccatttcgg gagcgtaccc 10560 tgcttcgaca agcgtctcaa agcctgcttt aaccaattca acagttcctc cgcacagaac 10620 cgcttgttct ccaaataaat ctgtctcagt ctcgtcttta aaagtggttt ctattatacc 10680 cgttctcccg ccaccaactc ctgctgcgta gcttaaagct acattcttag cgtttccgct 10740 tgcgtcttgg tatatagcga tcaaatctgg aataccacca cccttaacaa attcgctcct 10800 aacagtatgc cccggagcct taggtgcaat cataataacg tccaaatctg ccctggggac 10860 tacttgattg taatgaatgg caaatccatg actgaaggcc aaggtagcgc ccttcttaat 10920 gtttggttct atttcatttt tgtacaattg cgattgaaat tcatctggcg ttaaaatcat 10980 gactaaatca gcgccggcaa cagccgctgc aacatctgtg actttcaagc catgtgcttc 11040 agcctttgca acggtagcac taccttttct cagacctact gtcacgtcga ccccagaatc 11100 tttcaagtta caggcttgtg cgtgtccttg ggaaccatat cctataatag caaccttctt 11160 tccctggatg atgctcagat cgcagtcttt atcgtaaaac accttcatgt tttatttttt 11220 acttatattg ctggtagggt aaaaaaatat aactcctagg aataggttgt ctatatgttt 11280 ttgtcttgct tctataattg taacaaacaa ggaaagggaa aatactgggt gtaaaagcca 11340 ttgagtcaag ttaggtcatc ccttttatac aaaatttttc aatttttttt ccaagattct 11400 tgtacgatta attatttttt ttttgcgtcc tacagcgtga tgaaaatttc cgcctgctgc 11460 aagatgagcg ggaacgggcg aaatgtgcac gcgcacaact tacgaaacgc ggatgagtca 11520 ctgacagcca ccgcagaggt tctgactcct actgagctct attggaggtg gcagaaccgg 11580 taccggagga gaccgctata accggtttga atttattgtc acagtgtcac atcagcggca 11640 actcagaagt ttgacagcaa gcaagttcat cattcgaact agccttattg ttttagttca 11700 gtgacagcga actgccgtac tcgatgcttt atttctcacg gtagagcgga agaacagata 11760 ggggcagcgt gagaagagtt agaaagtaaa tttttatcac gtctgaagta ttcttattca 11820 taggaaattt tgcaaggttt tttagctcaa taacgggcta agttatataa ggtgttcacg 11880 cgattttctt gttatgtata cctcttctct gaggaatggt actactgtcc tgatgtaggc 11940 tccttaaatt ggtgggcaag aataacttat cgatattttg tatattggtc ttggagttca 12000 ccacgtaatg cctgtttaag accatcagtt aactctagta ttatttggtc ttggctactg 12060 gccgtttgct attattcaag tcttttgtgc cttcccgtcg ggtaagggag ttatttaggg 12120 atacagaatc taacgaaaac taaatctcaa tgattaactc catttaatcc ttttttgaaa 12180 ggcaaaagag gtcccttgtt cacttacaac gttcttagcc aaattcgctt atcacttact 12240 acttcacgat atacagaagt aaaaacatat aaaaagatgt ctgtttgttt agccatcaca 12300 aaaggtatcg cagtttcttc tataggcctc tactctggtc ttttggcttc cgcttcattg 12360 attacatcta ctactccact agaggtttta acaggatctc taaaaacatc gatatcgtct 12420 ctgcgttcca atcctacggt gaatatattt ccaagcaatt cactgaagaa gaaagagaag 12480 atgttgtgga acatgcatgc ccaggtcctg gttcttgtgg tggtatgtat actgccaaca 12540 caatggcttc tgccgctgaa gtgctaggtt tgaccattcc aaactcctct tccttcccag 12600 ccgtttccaa ggagaagtta gctgagtgtg acaacattgg tgaatacatc aagaagacaa 12660 tggaattggg tattttacct cgtgatatcc tcacaaaaga ggcttttgaa aacgccatta 12720 cttatgtcgt tgcaaccggt gggtccacta atgctgtttt gcatttggtg gctgttgctc 12780 actctgcggg tgtcaagttg tcaccagatg atttccaaag aatcagtgat actacaccat 12840 tgatcggtga cttcaaacct tctggtaaat acgtcatggc cgatttgatt aacgttggtg 12900 gtacccaatc tgtgattaag tatctatatg aaaacaacat gttgcacggt aacacaatga 12960 ctgttaccgg tgacactttg gcagaacgtg caaagaaagc accaagccta cctgaaggac 13020 aagagattat taagccactc tcccacccaa tcaaggccaa cggtcacttg caaattctgt 13080 acggttcatt ggcaccaggt ggagctgtgg gtaaaattac cggtaaggaa ggtacttact 13140 tcaagggtag agcacgtgtg ttcgaagagg aaggtgcctt tattgaagcc ttggaaagag 13200 gtgaaatcaa gaagggtgaa aaaaccgttg ttgttatcag atatgaaggt ccaagaggtg 13260 caccaggtat gcctgaaatg ctaaagcctt cctctgctct gatgggttac ggtttgggta 13320 aagatgttgc attgttgact gatggtagat tctctggtgg ttctcacggg ttcttaatcg 13380 gccacattgt tcccgaagcc gctgaaggtg gtcctatcgg gttggtcaga gacggcgatg 13440 agattatcat tgatgctgat aataacaaga ttgacctatt agtctctgat aaggaaatgg 13500 ctcaacgtaa acaaagttgg gttgcacctc cacctcgtta cacaagaggt actctatcca 13560 agtatgctaa gttggtttcc aacgcttcca acggttgtgt tttagatgct tgattaatta 13620 agagtaagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat 13680 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag 13740 taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga 13800 ccacacctct accggcatgc cgagcaaatg cctgcaaatc gctccccatt tcacccaatt 13860 gtagatatgc taactccagc aatgagttga tgaatctcgg tgtgtatttt atgtcctcag 13920 aggacaacac ctgtggtact agttctagag cggccgcccg caaattaaag ccttcgagcg 13980 tcccaaaacc ttctcaagca aggttttcag tataatgtta catgcgtaca cgcgtctgta 14040 cagaaaaaaa agaaaaattt gaaatataaa taacgttctt aatactaaca taactataaa 14100 aaaataaata gggacctaga cttcaggttg tctaactcct tccttttcgg ttagagcgga 14160 tgtgggggga gggcgtgaat gtaagcgtga cataactaat tacatgatta attaactaga 14220 gagctttcgt tttcatgagt tccccgaatt ctttcggaag cttgtcactt gctaaattaa 14280 tgttatcact gtagtcaacc gggacatcga tgatgacagg accttcagcg ttcatgcctt 14340 gacgcagaac atctgccagc tggtctggtg attctacgcg caagccagtt gctccgaagc 14400 tttccgcata tttcacgata tcgatatttc cgaaatcgac cgcagatgta cggttatatt 14460 ttttcaattg ctggaatgca accatgtcat atgtgctgtc gttccataca atgtgtacaa 14520 ttggtgcttt tagtcgaact gctgtctcta attccattgc tgagaataag aaaccgccgt 14580 caccagagac agaaaccact ttttctcccg gtttcaccaa tgaagcgccg attgcccaag 14640 gaagcgcaac gccgagtgtt tgcataccgt tactgatcat taatgttaac ggctcgtagc 14700 tgcggaaata acgtgacatc caaatggcgt gcgaaccgat atcgcaagtt actgtaacat 14760 gatcatcgac tgcattacgc aactctttaa cgatttcaag agggtgcgct ctgtctgatt 14820 tccaatctgc aggcacctgc tcaccttcat gcatatattg ttttaaatca gaaaggattt 14880 tctgctcacg ctctgcaaat tccactttca cagcatcgtg ttcgatatga ttgatcgtgg 14940 acggaatgtc accgatcaat tcaagatcag gctggtaagc atgatcaatg tcagcgataa 15000 tctcgtctaa atggataatt gtccggtctc cattgatatt ccagaatttc ggatcatatt 15060 caatcgggtc atagccgatc gtcagaacaa catctgcctg ctctagcagt aaatcgccag 15120 gctggttgcg gaacaaaccg atacggccaa aatattgatc ctctaaatct ctagaaaggg 15180 taccggcagc ttgatatgtt tcaacaaatg gaagctgaac ctttttcaaa agcttgcgaa 15240 ccgctttaat tgcttccggt cttccgcctt tcatgccgac caaaacgaca ggaagttttg 15300 ctgtttggat ttttgctatg gccgcactga ttgcatcatc tgctgcagga ccgagttttg 15360 gcgctgcaac agcacgcacg tttttcgtat ttgtgacttc attcacaaca tcttgcggaa 15420 agctcacaaa agcggcccca gcctgccctg ctgacgctat cctaaatgca tttgtaacag 15480 cttccggtat attttttaca tcttgaactt ctacactgta ttttgtaatc ggctggaata 15540 gcgccgcatt atccaaagat tgatgtgtcc gttttaaacg atctgcacgg atcacgtttc 15600 cagcaagcgc aacgacaggg tctccttcag tgttcgctgt cagcaggcct gttgccaagt 15660 tagaggcacc cggtcctgat gtgactaaca cgactcccgg ttttccagtt aaacggccga 15720 ctgcttgggc catgaatgct gcgttttgtt cgtgccgggc aacgataatt tcaggtcctt 15780 tatcttgtaa agcgtcaaat accgcatcaa tttttgcacc tggaatgcca aatacatgtg 15840 tgacaccttg ctccactaag caatcaacaa caagctccgc ccctctgttt ttcacaaggg 15900 atttttgttc ttttgttgct tttgtcaaca tcctcagcga tgattgattg attgattgta 15960 cagtttgttt ttcttaatat ctatttcgat gacttctata tgatattgca ctaacaagaa 16020 gatattataa tgcaattgat acaagacaag gagttatttg cttctctttt atatgattct 16080 gacaatccat attgcgttgg tagtcttttt tgctggaacg gttcagcgga aaagacgcat 16140 cgctcttttt gcttctagaa gaaatgccag caaaagaatc tcttgacagt gactgacagc 16200 aaaaatgtct ttttctaact agtaacaagg ctaagatatc agcctgaaat aaagggtggt 16260 gaagtaataa ttaaatcatc cgtataaacc tatacacata tatgaggaaa aataatacaa 16320 aagtgtttta aatacagata catacatgaa catatgcacg tatagcgccc aaatgtcggt 16380 aatggga 16387 <210> 131 <211> 1188 <212> DNA <213> Saccharomyces cerevisiae <400> 131 atgttgagaa ctcaagccgc cagattgatc tgcaactccc gtgtcatcac tgctaagaga 60 acctttgctt tggccacccg tgctgctgct tacagcagac cagctgcccg tttcgttaag 120 ccaatgatca ctacccgtgg tttgaagcaa atcaacttcg gtggtactgt tgaaaccgtc 180 tacgaaagag ctgactggcc aagagaaaag ttgttggact acttcaagaa cgacactttt 240 gctttgatcg gttacggttc ccaaggttac ggtcaaggtt tgaacttgag agacaacggt 300 ttgaacgtta tcattggtgt ccgtaaagat ggtgcttctt ggaaggctgc catcgaagac 360 ggttgggttc caggcaagaa cttgttcact gttgaagatg ctatcaagag aggtagttac 420 gttatgaact tgttgtccga tgccgctcaa tcagaaacct ggcctgctat caagccattg 480 ttgaccaagg gtaagacttt gtacttctcc cacggtttct ccccagtctt caaggacttg 540 actcacgttg aaccaccaaa ggacttagat gttatcttgg ttgctccaaa gggttccggt 600 agaactgtca gatctttgtt caaggaaggt cgtggtatta actcttctta cgccgtctgg 660 aacgatgtca ccggtaaggc tcacgaaaag gcccaagctt tggccgttgc cattggttcc 720 ggttacgttt accaaaccac tttcgaaaga gaagtcaact ctgacttgta cggtgaaaga 780 ggttgtttaa tgggtggtat ccacggtatg ttcttggctc aatacgacgt cttgagagaa 840 aacggtcact ccccatctga agctttcaac gaaaccgtcg aagaagctac ccaatctcta 900 tacccattga tcggtaagta cggtatggat tacatgtacg atgcttgttc caccaccgcc 960 agaagaggtg ctttggactg gtacccaatc ttcaagaatg ctttgaagcc tgttttccaa 1020 gacttgtacg aatctaccaa gaacggtacc gaaaccaaga gatctttgga attcaactct 1080 caacctgact acagagaaaa gctagaaaag gaattagaca ccatcagaaa catggaaatc 1140 tggaaggttg gtaaggaagt cagaaagttg agaccagaaa accaataa 1188 <210> 132 <211> 1014 <212> DNA <213> Pseudomonas fluorescens <400> 132 atgaaagttt tctacgataa agactgcgac ctgtcgatca tccaaggtaa gaaagttgcc 60 atcatcggct acggttccca gggccacgct caagcatgca acctgaagga ttccggcgta 120 gacgtgactg ttggcctgcg taaaggctcg gctaccgttg ccaaggctga agcccacggc 180 ttgaaagtga ccgacgttgc tgcagccgtt gccggtgccg acttggtcat gatcctgacc 240 ccggacgagt tccagtccca gctgtacaag aacgaaatcg agccgaacat caagaagggc 300 gccactctgg ccttctccca cggcttcgcg atccactaca accaggttgt gcctcgtgcc 360 gacctcgacg tgatcatgat cgcgccgaag gctccaggcc acaccgtacg ttccgagttc 420 gtcaagggcg gtggtattcc tgacctgatc gcgatctacc aggacgcttc cggcaacgcc 480 aagaacgttg ccctgtccta cgccgcaggc gtgggcggcg gccgtaccgg catcatcgaa 540 accaccttca aggacgagac tgaaaccgac ctgttcggtg agcaggctgt tctgtgtggc 600 ggtaccgtcg agctggtcaa agccggtttc gaaaccctgg ttgaagctgg ctacgctcca 660 gaaatggcct acttcgagtg cctgcacgaa ctgaagctga tcgttgacct catgtacgaa 720 ggcggtatcg ccaacatgaa ctactcgatc tccaacaacg ctgaatacgg cgagtacgtg 780 actggtccag aagtcatcaa cgccgaatcc cgtcaggcca tgcgcaatgc tctgaagcgc 840 atccaggacg gcgaatacgc gaagatgttc atcagcgaag gcgctaccgg ctacccatcg 900 atgaccgcca agcgtcgtaa caacgctgct cacggtatcg aaatcatcgg cgagcaactg 960 cgctcgatga tgccttggat cggtgccaac aaaatcgtcg acaaagccaa gaac 1014 <210> 133 <211> 250 <212> DNA <213> Saccharomyces cerevisiae <400> 133 ccgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 60 ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 120 cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 180 ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 240 aattacatga 250 <210> 134 <211> 1181 <212> DNA <213> Saccharomyces cerevisiae <400> 134 taaaacctct agtggagtag tagatgtaat caatgaagcg gaagccaaaa gaccagagta 60 gaggcctata gaagaaactg cgataccttt tgtgatggct aaacaaacag acatcttttt 120 atatgttttt acttctgtat atcgtgaagt agtaagtgat aagcgaattt ggctaagaac 180 gttgtaagtg aacaagggac ctcttttgcc tttcaaaaaa ggattaaatg gagttaatca 240 ttgagattta gttttcgtta gattctgtat ccctaaataa ctcccttacc cgacgggaag 300 gcacaaaaga cttgaataat agcaaacggc cagtagccaa gaccaaataa tactagagtt 360 aactgatggt cttaaacagg cattacgtgg tgaactccaa gaccaatata caaaatatcg 420 ataagttatt cttgcccacc aatttaagga gcctacatca ggacagtagt accattcctc 480 agagaagagg tatacataac aagaaaatcg cgtgaacacc ttatataact tagcccgtta 540 ttgagctaaa aaaccttgca aaatttccta tgaataagaa tacttcagac gtgataaaaa 600 tttactttct aactcttctc acgctgcccc tatctgttct tccgctctac cgtgagaaat 660 aaagcatcga gtacggcagt tcgctgtcac tgaactaaaa caataaggct agttcgaatg 720 atgaacttgc ttgctgtcaa acttctgagt tgccgctgat gtgacactgt gacaataaat 780 tcaaaccggt tatagcggtc tcctccggta ccggttctgc cacctccaat agagctcagt 840 aggagtcaga acctctgcgg tggctgtcag tgactcatcc gcgtttcgta agttgtgcgc 900 gtgcacattt cgcccgttcc cgctcatctt gcagcaggcg gaaattttca tcacgctgta 960 ggacgcaaaa aaaaaataat taatcgtaca agaatcttgg aaaaaaaatt gaaaaatttt 1020 gtataaaagg gatgacctaa cttgactcaa tggcttttac acccagtatt ttccctttcc 1080 ttgtttgtta caattataga agcaagacaa aaacatatag acaacctatt cctaggagtt 1140 atattttttt accctaccag caatataagt aaaaaactag t 1181 <210> 135 <211> 759 <212> DNA <213> Saccharomyces cerevisiae <400> 135 ggccctgcag gcctatcaag tgctggaaac tttttctctt ggaatttttg caacatcaag 60 tcatagtcaa ttgaattgac ccaatttcac atttaagatt tttttttttt catccgacat 120 acatctgtac actaggaagc cctgtttttc tgaagcagct tcaaatatat atatttttta 180 catatttatt atgattcaat gaacaatcta attaaatcga aaacaagaac cgaaacgcga 240 ataaataatt tatttagatg gtgacaagtg tataagtcct catcgggaca gctacgattt 300 ctctttcggt tttggctgag ctactggttg ctgtgacgca gcggcattag cgcggcgtta 360 tgagctaccc tcgtggcctg aaagatggcg ggaataaagc ggaactaaaa attactgact 420 gagccatatt gaggtcaatt tgtcaactcg tcaagtcacg tttggtggac ggcccctttc 480 caacgaatcg tatatactaa catgcgcgcg cttcctatat acacatatac atatatatat 540 atatatatat gtgtgcgtgt atgtgtacac ctgtatttaa tttccttact cgcgggtttt 600 tcttttttct caattcttgg cttcctcttt ctcgagtata taatttttca ggtaaaattt 660 agtacgatag taaaatactt ctcgaactcg tcacatatac gtgtacataa tgtctgaacc 720 agctcaaaag aaacaaaagg ttgctaacaa ctctctaga 759 <210> 136 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 136 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 137 <211> 1716 <212> DNA <213> Bacillus subtilis <400> 137 atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60 gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120 attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180 gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240 gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300 actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360 cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420 gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480 gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540 aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600 atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660 aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720 ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780 tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840 gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900 ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960 cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020 gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080 catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140 gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200 cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260 aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320 ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380 ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440 tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500 ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560 tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620 atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680 gaattcgggg aactcatgaa aacgaaagct ctctag 1716 <210> 138 <211> 571 <212> PRT <213> Bacillus subtilis <400> 138 Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg 1 5 10 15 Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His 20 25 30 Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu 35 40 45 Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala 50 55 60 Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val 65 70 75 80 Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu 85 90 95 Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn 100 105 110 Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn 115 120 125 Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp 130 135 140 Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser 145 150 155 160 Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val 165 170 175 Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys 180 185 190 Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile 195 200 205 Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg 210 215 220 Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu 225 230 235 240 Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu 245 250 255 Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly 260 265 270 Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp 275 280 285 Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr 290 295 300 Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln 305 310 315 320 Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile 325 330 335 Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile 340 345 350 Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala 355 360 365 Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu 370 375 380 Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser 385 390 395 400 His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr 405 410 415 Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp 420 425 430 Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val 435 440 445 Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala 450 455 460 Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr 465 470 475 480 Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser 485 490 495 Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe 500 505 510 Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val 515 520 525 Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro 530 535 540 Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys 545 550 555 560 Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu 565 570 <210> 139 <211> 448 <212> DNA <213> Saccharomyces cerevisiae <400> 139 cccattaccg acatttgggc gctatacgtg catatgttca tgtatgtatc tgtatttaaa 60 acacttttgt attatttttc ctcatatatg tgtataggtt tatacggatg atttaattat 120 tacttcacca ccctttattt caggctgata tcttagcctt gttactagtt agaaaaagac 180 atttttgctg tcagtcactg tcaagagatt cttttgctgg catttcttct agaagcaaaa 240 agagcgatgc gtcttttccg ctgaaccgtt ccagcaaaaa agactaccaa cgcaatatgg 300 attgtcagaa tcatataaaa gagaagcaaa taactccttg tcttgtatca attgcattat 360 aatatcttct tgttagtgca atatcatata gaagtcatcg aaatagatat taagaaaaac 420 aaactgtaca atcaatcaat caatcatc 448 <210> 140 <211> 16387 <212> DNA <213> artificial sequence <220> <223> plasmid construct <400> 140 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt cagcttccat ttgttgaaac atatcaagct 1200 gccggtaccc tttctagaga tttagaggat caatattttg gccgtatcgg tttgttccgc 1260 aaccagcctg gcgatttact gctagagcag gcagatgttg ttctgacgat cggctatgac 1320 ccgattgaat atgatccgaa attctggaat atcaatggag accggacaat tatccattta 1380 gacgagatta tcgctgacat tgatcatgct taccagcctg atcttgaatt gatcggtgac 1440 attccgtcca cgatcaatca tatcgaacac gatgctgtga aagtggaatt tgcagagcgt 1500 gagcagaaaa tcctttctga tttaaaacaa tatatgcatg aaggtgagca ggtgcctgca 1560 gattggaaat cagacagagc gcaccctctt gaaatcgtta aagagttgcg taatgcagtc 1620 gatgatcatg ttacagtaac ttgcgatatc ggttcgcacg ccatttggat gtcacgttat 1680 ttccgcagct acgagccgtt aacattaatg atcagtaacg gtatgcaaac actcggcgtt 1740 gcgcttcctt gggcaatcgg cgcttcattg gtgaaaccgg gagaaaaagt ggtttctgtc 1800 tctggtgacg gcggtttctt attctcagca atggaattag agacagcagt tcgactaaaa 1860 gcaccaattg tacacattgt atggaacgac agcacatatg acatggttgc attccagcaa 1920 ttgaaaaaat ataaccgtac atctgcggtc gatttcggaa atatcgatat cgtgaaatat 1980 gcggaaagct tcggagcaac tggcttgcgc gtagaatcac cagaccagct ggcagatgtt 2040 ctgcgtcaag gcatgaacgc tgaaggtcct gtcatcatcg atgtcccggt tgactacagt 2100 gataacatta atttagcaag tgacaagctt ccgaaagaat tcggggaact catgaaaacg 2160 aaagctctct agttaattaa tcatgtaatt agttatgtca cgcttacatt cacgccctcc 2220 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 2280 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt 2340 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt 2400 tttgggacgc tcgaaggctt taatttgcgg gcggccgctc tagaactagt accacaggtg 2460 ttgtcctctg aggacataaa atacacaccg agattcatca actcattgct ggagttagca 2520 tatctacaat tgggtgaaat ggggagcgat ttgcaggcat ttgctcggca tgccggtaga 2580 ggtgtggtca ataagagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa 2640 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta 2700 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc 2760 ttactcttaa ttaatcaagc atctaaaaca caaccgttgg aagcgttgga aaccaactta 2820 gcatacttgg atagagtacc tcttgtgtaa cgaggtggag gtgcaaccca actttgttta 2880 cgttgagcca tttccttatc agagactaat aggtcaatct tgttattatc agcatcaatg 2940 ataatctcat cgccgtctct gaccaacccg ataggaccac cttcagcggc ttcgggaaca 3000 atgtggccga ttaagaaccc gtgagaacca ccagagaatc taccatcagt caacaatgca 3060 acatctttac ccaaaccgta acccatcaga gcagaggaag gctttagcat ttcaggcata 3120 cctggtgcac ctcttggacc ttcatatctg ataacaacaa cggttttttc acccttcttg 3180 atttcacctc tttccaaggc ttcaataaag gcaccttcct cttcgaacac acgtgctcta 3240 cccttgaagt aagtaccttc cttaccggta attttaccca cagctccacc tggtgccaat 3300 gaaccgtaca gaatttgcaa gtgaccgttg gccttgattg ggtgggagag tggcttaata 3360 atctcttgtc cttcaggtag gcttggtgct ttctttgcac gttctgccaa agtgtcaccg 3420 gtaacagtca ttgtgttacc gtgcaacatg ttgttttcat atagatactt aatcacagat 3480 tgggtaccac caacgttaat caaatcggcc atgacgtatt taccagaagg tttgaagtca 3540 ccgatcaatg gtgtagtatc actgattctt tggaaatcat ctggtgacaa cttgacaccc 3600 gcagagtgag caacagccac caaatgcaaa acagcattag tggacccacc ggttgcaacg 3660 acataagtaa tggcgttttc aaaagcctct tttgtgagga tatcacgagg taaaataccc 3720 aattccattg tcttcttgat gtattcacca atgttgtcac actcagctaa cttctccttg 3780 gaaacggctg ggaaggaaga ggagtttgga atggtcaaac ctagcacttc agcggcagaa 3840 gccattgtgt tggcagtata cataccacca caagaaccag gacctgggca tgcatgttcc 3900 acaacatctt ctctttcttc ttcagtgaat tgcttggaaa tatattcacc gtaggattgg 3960 aacgcagaga cgatatcgat gtttttagag atcctgttaa aacctctagt ggagtagtag 4020 atgtaatcaa tgaagcggaa gccaaaagac cagagtagag gcctatagaa gaaactgcga 4080 taccttttgt gatggctaaa caaacagaca tctttttata tgtttttact tctgtatatc 4140 gtgaagtagt aagtgataag cgaatttggc taagaacgtt gtaagtgaac aagggacctc 4200 ttttgccttt caaaaaagga ttaaatggag ttaatcattg agatttagtt ttcgttagat 4260 tctgtatccc taaataactc ccttacccga cgggaaggca caaaagactt gaataatagc 4320 aaacggccag tagccaagac caaataatac tagagttaac tgatggtctt aaacaggcat 4380 tacgtggtga actccaagac caatatacaa aatatcgata agttattctt gcccaccaat 4440 ttaaggagcc tacatcagga cagtagtacc attcctcaga gaagaggtat acataacaag 4500 aaaatcgcgt gaacacctta tataacttag cccgttattg agctaaaaaa ccttgcaaaa 4560 tttcctatga ataagaatac ttcagacgtg ataaaaattt actttctaac tcttctcacg 4620 ctgcccctat ctgttcttcc gctctaccgt gagaaataaa gcatcgagta cggcagttcg 4680 ctgtcactga actaaaacaa taaggctagt tcgaatgatg aacttgcttg ctgtcaaact 4740 tctgagttgc cgctgatgtg acactgtgac aataaattca aaccggttat agcggtctcc 4800 tccggtaccg gttctgccac ctccaataga gctcagtagg agtcagaacc tctgcggtgg 4860 ctgtcagtga ctcatccgcg tttcgtaagt tgtgcgcgtg cacatttcgc ccgttcccgc 4920 tcatcttgca gcaggcggaa attttcatca cgctgtagga cgcaaaaaaa aaataattaa 4980 tcgtacaaga atcttggaaa aaaaattgaa aaattttgta taaaagggat gacctaactt 5040 gactcaatgg cttttacacc cagtattttc cctttccttg tttgttacaa ttatagaagc 5100 aagacaaaaa catatagaca acctattcct aggagttata tttttttacc ctaccagcaa 5160 tataagtaaa aaactagtat gaaggtgttt tacgataaag actgcgatct gagcatcatc 5220 cagggaaaga aggttgctat tataggatat ggttcccaag gacacgcaca agccttgaac 5280 ttgaaagatt ctggggtcga cgtgacagta ggtctgtata aaggtgctgc tgatgcagca 5340 aaggctgaag cacatggctt taaagtcaca gatgttgcag cggctgttgc tggcgctgat 5400 ttagtcatga ttttaattcc agatgaattt caatcgcaat tgtacaaaaa tgaaatagaa 5460 ccaaacatta agaagggcgc taccttggcc ttcagtcatg gatttgccat tcattacaat 5520 caagtagtcc ccagggcaga tttggacgtt attatgattg cacctaaggc tccggggcat 5580 actgttagga gcgaatttgt taagggtggt ggtattccag atttgatcgc tatataccaa 5640 gacgttagcg gaaacgctaa gaatgtagct ttaagctacg cagcaggagt tggtggcggg 5700 agaacgggta taatagaaac cacttttaaa gacgagactg agacagattt atttggagaa 5760 caagcggttc tgtgcggagg aactgttgaa ttggttaaag caggctttga gacgcttgtc 5820 gaagcagggt acgctcccga aatggcatac ttcgaatgtc tacatgaatt gaagttgata 5880 gtagacttaa tgtatgaagg tggtatagct aatatgaact attccatttc aaataatgca 5940 gaatatggtg agtatgtcac cggacctgaa gtcattaacg cagaatcaag acaagccatg 6000 agaaatgcct tgaaacgtat ccaggacggt gaatacgcta agatgttcat aagtgaaggc 6060 gctacgggtt acccgagtat gactgctaaa agaagaaaca atgcagcaca tggtatcgaa 6120 attattggtg aacagttaag gtctatgatg ccctggatcg gtgctaataa gatcgtagac 6180 aaggcgaaaa attaaggccc tgcaggccta tcaagtgctg gaaacttttt ctcttggaat 6240 ttttgcaaca tcaagtcata gtcaattgaa ttgacccaat ttcacattta agattttttt 6300 tttttcatcc gacatacatc tgtacactag gaagccctgt ttttctgaag cagcttcaaa 6360 tatatatatt ttttacatat ttattatgat tcaatgaaca atctaattaa atcgaaaaca 6420 agaaccgaaa cgcgaataaa taatttattt agatggtgac aagtgtataa gtcctcatcg 6480 ggacagctac gatttctctt tcggttttgg ctgagctact ggttgctgtg acgcagcggc 6540 attagcgcgg cgttatgagc taccctcgtg gcctgaaaga tggcgggaat aaagcggaac 6600 taaaaattac tgactgagcc atattgaggt caatttgtca actcgtcaag tcacgtttgg 6660 tggacggccc ctttccaacg aatcgtatat actaacatgc gcgcgcttcc tatatacaca 6720 tatacatata tatatatata tatatgtgtg cgtgtatgtg tacacctgta tttaatttcc 6780 ttactcgcgg gtttttcttt tttctcaatt cttggcttcc tctttctcga gtatataatt 6840 tttcaggtaa aatttagtac gatagtaaaa tacttctcga actcgtcaca tatacgtgta 6900 cataatgtct gaaccagctc aaaagaaaca aaaggttgct aacaactctc tagagcggcc 6960 gcccgcaaat taaagccttc gagcgtccca aaaccttctc aagcaaggtt ttcagtataa 7020 tgttacatgc gtacacgcgt ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg 7080 ttcttaatac taacataact ataaaaaaat aaatagggac ctagacttca ggttgtctaa 7140 ctccttcctt ttcggttaga gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa 7200 ctaattacat gattaattaa ttattggttt tctggtctca actttctgac ttccttacca 7260 accttccaga tttccatgtt tctgatggtg tctaattcct tttctagctt ttctctgtag 7320 tcaggttgag agttgaattc caaagatctc ttggtttcgg taccgttctt ggtagattcg 7380 tacaagtctt ggaaaacagg cttcaaagca ttcttgaaga ttgggtacca gtccaaagca 7440 cctcttctgg cggtggtgga acaagcatcg tacatgtaat ccataccgta cttaccgatc 7500 aatgggtata gagattgggt agcttcttcg acggtttcgt tgaaagcttc agatggggag 7560 tgaccgtttt ctctcaagac gtcgtattga gccaagaaca taccgtggat accacccatt 7620 aaacaacctc tttcaccgta caagtcagag ttgacttctc tttcgaaagt ggtttggtaa 7680 acgtaaccgg aaccaatggc aacggccaaa gcttgggcct tttcgtgagc cttaccggtg 7740 acatcgttcc agacggcgta agaagagtta ataccacgac cttccttgaa caaagatctg 7800 acagttctac cggaaccctt tggagcaacc aagataacat ctaagtcctt tggtggttca 7860 acgtgagtca agtccttgaa gactggggag aaaccgtggg agaagtacaa agtcttaccc 7920 ttggtcaaca atggcttgat agcaggccag gtttctgatt gagcggcatc ggacaacaag 7980 ttcataacgt aactacctct cttgatagca tcttcaacag tgaacaagtt cttgcctgga 8040 acccaaccgt cttcgatggc agccttccaa gaagcaccat ctttacggac accaatgata 8100 acgttcaaac cgttgtctct caagttcaaa ccttgaccgt aaccttggga accgtaaccg 8160 atcaaagcaa aagtgtcgtt cttgaagtag tccaacaact tttctcttgg ccagtcagct 8220 ctttcgtaga cggtttcaac agtaccaccg aagttgattt gcttcaacat cctcagctct 8280 agatttgaat atgtattact tggttatggt tatatatgac aaaagaaaaa gaagaacaga 8340 agaataacgc aaggaagaac aataactgaa attgatagag aagtattatg tctttgtctt 8400 tttataataa atcaagtgca gaaatccgtt agacaacatg agggataaaa tttaacgtgg 8460 gcgaagaaga aggaaaaaag tttttgtgag ggcgtaattg aagcgatctg ttgattgtag 8520 attttttttt tttgaggagt caaagtcaga agagaacaga caaatggtat taaccatcca 8580 atactttttt ggagcaacgc taagctcatg cttttccatt ggttacgtgc tcagttgtta 8640 gatatggaaa gagaggatgc tcacggcagc gtgactccaa ttgagcccga aagagaggat 8700 gccacgtttt cccgacggct gctagaatgg aaaaaggaaa aatagaagaa tcccattcct 8760 atcattattt acgtaatgac ccacacattt ttgagatttt caactattac gtattacgat 8820 aatcctgctg tcattatcat tattatctat atcgacgtat gcaacgtatg tgaagccaag 8880 taggcaatta tttagtactg tcagtattgt tattcatttc agatctatcc gcggtggagc 8940 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 9000 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 9060 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat 9120 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 9180 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 9240 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 9300 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 9360 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 9420 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 9480 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag 9540 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg 9600 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 9660 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 9720 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 9780 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 9840 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9900 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9960 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 10020 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 10080 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 10140 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 10200 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 10260 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 10320 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 10380 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 10440 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 10500 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 10560 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 10620 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 10680 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 10740 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 10800 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 10860 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 10920 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 10980 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 11040 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 11100 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 11160 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 11220 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 11280 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 11340 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 11400 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 11460 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 11520 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 11580 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 11640 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 11700 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 11760 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 11820 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 11880 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 11940 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 12000 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt 12060 ttctttccaa tttttttttt ttcgtcatta taaaaatcat tacgaccgag attcccgggt 12120 aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact 12180 tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc ccagcctgct 12240 tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag tcctcttcca 12300 acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata ctgttgaccc 12360 aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca atcgtaacct 12420 tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc tttgtcgctc 12480 ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc cttgcatgac 12540 aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc cgcctgcttc 12600 aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct 12660 gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat gtcagcaaat 12720 tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag cggcttaact 12780 gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa acaaattttg 12840 ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc caatgaagca 12900 cacaagtttg tttgcttttc gtgcatgata ttaaatagct tggcagcaac aggactagga 12960 tgagtagcag cacgttcctt atatgtagct ttcgacatga tttatcttcg tttcctgcag 13020 gtttttgttc tgtgcagttg ggttaagaat actgggcaat ttcatgtttc ttcaacacta 13080 catatgcgta tatataccaa tctaagtctg tgctccttcc ttcgttcttc cttctgttcg 13140 gagattaccg aatcaaaaaa atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa 13200 aatgatgaat tgaaaagctt gcatgcctgc aggtcgactc tagtatactc cgtctactgt 13260 acgatacact tccgctcagg tccttgtcct ttaacgaggc cttaccactc ttttgttact 13320 ctattgatcc agctcagcaa aggcagtgtg atctaagatt ctatcttcgc gatgtagtaa 13380 aactagctag accgagaaag agactagaaa tgcaaaaggc acttctacaa tggctgccat 13440 cattattatc cgatgtgacg ctgcattttt tttttttttt tttttttttt tttttttttt 13500 tttttttttt tttttttgta caaatatcat aaaaaaagag aatcttttta agcaaggatt 13560 ttcttaactt cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa 13620 tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat ggctttacct 13680 tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata 13740 gggttgacct tattctttgg caaatctgga gcggaaccat ggcatggttc gtacaaacca 13800 aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggag 13860 cctgggataa cggaggcttc atcggagatg atatcaccaa acatgttgct ggtgattata 13920 ataccattta ggtgggttgg gttcttaact aggatcatgg cggcagaatc aatcaattga 13980 tgttgaactt tcaatgtagg gaattcgttc ttgatggttt cctccacagt ttttctccat 14040 aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca 14100 tgttgtaggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat 14160 tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata 14220 cctcccacta attctctaac aacaacgaag tcagtacctt tagcaaattg tggcttgatt 14280 ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat 14340 tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc ggtaccccat 14400 ttaggaccac ccacagcacc taacaaaacg gcatcagcct tcttggaggc ttccagcgcc 14460 tcatctggaa gtggaacacc tgtagcatcg atagcagcac caccaattaa atgattttcg 14520 aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg 14580 gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac 14640 attacaatgg tatatccttg aaatatatat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 14700 tgcagcttct caatgatatt cgaatacgct ttgaggagat acagcctaat atccgacaaa 14760 ctgttttaca gatttacgat cgtacttgtt acccatcatt gaattttgaa catccgaacc 14820 tgggagtttt ccctgaaaca gatagtatat ttgaacctgt ataataatat atagtctagc 14880 gctttacgga agacaatgta tgtatttcgg ttcctggaga aactattgca tctattgcat 14940 aggtaatctt gcacgtcgca tccccggttc attttctgcg tttccatctt gcacttcaat 15000 agcatatctt tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga 15060 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg 15120 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa 15180 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 15240 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa 15300 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg 15360 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa 15420 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg 15480 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta 15540 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat 15600 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat 15660 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa 15720 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag 15780 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat 15840 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt 15900 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg 15960 gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga 16020 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac 16080 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga 16140 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg 16200 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc 16260 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct 16320 catccttcaa tgctatcatt tcctttgata ttggatcata tgcatagtac cgagaaacta 16380 gaggatc 16387 <210> 141 <211> 2237 <212> DNA <213> Artificial Sequence <220> <223> PDC1 Fragment A-ilvDSm <400> 141 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acactttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattatt 2237 <210> 142 <211> 4420 <212> DNA <213> Artificial Sequence <220> <223> PDC1 A-ilvDSm-BUC cassette <400> 142 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acactttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattattagt taaagtttta taagcatttt tatgtaacga aaaataaatt 2280 ggttcatatt attactgcac tgtcacttac catggaaaga ccagacaaga agttgccgac 2340 agtctgttga attggcctgg ttaggcttaa gtctgggtcc gcttctttac aaatttggag 2400 aatttctctt aaacgatatg tatattcttt tcgttggaaa agatgtcttc caaaaaaaaa 2460 accgatgaat tagtggaacc aaggaaaaaa aaagaggtat ccttgattaa ggaacactgt 2520 ttaaacagtg tggtttccaa aaccctgaaa ctgcattagt gtaatagaag actagacacc 2580 tcgatacaaa taatggttac tcaattcaaa actgccagcg aattcgactc tgcaattgct 2640 caagacaagc tagttgtcgt agatttctac gccacttggt gcggtccatg taaaatgatt 2700 gctccaatga ttgaaaaatg tggctgtggt ttcagggtcc ataaagcttt tcaattcatc 2760 tttttttttt ttgttctttt ttttgattcc ggtttctttg aaattttttt gattcggtaa 2820 tctccgagca gaaggaagaa cgaaggaagg agcacagact tagattggta tatatacgca 2880 tatgtggtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg cacagaacaa 2940 aaacctgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa cgtgctgcta 3000 ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag caaacaaact 3060 tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt gaagcattag 3120 gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt tccatggagg 3180 gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc ttcgaagaca 3240 gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt gtatacagaa 3300 tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt attgttagcg 3360 gtttgaagca ggcggcggaa gaagtaacaa aggaacctag aggccttttg atgttagcag 3420 aattgtcatg caagggctcc ctagctactg gagaatatac taagggtact gttgacattg 3480 cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg ggtggaagag 3540 atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac aagggagacg 3600 cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct gacattatta 3660 ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt gaacgttaca 3720 gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa aaaactgtat 3780 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt 3840 tattacccgg gaatctcggt cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa 3900 agaaaaagct tcatggcctt ccactttccc aaacaacacc tacggtatct ctcaagtctt 3960 atggggttcc attggtttca ccactggtgc taccttgggt gctgctttcg ctgctgaaga 4020 aattgatcca aagaagagag ttatcttatt cattggtgac ggttctttgc aattgactgt 4080 tcaagaaatc tccaccatga tcagatgggg cttgaagcca tacttgttcg tcttgaacaa 4140 cgatggttac accattgaaa agttgattca cggtccaaag gctcaataca acgaaattca 4200 aggttgggac cacctatcct tgttgccaac tttcggtgct aaggactatg aaacccacag 4260 agtcgctacc accggtgaat gggacaagtt gacccaagac aagtctttca acgacaactc 4320 taagatcaga atgattgaaa tcatgttgcc agtcttcgat gctccacaaa acttggttga 4380 acaagctaag ttgactgctg ctaccaacgc taagcaataa 4420 <210> 143 <211> 3521 <212> DNA <213> Artificial Sequence <220> <223> PDC5 A-sadB-BUC cassette <400> 143 aaggaaataa agcaaataac aataacacca ttattttaat tttttttcta ttactgtcgc 60 taacacctgt atggttgcaa ccaggtgaga atccttctga tgcatacttt atgcgtttat 120 gcgttttgcg ccccttggaa aaaaattgat tctcatcgta aatgcatact acatgcgttt 180 atgggaaaag cctccatatc caaaggtcgc gtttctttta gaaaaactaa tacgtaaacc 240 tgcattaagg taagattata tcagaaaatg tgttgcaaga aatgcattat gcaatttttt 300 gattatgaca atctctcgaa agaaatttca tatgatgaga cttgaataat gcagcggcgc 360 ttgctaaaag aacttgtata taagagctgc cattctcgat caatatactg tagtaagtcc 420 tttcctctct ttcttattac acttatttca cataatcaat ctcaaagaga acaacacaat 480 acaataacaa gaagaacaaa atgaaagctc tggtttatca cggtgaccac aagatctcgc 540 ttgaagacaa gcccaagccc acccttcaaa agcccacgga tgtagtagta cgggttttga 600 agaccacgat ctgcggcacg gatctcggca tctacaaagg caagaatcca gaggtcgccg 660 acgggcgcat cctgggccat gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca 720 cgcagttcaa gaaaggcgac aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg 780 actactgcaa gaagcagctt tactcccatt gccgcgacgg cgggtggatc ctgggttaca 840 tgatcgatgg cgtgcaggcc gaatacgtcc gcatcccgca tgccgacaac agcctctaca 900 agatccccca gacaattgac gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg 960 gccacgaaat cggcgtccag tatgggaatg tccagccggg cgatgcggtg gctattgtcg 1020 gcgcgggccc cgtcggcatg tccgtactgt tgaccgccca gttctactcc ccctcgacca 1080 tcatcgtgat cgacatggac gagaatcgcc tccagctcgc caaggagctc ggggcaacgc 1140 acaccatcaa ctccggcacg gagaacgttg tcgaagccgt gcataggatt gcggcagagg 1200 gagtcgatgt tgcgatcgag gcggtgggca taccggcgac ttgggacatc tgccaggaga 1260 tcgtcaagcc cggcgcgcac atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg 1320 agattcagaa gctctggatc aagaacctga cgatcaccac gggactggtg aacacgaaca 1380 cgacgcccat gctgatgaag gtcgcctcga ccgacaagct tccgttgaag aagatgatta 1440 cccatcgctt cgagctggcc gagatcgagc acgcctatca ggtattcctc aatggcgcca 1500 aggagaaggc gatgaagatc atcctctcga acgcaggcgc tgcctgagct aattaacata 1560 aaactcatga ttcaacgttt gtgtattttt ttacttttga aggttataga tgtttaggta 1620 aataattggc atagatatag ttttagtata ataaatttct gatttggttt aaaatatcaa 1680 ctattttttt tcacatatgt tcttgtaatt acttttctgt cctgtcttcc aggttaaaga 1740 ttagcttcta atattttagg tggtttatta tttaatttta tgctgattaa tttatttact 1800 tgtttaaacg gccggccaat gtggctgtgg tttcagggtc cataaagctt ttcaattcat 1860 cttttttttt tttgttcttt tttttgattc cggtttcttt gaaatttttt tgattcggta 1920 atctccgagc agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc 1980 atatgtggtg ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca 2040 aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct 2100 actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac 2160 ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta 2220 ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag 2280 ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaagac 2340 agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga 2400 atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc 2460 ggtttgaagc aggcggcgga agaagtaaca aaggaaccta gaggcctttt gatgttagca 2520 gaattgtcat gcaagggctc cctagctact ggagaatata ctaagggtac tgttgacatt 2580 gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga 2640 gatgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagac 2700 gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc tgacattatt 2760 attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac 2820 agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta 2880 ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag 2940 ttattacccg ggaatctcgg tcgtaatgat ttctataatg acgaaaaaaa aaaaattgga 3000 aagaaaaagc ttcatggcct tctactttcc caacagatgt atacgctatc gtccaagtct 3060 tgtggggttc cattggtttc acagtcggcg ctctattggg tgctactatg gccgctgaag 3120 aacttgatcc aaagaagaga gttattttat tcattggtga cggttctcta caattgactg 3180 ttcaagaaat ctctaccatg attagatggg gtttgaagcc atacattttt gtcttgaata 3240 acaacggtta caccattgaa aaattgattc acggtcctca tgccgaatat aatgaaattc 3300 aaggttggga ccacttggcc ttattgccaa cttttggtgc tagaaactac gaaacccaca 3360 gagttgctac cactggtgaa tgggaaaagt tgactcaaga caaggacttc caagacaact 3420 ctaagattag aatgattgaa gttatgttgc cagtctttga tgctccacaa aacttggtta 3480 aacaagctca attgactgcc gctactaacg ctaaacaata a 3521 <210> 144 <211> 2686 <212> DNA <213> Artificial Sequence <220> <223> pUC19 <400> 144 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686 <210> 145 <211> 1685 <212> DNA <213> Artificial Sequence <220> <223> gpd2::loxP-URA3-loxP cassette <400> 145 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag ataacttcgt atagcataca ttatacgaag 120 ttatgcagat tgtactgaga gtgcaccata ccacagcttt tcaattcaat tcatcatttt 180 ttttttattc ttttttttga tttcggtttc tttgaaattt ttttgattcg gtaatctccg 240 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta 300 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct 360 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc 420 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg 480 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca 540 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag 600 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat 660 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag 720 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga 780 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt 840 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga 900 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag 960 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg 1020 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg 1080 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag 1140 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag 1200 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac 1260 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg 1320 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 1380 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 1440 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 1500 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 1560 gataacttcg tatagcatac attatacgaa gttatccagt gatgatacaa cgagttagcc 1620 aaggtgacac tctccccccc cctccccctc tgatctttcc tgttgcctct ttttccccca 1680 accaa 1685                          SEQUENCE LISTING <110> Butamax (TM) Advanced Biofuels   <120> SUPPLEMENTATION OF FATTY ACIDS FOR IMPROVING ALCOHOL PRODUCTIVITY <130> CL4727 <150> US 61 / 368,451 <151> 2010-07-28 <160> 145 <170> PatentIn version 3.5 <210> 1 <211> 11844 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 1 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt cagcttccat ttgttgaaac atatcaagct 1200 gccggtaccc tttctagaga tttagaggat caatattttg gccgtatcgg tttgttccgc 1260 aaccagcctg gcgatttact gctagagcag gcagatgttg ttctgacgat cggctatgac 1320 ccgattgaat atgatccgaa attctggaat atcaatggag accggacaat tatccattta 1380 gacgagatta tcgctgacat tgatcatgct taccagcctg atcttgaatt gatcggtgac 1440 attccgtcca cgatcaatca tatcgaacac gatgctgtga aagtggaatt tgcagagcgt 1500 gagcagaaaa tcctttctga tttaaaacaa tatatgcatg aaggtgagca ggtgcctgca 1560 gattggaaat cagacagagc gcaccctctt gaaatcgtta aagagttgcg taatgcagtc 1620 gatgatcatg ttacagtaac ttgcgatatc ggttcgcacg ccatttggat gtcacgttat 1680 ttccgcagct acgagccgtt aacattaatg atcagtaacg gtatgcaaac actcggcgtt 1740 gcgcttcctt gggcaatcgg cgcttcattg gtgaaaccgg gagaaaaagt ggtttctgtc 1800 tctggtgacg gcggtttctt attctcagca atggaattag agacagcagt tcgactaaaa 1860 gcaccaattg tacacattgt atggaacgac agcacatatg acatggttgc attccagcaa 1920 ttgaaaaaat ataaccgtac atctgcggtc gatttcggaa atatcgatat cgtgaaatat 1980 gcggaaagct tcggagcaac tggcttgcgc gtagaatcac cagaccagct ggcagatgtt 2040 ctgcgtcaag gcatgaacgc tgaaggtcct gtcatcatcg atgtcccggt tgactacagt 2100 gataacatta atttagcaag tgacaagctt ccgaaagaat tcggggaact catgaaaacg 2160 aaagctctct agttaattaa tcatgtaatt agttatgtca cgcttacatt cacgccctcc 2220 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 2280 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt 2340 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt 2400 tttgggacgc tcgaaggctt taatttgcgg gcggccgcac ctggtaaaac ctctagtgga 2460 gtagtagatg taatcaatga agcggaagcc aaaagaccag agtagaggcc tatagaagaa 2520 actgcgatac cttttgtgat ggctaaacaa acagacatct ttttatatgt ttttacttct 2580 gtatatcgtg aagtagtaag tgataagcga atttggctaa gaacgttgta agtgaacaag 2640 ggacctcttt tgcctttcaa aaaaggatta aatggagtta atcattgaga tttagttttc 2700 gttagattct gtatccctaa ataactccct tacccgacgg gaaggcacaa aagacttgaa 2760 taatagcaaa cggccagtag ccaagaccaa ataatactag agttaactga tggtcttaaa 2820 caggcattac gtggtgaact ccaagaccaa tatacaaaat atcgataagt tattcttgcc 2880 caccaattta aggagcctac atcaggacag tagtaccatt cctcagagaa gaggatataca 2940 taacaagaaa atcgcgtgaa caccttatat aacttagccc gttattgagc taaaaaacct 3000 tgcaaaattt cctatgaata agaatacttc agacgtgata aaaatttact ttctaactct 3060 tctcacgctg cccctatctg ttcttccgct ctaccgtgag aaataaagca tcgagtacgg 3120 cagttcgctg tcactgaact aaaacaataa ggctagttcg aatgatgaac ttgcttgctg 3180 tcaaacttct gagttgccgc tgatgtgaca ctgtgacaat aaattcaaac cggttatagc 3240 ggtctcctcc ggtaccggtt ctgccacctc caatagagct cagtaggagt cagaacctct 3300 gcggtggctg tcagtgactc atccgcgttt cgtaagttgt gcgcgtgcac atttcgcccg 3360 ttcccgctca tcttgcagca ggcggaaatt ttcatcacgc tgtaggacgc aaaaaaaaaa 3420 taattaatcg tacaagaatc ttggaaaaaa aattgaaaaa ttttgtataa aagggatgac 3480 ctaacttgac tcaatggctt ttacacccag tattttccct ttccttgttt gttacaatta 3540 tagaagcaag acaaaaacat atagacaacc tattcctagg agttatattt ttttacccta 3600 ccagcaatat aagtaaaaaa ctgtttaaac agtatggcag ttacaatgta ttatgaagat 3660 gatgtagaag tatcagcact tgctggaaag caaattgcag taatcggtta tggttcacaa 3720 ggacatgctc acgcacagaa tttgcgtgat tctggtcaca acgttatcat tggtgtgcgc 3780 cacggaaaat cttttgataa agcaaaagaa gatggctttg aaacatttga agtaggagaa 3840 gcagtagcta aagctgatgt tattatggtt ttggcaccag atgaacttca acaatccatt 3900 tatgaagagg acatcaaacc aaacttgaaa gcaggttcag cacttggttt tgctcacgga 3960 tttaatatcc attttggcta tattaaagta ccagaagacg ttgacgtctt tatggttgcg 4020 cctaaggctc caggtcacct tgtccgtcgg acttatactg aaggttttgg tacaccagct 4080 ttgtttgttt cacaccaaaa tgcaagtggt catgcgcgtg aaatcgcaat ggattgggcc 4140 aaaggaattg gttgtgctcg agtgggaatt attgaaacaa cttttaaaga agaaacagaa 4200 gaagatttgt ttggagaaca agctgttcta tgtggaggtt tgacagcact tgttgaagcc 4260 ggttttgaaa cactgacaga agctggatac gctggcgaat tggcttactt tgaagttttg 4320 cacgaaatga aattgattgt tgacctcatg tatgaaggtg gttttactaa aatgcgtcaa 4380 tccatctcaa atactgctga gtttggcgat tatgtgactg gtccacggat tattactgac 4440 gaagttaaaa agaatatgaa gcttgttttg gctgatattc aatctggaaa atttgctcaa 4500 gatttcgttg atgacttcaa agcggggcgt ccaaaattaa tagcctatcg cgaagctgca 4560 aaaaatcttg aaattgaaaa aattggggca gagctacgtc aagcaatgcc attcacacaa 4620 tctggtgatg acgatgcctt taaaatctat cagtaaggcc ctgcaggcct atcaagtgct 4680 ggaaactttt tctcttggaa tttttgcaac atcaagtcat agtcaattga attgacccaa 4740 tttcacattt aagatttttt ttttttcatc cgacatacat ctgtacacta ggaagccctg 4800 tttttctgaa gcagcttcaa atatatatat tttttacata tttattatga ttcaatgaac 4860 aatctaatta aatcgaaaac aagaaccgaa acgcgaataa ataatttatt tagatggtga 4920 caagtgtata agtcctcatc gggacagcta cgatttctct ttcggttttg gctgagctac 4980 tggttgctgt gacgcagcgg cattagcgcg gcgttatgag ctaccctcgt ggcctgaaag 5040 atggcgggaa taaagcggaa ctaaaaatta ctgactgagc catattgagg tcaatttgtc 5100 aactcgtcaa gtcacgtttg gtggacggcc cctttccaac gaatcgtata tactaacatg 5160 cgcgcgcttc ctatatacac atatacatat atatatatat atatatgtgt gcgtgtatgt 5220 gtacacctgt atttaatttc cttactcgcg ggtttttctt ttttctcaat tcttggcttc 5280 ctctttctcg agcggaccgg atcctccgcg gtgccggcag atctatttaa atggcgcgcc 5340 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 5400 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 5460 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 5520 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 5580 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 5640 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 5700 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 5760 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 5820 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 5880 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 5940 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 6000 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 6060 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 6120 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 6180 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 6240 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 6300 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 6360 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 6420 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 6480 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 6540 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 6600 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 6660 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 6720 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 6780 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 6840 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 6900 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 6960 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 7020 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 7080 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 7140 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 7200 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 7260 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 7320 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 7380 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 7440 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7500 tgattacgcc aagctttttc tttccaattt tttttttttc gtcattataa aaatcattac 7560 gaccgagatt cccgggtaat aactgatata attaaattga agctctaatt tgtgagttta 7620 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat 7680 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg 7740 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg 7800 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa 7860 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa 7920 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata 7980 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt 8040 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg 8100 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat 8160 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg 8220 cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt 8280 ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac 8340 gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg 8400 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt 8460 atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc 8520 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc 8580 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa 8640 aaaaagaata aaaaaaaaat gatgaattga aaagcttgca tgcctgcagg tcgactctag 8700 tatactccgt ctactgtacg atacacttcc gctcaggtcc ttgtccttta acgaggcctt 8760 accactcttt tgttactcta ttgatccagc tcagcaaagg cagtgtgatc taagattcta 8820 tcttcgcgat gtagtaaaac tagctagacc gagaaagaga ctagaaatgc aaaaggcact 8880 tctacaatgg ctgccatcat tattatccga tgtgacgctg catttttttt tttttttttt 8940 tttttttttt tttttttttt tttttttttt ttttgtacaa atatcataaa aaaagagaat 9000 ctttttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac 9060 tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat 9120 cttcaatggc tttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag 9180 acaagatagt ggcgataggg ttgaccttat tctttggcaa atctggagcg gaaccatggc 9240 atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg 9300 gcaacaaacc caaggagcct gggataacgg aggcttcatc ggagatgata tcaccaaaca 9360 tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg 9420 cagaatcaat caattgatgt tgaactttca atgtagggaa ttcgttcttg atggtttcct 9480 ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa 9540 taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca 9600 cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc 9660 tcttaccaaa gtaaatacct cccactaatt ctctaacaac aacgaagtca gtacctttag 9720 caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc 9780 ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa 9840 cactaccggt accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct 9900 tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata gcagcaccac 9960 caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa 10020 gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga 10080 tcttcttagg ggcagacatt acaatggtat atccttgaaa tatatataaa aaaaaaaaaa 10140 aaaaaaaaaa aaaaaaatgc agcttctcaa tgatattcga atacgctttg aggagataca 10200 gcctaatatc cgacaaactg ttttacagat ttacgatcgt acttgttacc catcattgaa 10260 ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg aacctgtata 10320 ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc ctggagaaac 10380 tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt 10440 ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc attttgtaga 10500 acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 10560 agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 10620 gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10680 tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10740 tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10800 actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10860 gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10920 tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10980 aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 11040 gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 11100 ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 11160 aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 11220 gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 11280 ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 11340 attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 11400 tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 11460 ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 11520 gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 11580 tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 11640 gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11700 tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11760 ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatatgc 11820 atagtaccga gaaactagag gatc 11844 <210> 2 <211> 15539 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1680 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1740 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1800 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1860 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 1980 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 2040 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac 2100 ggcgcgccac tggtagagag cgactttgta tgccccaatt gcgaaacccg cgatatcctt 2160 ctcgattctt tagtacccga ccaggacaag gaaaaggagg tcgaaacgtt tttgaagaaa 2220 caagaggaac tacacggaag ctctaaagat ggcaaccagc cagaaactaa gaaaatgaag 2280 ttgatggatc caactggcac cgctggcttg aacaacaata ccagccttcc aacttctgta 2340 aataacggcg gtacgccagt gccaccagta ccgttacctt tcggtatacc tcctttcccc 2400 atgtttccaa tgcccttcat gcctccaacg gctactatca caaatcctca tcaagctgac 2460 gcaagcccta agaaatgaat aacaatactg acagtactaa ataattgcct acttggcttc 2520 acatacgttg catacgtcga tatagataat aatgataatg acagcaggat tatcgtaata 2580 cgtaatagct gaaaatctca aaaatgtgtg ggtcattacg taaataatga taggaatggg 2640 attcttctat ttttcctttt tccattctag cagccgtcgg gaaaacgtgg catcctctct 2700 ttcgggctca attggagtca cgctgccgtg agcatcctct ctttccatat ctaacaactg 2760 agcacgtaac caatggaaaa gcatgagctt agcgttgctc caaaaaagta ttggatggtt 2820 aataccattt gtctgttctc ttctgacttt gactcctcaa aaaaaaaaat ctacaatcaa 2880 cagatcgctt caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa 2940 ttttatccct catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga 3000 cataatactt ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc 3060 tttttctttt gtcatatata accataacca agtaatacat attcaaacta gtatgactga 3120 caaaaaaact cttaaagact taagaaatcg tagttctgtt tacgattcaa tggttaaatc 3180 acctaatcgt gctatgttgc gtgcaactgg tatgcaagat gaagactttg aaaaacctat 3240 cgtcggtgtc atttcaactt gggctgaaaa cacaccttgt aatatccact tacatgactt 3300 tggtaaacta gccaaagtcg gtgttaagga agctggtgct tggccagttc agttcggaac 3360 aatcacggtt tctgatggaa tcgccatggg aacccaagga atgcgtttct ccttgacatc 3420 tcgtgatatt attgcagatt ctattgaagc agccatggga ggtcataatg cggatgcttt 3480 tgtagccatt ggcggttgtg ataaaaacat gcccggttct gttatcgcta tggctaacat 3540 ggatatccca gccatttttg cttacggcgg aacaattgca cctggtaatt tagacggcaa 3600 agatatcgat ttagtctctg tctttgaagg tgtcggccat tggaaccacg gcgatatgac 3660 caaagaagaa gttaaagctt tggaatgtaa tgcttgtccc ggtcctggag gctgcggtgg 3720 tatgtatact gctaacacaa tggcgacagc tattgaagtt ttgggactta gccttccggg 3780 ttcatcttct cacccggctg aatccgcaga aaagaaagca gatattgaag aagctggtcg 3840 cgctgttgtc aaaatgctcg aaatgggctt aaaaccttct gacattttaa cgcgtgaagc 3900 ttttgaagat gctattactg taactatggc tctgggaggt tcaaccaact caacccttca 3960 cctcttagct attgcccatg ctgctaatgt ggaattgaca cttgatgatt tcaatacttt 4020 ccaagaaaaa gttcctcatt tggctgattt gaaaccttct ggtcaatatg tattccaaga 4080 cctttacaag gtcggagggg taccagcagt tatgaaatat ctccttaaaa atggcttcct 4140 tcatggtgac cgtatcactt gtactggcaa aacagtcgct gaaaatttga aggcttttga 4200 tgatttaaca cctggtcaaa aggttattat gccgcttgaa aatcctaaac gtgaagatgg 4260 tccgctcatt attctccatg gtaacttggc tccagacggt gccgttgcca aagtttctgg 4320 tgtaaaagtg cgtcgtcatg tcggtcctgc taaggtcttt aattctgaag aagaagccat 4380 tgaagctgtc ttgaatgatg atattgttga tggtgatgtt gttgtcgtac gttttgtagg 4440 accaaagggc ggtcctggta tgcctgaaat gctttccctt tcatcaatga ttgttggtaa 4500 agggcaaggt gaaaaagttg cccttctgac agatggccgc ttctcaggtg gtacttatgg 4560 tcttgtcgtg ggtcatatcg ctcctgaagc acaagatggc ggtccaatcg cctacctgca 4620 aacaggagac atagtcacta ttgaccaaga cactaaggaa ttacactttg atatctccga 4680 tgaagagtta aaacatcgtc aagagaccat tgaattgcca ccgctctatt cacgcggtat 4740 ccttggtaaa tatgctcaca tcgtttcgtc tgcttctagg ggagccgtaa cagacttttg 4800 gaagcctgaa gaaactggca aaaaatgttg tcctggttgc tgtggttaag cggccgcgtt 4860 aattcaaatt aattgatata gttttttaat gagtattgaa tctgtttaga aataatggaa 4920 tattattttt atttatttat ttatattatt ggtcggctct tttcttctga aggtcaatga 4980 caaaatgata tgaaggaaat aatgatttct aaaattttac aacgtaagat atttttacaa 5040 aagcctagct catcttttgt catgcactat tttactcacg cttgaaatta acggccagtc 5100 cactgcggag tcatttcaaa gtcatcctaa tcgatctatc gtttttgata gctcattttg 5160 gagttcgcga ttgtcttctg ttattcacaa ctgttttaat ttttatttca ttctggaact 5220 cttcgagttc tttgtaaagt ctttcatagt agcttacttt atcctccaac atatttaact 5280 tcatgtcaat ttcggctctt aaattttcca catcatcaag ttcaacatca tcttttaact 5340 tgaatttatt ctctagctct tccaaccaag cctcattgct ccttgattta ctggtgaaaa 5400 gtgatacact ttgcgcgcaa tccaggtcaa aactttcctg caaagaattc accaatttct 5460 cgacatcata gtacaatttg ttttgttctc ccatcacaat ttaatatacc tgatggattc 5520 ttatgaagcg ctgggtaatg gacgtgtcac tctacttcgc ctttttccct actcctttta 5580 gtacggaaga caatgctaat aaataagagg gtaataataa tattattaat cggcaaaaaa 5640 gattaaacgc caagcgttta attatcagaa agcaaacgtc gtaccaatcc ttgaatgctt 5700 cccaattgta tattaagagt catcacagca acatattctt gttattaaat taattattat 5760 tgatttttga tattgtataa aaaaaccaaa tatgtataaa aaaagtgaat aaaaaatacc 5820 aagtatggag aaatatatta gaagtctata cgttaaacca cccgggcccc ccctcgaggt 5880 cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag 5940 agcggccgct ctagaactag taccacaggt gttgtcctct gaggacataa aatacacacc 6000 gagattcatc aactcattgc tggagttagc atatctacaa ttgggtgaaa tggggagcga 6060 tttgcaggca tttgctcggc atgccggtag aggtgtggtc aataagagcg acctcatgct 6120 atacctgaga aagcaacctg acctacagga aagagttact caagaataag aattttcgtt 6180 ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata acttatttaa 6240 taataaaaat cataaatcat aagaaattcg cttactctta attaatcaaa aagttaaaat 6300 tgtacgaata gattcaccac ttcttaacaa atcaaaccct tcattgattt tctcgaatgg 6360 caatacatgt gtaattaaag gatcaagagc aaacttcttc gccataaagt cggcaacaag 6420 ttttggaaca ctatccttgc tcttaaaacc gccaaatata gctcccttcc atgtacgacc 6480 gcttagcaac agcataggat tcatcgacaa attttgtgaa tcaggaggaa cacctacgat 6540 cacactgact ccatatgcct cttgacagca ggacaacgca gttaccatag tatcaagacg 6600 gcctataact tcaaaagaga aatcaactcc accgtttgac atttcagtaa ggacttcttg 6660 tattggtttc ttataatctt gagggttaac acattcagta gccccgacct ccttagcttt 6720 tgcaaatttg tccttattga tgtctacacc tataatcctc gctgcgcctg cagctttaca 6780 ccccataata acgcttagtc ctactcctcc taaaccgaat actgcacaag tcgaaccctg 6840 tgtaaccttt gcaactttaa ctgcggaacc gtaaccggtg gaaaatccgc accctatcaa 6900 gcaaactttt tccagtggtg aagctgcatc gattttagcg acagatatct cgtccaccac 6960 tgtgtattgg gaaaatgtag aagtaccaag gaaatggtgt ataggtttcc ctctgcatgt 7020 aaatctgctt gtaccatcct gcatagtacc tctaggcata gacaaatcat ttttaaggca 7080 gaaattaccc tcaggatgtt tgcagactct acacttacca cattgaggag tgaacagtgg 7140 gatcacttta tcaccaggac gaacagtggt aacaccttca cctatggatt caacgattcc 7200 ggcagcctcg tgtcccgcga ttactggcaa aggagtaact agagtgccac tcaccacatg 7260 gtcgtcggat ctacagattc cggtggcaac catcttgatt ctaacctcgt gtgcttttgg 7320 tggcgctact tctacttctt ctatgctaaa cggctttttc tcttcccaca aaactgccgc 7380 tttacactta ataactttac cggctgttga catcctcagc tagctattgt aatatgtgtg 7440 tttgtttgga ttattaagaa gaataattac aaaaaaaatt acaaaggaag gtaattacaa 7500 cagaattaag aaaggacaag aaggaggaag agaatcagtt cattatttct tctttgttat 7560 ataacaaacc caagtagcga tttggccata cattaaaagt tgagaaccac cctccctggc 7620 aacagccaca actcgttacc attgttcatc acgatcatga aactcgctgt cagctgaaat 7680 ttcacctcag tggatctctc tttttattct tcatcgttcc actaaccttt ttccatcagc 7740 tggcagggaa cggaaagtgg aatcccattt agcgagcttc ctcttttctt caagaaaaga 7800 cgaagcttgt gtgtgggtgc gcgcgctagt atctttccac attaagaaat ataccataaa 7860 ggttacttag acatcactat ggctatatat atatatatat atatatgtaa cttagcacca 7920 tcgcgcgtgc atcactgcat gtgttaaccg aaaagtttgg cgaacacttc accgacacgg 7980 tcatttagat ctgtcgtctg cattgcacgt cccttagcct taaatcctag gcgggagcat 8040 tctcgtgtaa ttgtgcagcc tgcgtagcaa ctcaacatag cgtagtctac ccagtttttc 8100 aagggtttat cgttagaaga ttctcccttt tcttcctgct cacaaatctt aaagtcatac 8160 attgcacgac taaatgcaag catgcggatc ccccgggctg caggaattcg atatcaagct 8220 tatcgatacc gtcgactggc cattaatctt tcccatatta gatttcgcca agccatgaaa 8280 gttcaagaaa ggtctttaga cgaattaccc ttcatttctc aaactggcgt caagggatcc 8340 tggtatggtt ttatcgtttt atttctggtt cttatagcat cgttttggac ttctctgttc 8400 ccattaggcg gttcaggagc cagcgcagaa tcattctttg aaggatactt atcctttcca 8460 attttgattg tctgttacgt tggacataaa ctgtatacta gaaattggac tttgatggtg 8520 aaactagaag atatggatct tgataccggc agaaaacaag tagatttgac tcttcgtagg 8580 gaagaaatga ggattgagcg agaaacatta gcaaaaagat ccttcgtaac aagattttta 8640 catttctggt gttgaaggga aagatatgag ctatacagcg gaatttccat atcactcaga 8700 ttttgttatc taattttttc cttcccacgt ccgcgggaat ctgtgtatat tactgcatct 8760 agatatatgt tatcttatct tggcgcgtac atttaatttt caacgtattc tataagaaat 8820 tgcgggagtt tttttcatgt agatgatact gactgcacgc aaatataggc atgatttata 8880 ggcatgattt gatggctgta ccgataggaa cgctaagagt aacttcagaa tcgttatcct 8940 ggcggaaaaa attcatttgt aaactttaaa aaaaaaagcc aatatcccca aaattattaa 9000 gagcgcctcc attattaact aaaatttcac tcagcatcca caatgtatca ggtatctact 9060 acagatatta catgtggcga aaaagacaag aacaatgcaa tagcgcatca agaaaaaaca 9120 caaagctttc aatcaatgaa tcgaaaatgt cattaaaata gtatataaat tgaaactaag 9180 tcataaagct ataaaaagaa aatttattta aatgcaagat ttaaagtaaa ttcacggccc 9240 tgcaggcctc agctcttgtt ttgttctgca aataacttac ccatcttttt caaaacttta 9300 ggtgcaccct cctttgctag aataagttct atccaataca tcctatttgg atctgcttga 9360 gcttctttca tcacggatac gaattcattt tctgttctca caattttgga cacaactctg 9420 tcttccgttg ccccgaaact ttctggcagt tttgagtaat tccacatagg aatgtcatta 9480 taactctggt tcggaccatg aatttccctc tcaaccgtgt aaccatcgtt attaatgata 9540 aagcagattg ggtttatctt ctctctaatg gctagtccta attcttggac agtcagttgc 9600 aatgatccat ctccgataaa caataaatgt ctagattctt tatctgcaat ttggctgcct 9660 agagctgcgg ggaaagtgta tcctatagat ccccacaagg gttgaccaat aaaatgtgat 9720 ttcgatttca gaaatataga tgaggcaccg aagaaagaag tgccttgttc agccacgatc 9780 gtctcattac tttgggtcaa attttcgaca gcttgccaca gtctatcttg tgacaacagc 9840 gcgttagaag gtacaaaatc ttcttgcttt ttatctatgt acttgccttt atattcaatt 9900 tcggacaagt caagaagaga tgatatcagg gattcgaagt cgaaattttg gattctttcg 9960 ttgaaaattt taccttcatc gatattcaag gaaatcattt tattttcatt aagatggtga 10020 gtaaatgcac ccgtactaga atcggtaagc tttacaccca acataagaat aaaatcagca 10080 gattccacaa attccttcaa gtttggctct gacagagtac cgttgtaaat ccccaaaaat 10140 gagggcaatg cttcatcaac agatgattta ccaaagttca aagtagtaat aggtaactta 10200 gtctttgaaa taaactgagt aacagtcttc tctaggccga acgatataat ttcatggcct 10260 gtgattacaa ttggtttctt ggcattcttc agactttcct gtattttgtt cagaatctct 10320 tgatcagatg tattcgacgt ggaattttcc ttcttaagag gcaaggatgg tttttcagcc 10380 ttagcggcag ctacatctac aggtaaattg atgtaaaccg gctttctttc ctttagtaag 10440 gcagacaaca ctctatcaat ttcaacagtt gcattctcgg ctgtcaataa agtcctggca 10500 gcagtaaccg gttcgtgcat cttcataaag tgcttgaaat caccatcagc caacgtatgg 10560 tgaacaaact taccttcgtt ctgcactttc gaggtaggag atcccacgat ctcaacaaca 10620 ggcaggttct cagcatagga gcccgctaag ccattaactg cggataattc gccaacacca 10680 aatgtagtca agaatgccgc agcctttttc gttcttgcgt acccgtcggc catataggag 10740 gcatttaact cattagcatt tcccacccat ttcatatctt tgtgtgaaat aatttgatct 10800 agaaattgca aattgtagtc acctggtact ccgaatattt cttctatacc taattcgtgt 10860 aatctgtcca acagatagtc acctactgta tacattttgt ttactagttt atgtgtgttt 10920 attcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 10980 tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 11040 cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 11100 tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 11160 cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 11220 ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 11280 cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 11340 tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 11400 cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 11460 cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 11520 ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaactga 11580 aaaagcgtgt tttttattca aaatgattct aactccctta cgtaatcaag gaatcttttt 11640 gccttggcct ccgcgtcatt aaacttcttg ttgttgacgc taacattcaa cgctagtata 11700 tattcgtttt tttcaggtaa gttcttttca acgggtctta ctgatgaggc agtcgcgtct 11760 gaacctgtta agaggtcaaa tatgtcttct tgaccgtacg tgtcttgcat gttattagct 11820 ttgggaattt gcatcaagtc ataggaaaat ttaaatcttg gctctcttgg gctcaaggtg 11880 acaaggtcct cgaaaatagg gcgcgcccca ccgcggtgga gctccagctt ttgttccctt 11940 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 12000 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 12060 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 12120 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 12180 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 12240 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 12300 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 12360 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 12420 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 12480 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 12540 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 12600 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 12660 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 12720 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 12780 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 12840 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 12900 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 12960 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 13020 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 13080 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 13140 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 13200 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 13260 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 13320 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 13380 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 13440 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 13500 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 13560 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 13620 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 13680 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 13740 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 13800 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 13860 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 13920 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 13980 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 14040 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 14100 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca 14160 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga 14220 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta 14280 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt 14340 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt 14400 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact 14460 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc 14520 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga 14580 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa 14640 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg 14700 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc 14760 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat 14820 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta 14880 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga 14940 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt 15000 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct 15060 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga 15120 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg 15180 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat 15240 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg 15300 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc 15360 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc 15420 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat catactaaga 15480 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 15539 <210> 3 <211> 4586 <212> DNA <213> Template <400> 3 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 60 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 120 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 180 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 240 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 300 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 360 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 420 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 480 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 540 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 600 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 660 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 720 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 780 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 840 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 900 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 960 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1020 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1080 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1140 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1200 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1260 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1320 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1380 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1440 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1500 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 1560 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 1620 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 1680 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 1740 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 1800 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 1860 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1920 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1980 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2040 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2100 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2160 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2220 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2280 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2340 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2400 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2460 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 2520 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 2580 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 2640 attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgcat tgcggattac 2700 gtattctaat gttcagataa cttcgtatag catacattat acgaagttat ctagggattc 2760 ataaccattt tctcaatcga attacacaga acacaccgta caaacctctc tatcataact 2820 acttaatagt cacacacgta ctcgtctaaa tacacatcat cgtcctacaa gttcatcaaa 2880 gtgttggaca gacaactata ccagcatgga tctcttgtat cggttctttt ctcccgctct 2940 ctcgcaataa caatgaacac tgggtcaatc atagcctaca caggtgaaca gagtagcgtt 3000 tatacagggt ttatacggtg attcctacgg caaaaatttt tcatttctaa aaaaaaaaag 3060 aaaaattttt ctttccaacg ctagaaggaa aagaaaaatc taattaaatt gatttggtga 3120 ttttctgaga gttccctttt tcatatatcg aattttgaat ataaaggag atcgaaaaaa 3180 tttttctatt caatctgttt tctggtttta tttgatagtt tttttgtgta ttattattat 3240 ggattagtac tggtttatat gggtttttct gtataacttc tttttatttt agtttgttta 3300 atcttatttt gagttacatt atagttccct aactgcaaga gaagtaacat taaaactcga 3360 gatgggtaag gaaaagactc acgtttcgag gccgcgatta aattccaaca tggatgctga 3420 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 3480 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 3540 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc 3600 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 3660 cggcaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 3720 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa 3780 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga 3840 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 3900 gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 3960 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 4020 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 4080 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 4140 gtttcatttg atgctcgatg agtttttcta agtttaactt gatactacta gattttttct 4200 cttcatttat aaaatttttg gttataattg aagctttaga agtatgaaaa aatccttttt 4260 tttcattctt tgcaaccaaa ataagaagct tcttttattc attgaaatga tgaatataaa 4320 cctaacaaaa gaaaaagact cgaatatcaa acattaaaaa aaaataaaag aggttatctg 4380 ttttcccatt tagttggagt ttgcattttc taatagatag aactctcaat taatgtggat 4440 ttagtttctc tgttcgtttt tttttgtttt gttctcactg tatttacatt tctatttagt 4500 atttagttat tcatataatc tataacttcg tatagcatac attatacgaa gttatccagt 4560 gatgatacaa cgagttagcc aaggtg 4586 <210> 4 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 4 ttccggtttc tttgaaattt ttttgattcg gtaatctccg agcagaagga gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 5 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 5 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta caccttggct 60 aactcgttgt atcatcactg g 81 <210> 6 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 6 gcctcgagtt ttaatgttac ttctcttgca gttaggga 38 <210> 7 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 gctaaattcg agtgaaacac aggaagacca g 31 <210> 8 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 8 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 9 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 9 ttggttgggg gaaaaagagg caacaggaaa gatcagaggg ggaggggggg ggagagtgtc 60 accttggcta actcgttgta tcatcactgg 90 <210> 10 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 10 tcggtgcggg cctcttcgct a 21 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 11 aatgtgagtt agctcactca t 21 <210> 12 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 12 aattggatcc ggcgcgccgt ttaaacggcc ggccaatgtg gctgtggttt cagggtc 57 <210> 13 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 13 aatttctaga ttaattaagc ggccgcaagg ccatgaagct ttttctttc 49 <210> 14 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 14 ttctcgacgt gggccttttt cttg 24 <210> 15 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 15 tgcagcttta aataatcggt gtcactactt tgccttcgtt tatcttgcc 49 <210> 16 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 16 gagcaggcaa gataaacgaa ggcaaagtag tgacaccgat tatttaaag 49 <210> 17 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 tatggaccct gaaaccacag ccacattgta accaccacga cggttgttg 49 <210> 18 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 18 tttagcaaca accgtcgtgg tggttacaat gtggctgtgg tttcagggt 49 <210> 19 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 19 ccagaaaccc tatacctgtg tggacgtaag gccatgaagc tttttcttt 49 <210> 20 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 20 attggaaaga aaaagcttca tggccttacg tccacacagg tatagggtt 49 <210> 21 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 21 cataagaaca cctttggtgg ag 22 <210> 22 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 22 aggattatca ttcataagtt tc 22 <210> 23 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 23 ttcttggagc tgggacatgt ttg 23 <210> 24 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 24 tgatgatatt tcataaataa tg 22 <210> 25 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 25 atgcgtccat ctttacagtc ctg 23 <210> 26 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 26 tacgtacgga ccaatcgaag tg 22 <210> 27 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 27 aattcgtttg agtacactac taatggcttt gttggcaata tgtttttgc 49 <210> 28 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 28 atatagcaaa aacatattgc caacaaagcc attagtagtg tactcaaac 49 <210> 29 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 29 tatggaccct gaaaccacag ccacattctt gttatttata aaaagacac 49 <210> 30 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 30 ctcccgtgtc tttttataaa taacaagaat gtggctgtgg tttcagggt 49 <210> 31 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 31 taccgtaggc gtccttagga aagatagaag gccatgaagc tttttcttt 49 <210> 32 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 32 attggaaaga aaaagcttca tggccttcta tctttcctaa ggacgccta 49 <210> 33 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 33 ttattgtttg gcatttgtag c 21 <210> 34 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 34 ccaagcatct cataaaccta tg 22 <210> 35 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 35 tgtgcagatg cagatgtgag ac 22 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 36 agttattgat accgtac 17 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 37 cgagataccg taggcgtcc 19 <210> 38 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 38 ttatgtatgc tcttctgact tttc 24 <210> 39 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 39 aataattaga gattaaatcg ctcatttttt gccagtttct tcaggcttc 49 <210> 40 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 40 agcctgaaga aactggcaaa aaatgagcga tttaatctct aattattag 49 <210> 41 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 41 tatggaccct gaaaccacag ccacattttt caatcattgg agcaatcat 49 <210> 42 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 42 taaaatgatt gctccaatga ttgaaaaatg tggctgtggt ttcagggtc 49 <210> 43 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 43 accgtaggtg ttgtttggga aagtggaagg ccatgaagct ttttctttc 49 <210> 44 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 44 ttggaaagaa aaagcttcat ggccttccac tttcccaaac aacacctac 49 <210> 45 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 45 ttattgctta gcgttggtag cag 23 <210> 46 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 46 tttttggtgg ttccggcttc c 21 <210> 47 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 47 aaagttggca tagcggaaac tt 22 <210> 48 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 48 gtcattgaca ccatct 16 <210> 49 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 49 agagataccg taggtgttg 19 <210> 50 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 50 aattggcgcg ccatgaaagc tctggtttat cac 33 <210> 51 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 51 tgaatcatga gttttatgtt aattagctca ggcagcgcct gcgttcgag 49 <210> 52 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 52 atcctctcga acgcaggcgc tgcctgagct aattaacata aaactcatg 49 <210> 53 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 53 aattgtttaa acaagtaaat aaattaatca gcat 34 <210> 54 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 54 acacaataca ataacaagaa gaacaaaatg aaagctctgg tttatcacg 49 <210> 55 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 55 agcgtataca tctgttggga aagtagaagg ccatgaagct ttttctttc 49 <210> 56 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 56 ttggaaagaa aaagcttcat ggccttctac tttcccaaca gatgtatac 49 <210> 57 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 57 ttattgttta gcgttagtag cg 22 <210> 58 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 58 cataatcaat ctcaaagaga acaacacaat acaataacaa gaagaacaaa atgaaagctc 60 tggtttatca cg 72 <210> 59 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 59 taggcataat caccgaagaa g 21 <210> 60 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 60 aaaatggtaa gcagctgaaa g 21 <210> 61 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 61 agttgttaga actgttg 17 <210> 62 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 62 gacgatagcg tatacatct 19 <210> 63 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 63 cttagcctct agccatagcc at 22 <210> 64 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 64 ttagttttgc tggccgcatc ttc 23 <210> 65 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 65 cccattaata tactattgag a 21 <210> 66 <211> 7523 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 66 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 60 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga gccggaagca 120 taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt gcgttgcgct 180 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 240 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 300 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 360 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 420 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 480 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 540 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 600 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 660 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 720 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 780 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 840 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 900 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 960 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1020 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1080 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1140 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1200 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1260 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1320 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1380 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1440 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1500 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1560 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1620 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1680 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1740 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1800 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1860 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1920 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1980 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2040 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2100 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2160 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg 2220 cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg 2280 agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc 2340 tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga 2400 atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa 2460 gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca 2520 aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac 2580 tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt 2640 ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg 2700 cattttttca agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac 2760 tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt 2820 ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt 2880 cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga 2940 taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg 3000 ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg 3060 tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt 3120 tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata 3180 ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct 3240 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct 3300 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa 3360 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat 3420 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct 3480 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat 3540 attggatcat ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 3600 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3660 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3720 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3780 ttgtactgag agtgcaccat aaattcccgt tttaagagct tggtgagcgc taggagtcac 3840 tgccaggtat cgtttgaaca cggcattagt cagggaagtc ataacacagt cctttcccgc 3900 aattttcttt ttctattact cttggcctcc tctagtacac tctatatttt tttatgcctc 3960 ggtaatgatt ttcatttttt tttttcccct agcggatgac tctttttttt tcttagcgat 4020 tggcattatc acataatgaa ttatacatta tataaagtaa tgtgatttct tcgaagaata 4080 tactaaaaaa tgagcaggca agataaacga aggcaaagat gacagagcag aaagccctag 4140 taaagcgtat tacaaatgaa accaagattc agattgcgat ctctttaaag ggtggtcccc 4200 tagcgataga gcactcgatc ttcccagaaa aagaggcaga agcagtagca gaacaggcca 4260 cacaatcgca agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg 4320 ctctggccaa gcattccggc tggtcgctaa tcgttgagtg cattggtgac ttacacatag 4380 acgaccatca caccactgaa gactgcggga ttgctctcgg tcaagctttt aaagaggccc 4440 tactggcgcg tggagtaaaa aggtttggat caggatttgc gcctttggat gaggcacttt 4500 ccagagcggt ggtagatctt tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa 4560 gggagaaagt aggagatctc tcttgcgaga tgatcccgca ttttcttgaa agctttgcag 4620 aggctagcag aattaccctc cacgttgatt gtctgcgagg caagaatgat catcaccgta 4680 gtgagagtgc gttcaaggct cttgcggttg ccataagaga agccacctcg cccaatggta 4740 ccaacgatgt tccctccacc aaaggtgttc ttatgtagtg acaccgatta tttaaagctg 4800 cagcatacga tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta 4860 tacgaacagt atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg 4920 aacgaggcgc gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac 4980 ggatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 5040 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 5100 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 5160 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 5220 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 5280 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 5340 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 5400 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 5460 ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca 5520 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 5580 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 5640 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc 5700 cccccctcga ggtattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc 5760 tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca 5820 ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt 5880 ggcagtaacc tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg 5940 ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt 6000 ttgatctatt aacagatata taaatggaaa agctgcataa ccactttaac taatactttc 6060 aacattttca gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg 6120 ttaatatacc tctatacttt aacgtcaagg agaaaaatgt ccaatttact gcccgtacac 6180 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg 6240 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt 6300 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct 6360 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc 6420 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt 6480 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt 6540 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc 6600 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat 6660 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact 6720 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt 6780 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct 6840 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc 6900 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact 6960 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga 7020 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag 7080 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg 7140 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagga gtaagcgaat 7200 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 7260 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 7320 taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 7380 ggcatgccga gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa 7440 ctccagcaat gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg 7500 tggtccgcca ccgcggtgga gct 7523 <210> 67 <211> 15456 <212> DNA <213> Artificial sequence <220> <223> Template <400> 67 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca 60 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag 120 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca 180 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag 240 cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg 300 tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt 360 cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata 420 gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag 480 tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca 540 ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat 600 gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc 660 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa 720 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 780 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac 840 tatgcggcat cagagcagat tgtactgaga gtgcaccata aattcccgtt ttaagagctt 900 ggtgagcgct aggagtcact gccaggtatc gtttgaacac ggcattagtc agggaagtca 960 taacacagtc ctttcccgca attttctttt tctattactc ttggcctcct ctagtacact 1020 ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct agcggatgac 1080 tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta tataaagtaa 1140 tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga aggcaaagat 1200 gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc agattgcgat 1260 ctctttaaag ggtggtcccc tagcgataga gcactcgatc ttcccagaaa aagaggcaga 1320 agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag gtatagggtt 1380 tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa tcgttgagtg 1440 cattggtgac ttacacatag acgaccatca caccactgaa gactgcggga ttgctctcgg 1500 tcaagctttt aaagaggccc taggggccgt gcgtggagta aaaaggtttg gatcaggatt 1560 tgcgcctttg gatgaggcac tttccagagc ggtggtagat ctttcgaaca ggccgtacgc 1620 agttgtcgaa cttggtttgc aaagggagaa agtaggagat ctctcttgcg agatgatccc 1680 gcattttctt gaaagctttg cagaggctag cagaattacc ctccacgttg attgtctgcg 1740 aggcaagaat gatcatcacc gtagtgagag tgcgttcaag gctcttgcgg ttgccataag 1800 agaagccacc tcgcccaatg gtaccaacga tgttccctcc accaaaggtg ttcttatgta 1860 gtgacaccga ttatttaaag ctgcagcata cgatatatat acatgtgtat atatgtatac 1920 ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac aaggtaatgc 1980 atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt ttgctttttc 2040 tttttttttc tcttgaactc gacggatcta tgcggtgtga aataccgcac agatgcgtaa 2100 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 2160 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 2220 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 2280 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 2340 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 2400 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 2460 ggaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 2520 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 2580 tcgccattca ggctgcgcaa ctgttgggaa gggcgcggtg cgggcctctt cgctattacg 2640 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 2700 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg 2760 cgaattgggt accgggcccc ccctcgaggt cgacggcgcg ccactggtag agagcgactt 2820 tgtatgcccc aattgcgaaa cccgcgatat ccttctcgat tctttagtac ccgaccagga 2880 caaggaaaag gaggtcgaaa cgtttttgaa gaaacaagag gaactacacg gaagctctaa 2940 agatggcaac cagccagaaa ctaagaaaat gaagttgatg gatccaactg gcaccgctgg 3000 cttgaacaac aataccagcc ttccaacttc tgtaaataac ggcggtacgc cagtgccacc 3060 agtaccgtta cctttcggta tacctccttt ccccatgttt ccaatgccct tcatgcctcc 3120 aacggctact atcacaaatc ctcatcaagc tgacgcaagc cctaagaaat gaataacaat 3180 actgacagta ctaaataatt gcctacttgg cttcacatac gttgcatacg tcgatataga 3240 taataatgat aatgacagca ggattatcgt aatacgtaat agctgaaaat ctcaaaaatg 3300 tgtgggtcat tacgtaaata atgataggaa tgggattctt ctatttttcc tttttccatt 3360 ctagcagccg tcgggaaaac gtggcatcct ctctttcggg ctcaattgga gtcacgctgc 3420 cgtgagcatc ctctctttcc atatctaaca actgagcacg taaccaatgg aaaagcatga 3480 gcttagcgtt gctccaaaaa agtattggat ggttaatacc atttgtctgt tctcttctga 3540 ctttgactcc tcaaaaaaaa aaatctacaa tcaacagatc gcttcaatta cgccctcaca 3600 aaaacttttt tccttcttct tcgcccacgt taaattttat ccctcatgtt gtctaacgga 3660 tttctgcact tgatttatta taaaaagaca aagacataat acttctctat caatttcagt 3720 tattgttctt ccttgcgtta ttcttctgtt cttctttttc ttttgtcata tataaccata 3780 accaagtaat acatattcaa actagtatga ctgacaaaaa aactcttaaa gacttaagaa 3840 atcgtagttc tgtttacgat tcaatggtta aatcacctaa tcgtgctatg ttgcgtgcaa 3900 ctggtatgca agatgaagac tttgaaaaac ctatcgtcgg tgtcatttca acttgggctg 3960 aaaacacacc ttgtaatatc cacttacatg actttggtaa actagccaaa gtcggtgtta 4020 aggaagctgg tgcttggcca gttcagttcg gaacaatcac ggtttctgat ggaatcgcca 4080 tgggaaccca aggaatgcgt ttctccttga catctcgtga tattattgca gattctattg 4140 aagcagccat gggaggtcat aatgcggatg cttttgtagc cattggcggt tgtgataaaa 4200 acatgcccgg ttctgttatc gctatggcta acatggatat cccagccatt tttgcttacg 4260 gcggaacaat tgcacctggt aatttagacg gcaaagatat cgatttagtc tctgtctttg 4320 aaggtgtcgg ccattggaac cacggcgata tgaccaaaga agaagttaaa gctttggaat 4380 gtaatgcttg tcccggtcct ggaggctgcg gtggtatgta tactgctaac acaatggcga 4440 cagctattga agttttggga cttagccttc cgggttcatc ttctcacccg gctgaatccg 4500 cagaaaagaa agcagatatt gaagaagctg gtcgcgctgt tgtcaaaatg ctcgaaatgg 4560 gcttaaaacc ttctgacatt ttaacgcgtg aagcttttga agatgctatt actgtaacta 4620 tggctctggg aggttcaacc aactcaaccc ttcacctctt agctattgcc catgctgcta 4680 atgtggaatt gacacttgat gatttcaata ctttccaaga aaaagttcct catttggctg 4740 atttgaaacc ttctggtcaa tatgtattcc aagaccttta caaggtcgga ggggtaccag 4800 cagttatgaa atatctcctt aaaaatggct tccttcatgg tgaccgtatc acttgtactg 4860 gcaaaacagt cgctgaaaat ttgaaggctt ttgatgattt aacacctggt caaaaggtta 4920 ttatgccgct tgaaaatcct aaacgtgaag atggtccgct cattattctc catggtaact 4980 tggctccaga cggtgccgtt gccaaagttt ctggtgtaaa agtgcgtcgt catgtcggtc 5040 ctgctaaggt ctttaattct gaagaagaag ccattgaagc tgtcttgaat gatgatattg 5100 ttgatggtga tgttgttgtc gtacgttttg taggaccaaa gggcggtcct ggtatgcctg 5160 aaatgctttc cctttcatca atgattgttg gtaaagggca aggtgaaaaa gttgcccttc 5220 tgacagatgg ccgcttctca ggtggtactt atggtcttgt cgtgggtcat atcgctcctg 5280 aagcacaaga tggcggtcca atcgcctacc tgcaaacagg agacatagtc actattgacc 5340 aagacactaa ggaattacac tttgatatct ccgatgaaga gttaaaacat cgtcaagaga 5400 ccattgaatt gccaccgctc tattcacgcg gtatccttgg taaatatgct cacatcgttt 5460 cgtctgcttc taggggagcc gtaacagact tttggaagcc tgaagaaact ggcaaaaaat 5520 gttgtcctgg ttgctgtggt taagcggccg cgttaattca aattaattga tatagttttt 5580 taatgagtat tgaatctgtt tagaaataat ggaatattat ttttatttat ttatttatat 5640 tattggtcgg ctcttttctt ctgaaggtca atgacaaaat gatatgaagg aaataatgat 5700 ttctaaaatt ttacaacgta agatattttt acaaaagcct agctcatctt ttgtcatgca 5760 ctattttact cacgcttgaa attaacggcc agtccactgc ggagtcattt caaagtcatc 5820 ctaatcgatc tatcgttttt gatagctcat tttggagttc gcgattgtct tctgttattc 5880 acaactgttt taatttttat ttcattctgg aactcttcga gttctttgta aagtctttca 5940 tagtagctta ctttatcctc caacatattt aacttcatgt caatttcggc tcttaaattt 6000 tccacatcat caagttcaac atcatctttt aacttgaatt tattctctag ctcttccaac 6060 caagcctcat tgctccttga tttactggtg aaaagtgata cactttgcgc gcaatccagg 6120 tcaaaacttt cctgcaaaga attcaccaat ttctcgacat catagtacaa tttgttttgt 6180 tctcccatca caatttaata tacctgatgg attcttatga agcgctgggt aatggacgtg 6240 tcactctact tcgccttttt ccctactcct tttagtacgg aagacaatgc taataaataa 6300 gagggtaata ataatattat taatcggcaa aaaagattaa acgccaagcg tttaattatc 6360 agaaagcaaa cgtcgtacca atccttgaat gcttcccaat tgtatattaa gagtcatcac 6420 agcaacatat tcttgttatt aaattaatta ttattgattt ttgatattgt ataaaaaaac 6480 caaatatgta taaaaaaagt gaataaaaaa taccaagtat ggagaaatat attagaagtc 6540 tatacgttaa accacccggg ccccccctcg aggtcgacgg tatcgataag cttgatatcg 6600 aattcctgca gcccggggga tccactagtt ctagagcggc cgctctagaa ctagtaccac 6660 aggtgttgtc ctctgaggac ataaaataca caccgagatt catcaactca ttgctggagt 6720 tagcatatct acaattgggt gaaatgggga gcgatttgca ggcatttgct cggcatgccg 6780 gtagaggtgt ggtcaataag agcgacctca tgctatacct gagaaagcaa cctgacctac 6840 aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat 6900 ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa 6960 ttcgcttact cttaattaat caggcagcgc ctgcgttcga gaggatgatc ttcatcgcct 7020 tctccttggc gccattgagg aatacctgat aggcgtgctc gatctcggcc agctcgaagc 7080 gatgggtaat catcttcttc aacggaagct tgtcggtcga ggcgaccttc atcagcatgg 7140 gcgtcgtgtt cgtgttcacc agtcccgtgg tgatcgtcag gttcttgatc cagagcttct 7200 gaatctcgaa gtcaaccttg acgccatgca cgccgacgtt ggcgatgtgc gcgccgggct 7260 tgacgatctc ctggcagatg tcccaagtcg ccggtatgcc caccgcctcg atcgcaacat 7320 cgactccctc tgccgcaatc ctatgcacgg cttcgacaac gttctccgtg ccggagttga 7380 tggtgtgcgt tgccccgagc tccttggcga gctggaggcg attctcgtcc atgtcgatca 7440 cgatgatggt cgagggggag tagaactggg cggtcaacag tacggacatg ccgacggggc 7500 ccgcgccgac aatagccacc gcatcgcccg gctggacatt cccatactgg acgccgattt 7560 cgtggccggt gggcaggatg tcgctcagca ggacggcgat ttcgtcgtca attgtctggg 7620 ggatcttgta gaggctgttg tcggcatgcg ggatgcggac gtattcggcc tgcacgccat 7680 cgatcatgta acccaggatc cacccgccgt cgcggcaatg ggagtaaagc tgcttcttgc 7740 agtagtcgca cgagccgcaa gaagtgacgc aggaaatcag gaccttgtcg cctttcttga 7800 actgcgtgac actctcgccc acttcctcga tgacgcctac cccttcatgg cccaggatgc 7860 gcccgtcggc gacctctgga ttcttgcctt tgtagatgcc gagatccgtg ccgcagatcg 7920 tggtcttcaa aacccgtact actacatccg tgggcttttg aagggtgggc ttgggcttgt 7980 cttcaagcga gatcttgtgg tcaccgtgat aaaccagagc tttcatcctc agctattgta 8040 atatgtgtgt ttgtttggat tattaagaag aataattaca aaaaaaatta caaaggaagg 8100 taattacaac agaattaaga aaggacaaga aggaggaaga gaatcagttc attatttctt 8160 ctttgttata taacaaaccc aagtagcgat ttggccatac attaaaagtt gagaaccacc 8220 ctccctggca acagccacaa ctcgttacca ttgttcatca cgatcatgaa actcgctgtc 8280 agctgaaatt tcacctcagt ggatctctct ttttattctt catcgttcca ctaacctttt 8340 tccatcagct ggcagggaac ggaaagtgga atcccattta gcgagcttcc tcttttcttc 8400 aagaaaagac gaagcttgtg tgtgggtgcg cgcgctagta tctttccaca ttaagaaata 8460 taccataaag gttacttaga catcactatg gctatatata tatatatata tatatatgta 8520 acttagcacc atcgcgcgtg catcactgca tgtgttaacc gaaaagtttg gcgaacactt 8580 caccgacacg gtcatttaga tctgtcgtct gcattgcacg tcccttagcc ttaaatccta 8640 ggcgggagca ttctcgtgta attgtgcagc ctgcgtagca actcaacata gcgtagtcta 8700 cccagttttt caagggttta tcgttagaag attctccctt ttcttcctgc tcacaaatct 8760 taaagtcata cattgcacga ctaaatgcaa gcatgcggat cccccgggct gcaggaattc 8820 gatatcaagc ttatcgatac cgtcgactgg ccattaatct ttcccatatt agatttcgcc 8880 aagccatgaa agttcaagaa aggtctttag acgaattacc cttcatttct caaactggcg 8940 tcaagggatc ctggtatggt tttatcgttt tatttctggt tcttatagca tcgttttgga 9000 cttctctgtt cccattaggc ggttcaggag ccagcgcaga atcattcttt gaaggatact 9060 tatcctttcc aattttgatt gtctgttacg ttggacataa actgtatact agaaattgga 9120 ctttgatggt gaaactagaa gatatggatc ttgataccgg cagaaaacaa gtagatttga 9180 ctcttcgtag ggaagaaatg aggattgagc gagaaacatt agcaaaaaga tccttcgtaa 9240 caagattttt acatttctgg tgttgaaggg aaagatatga gctatacagc ggaatttcca 9300 tatcactcag attttgttat ctaatttttt ccttcccacg tccgcgggaa tctgtgtata 9360 ttactgcatc tagatatatg ttatcttatc ttggcgcgta catttaattt tcaacgtatt 9420 ctataagaaa ttgcgggagt ttttttcatg tagatgatac tgactgcacg caaatatagg 9480 catgatttat aggcatgatt tgatggctgt accgatagga acgctaagag taacttcaga 9540 atcgttatcc tggcggaaaa aattcatttg taaactttaa aaaaaaaagc caatatcccc 9600 aaaattatta agagcgcctc cattattaac taaaatttca ctcagcatcc acaatgtatc 9660 aggtatctac tacagatatt acatgtggcg aaaaagacaa gaacaatgca atagcgcatc 9720 aagaaaaaac acaaagcttt caatcaatga atcgaaaatg tcattaaaat agtatataaa 9780 ttgaaactaa gtcataaagc tataaaaaga aaatttattt aaatgcaaga tttaaagtaa 9840 attcacggcc ctgcaggcct cagctcttgt tttgttctgc aaataactta cccatctttt 9900 tcaaaacttt aggtgcaccc tcctttgcta gaataagttc tatccaatac atcctatttg 9960 gatctgcttg agcttctttc atcacggata cgaattcatt ttctgttctc acaattttgg 10020 acacaactct gtcttccgtt gccccgaaac tttctggcag ttttgagtaa ttccacatag 10080 gaatgtcatt ataactctgg ttcggaccat gaatttccct ctcaaccgtg taaccatcgt 10140 tattaatgat aaagcagatt gggtttatct tctctctaat ggctagtcct aattcttgga 10200 cagtcagttg caatgatcca tctccgataa acaataaatg tctagattct ttatctgcaa 10260 tttggctgcc tagagctgcg gggaaagtgt atcctataga tccccacaag ggttgaccaa 10320 taaaatgtga tttcgatttc agaaatatag atgaggcacc gaagaaagaa gtgccttgtt 10380 cagccacgat cgtctcatta ctttgggtca aattttcgac agcttgccac agtctatctt 10440 gtgacaacag cgcgttagaa ggtacaaaat cttcttgctt tttatctatg tacttgcctt 10500 tatattcaat ttcggacaag tcaagaagag atgatatcag ggattcgaag tcgaaatttt 10560 ggattctttc gttgaaaatt ttaccttcat cgatattcaa ggaaatcatt ttattttcat 10620 taagatggtg agtaaatgca cccgtactag aatcggtaag ctttacaccc aacataagaa 10680 taaaatcagc agattccaca aattccttca agtttggctc tgacagagta ccgttgtaaa 10740 tccccaaaaa tgagggcaat gcttcatcaa cagatgattt accaaagttc aaagtagtaa 10800 taggtaactt agtctttgaa ataaactgag taacagtctt ctctaggccg aacgatataa 10860 tttcatggcc tgtgattaca attggtttct tggcattctt cagactttcc tgtattttgt 10920 tcagaatctc ttgatcagat gtattcgacg tggaattttc cttcttaaga ggcaaggatg 10980 gtttttcagc cttagcggca gctacatcta caggtaaatt gatgtaaacc ggctttcttt 11040 cctttagtaa ggcagacaac actctatcaa tttcaacagt tgcattctcg gctgtcaata 11100 aagtcctggc agcagtaacc ggttcgtgca tcttcataaa gtgcttgaaa tcaccatcag 11160 ccaacgtatg gtgaacaaac ttaccttcgt tctgcacttt cgaggtagga gatcccacga 11220 tctcaacaac aggcaggttc tcagcatagg agcccgctaa gccattaact gcggataatt 11280 cgccaacacc aaatgtagtc aagaatgccg cagccttttt cgttcttgcg tacccgtcgg 11340 ccatatagga ggcatttaac tcattagcat ttcccaccca tttcatatct ttgtgtgaaa 11400 taatttgatc tagaaattgc aaattgtagt cacctggtac tccgaatatt tcttctatac 11460 ctaattcgtg taatctgtcc aacagatagt cacctactgt atacattttg tttactagtt 11520 tatgtgtgtt tattcgaaac taagttcttg gtgttttaaa actaaaaaaa agactaacta 11580 taaaagtaga atttaagaag tttaagaaat agatttacag aattacaatc aatacctacc 11640 gtctttatat acttattagt caagtagggg aataatttca gggaactggt ttcaaccttt 11700 tttttcagct ttttccaaat cagagagagc agaaggtaat agaaggtgta agaaaatgag 11760 atagatacat gcgtgggtca attgccttgt gtcatcattt actccaggca ggttgcatca 11820 ctccattgag gttgtgcccg ttttttgcct gtttgtgccc ctgttctctg tagttgcgct 11880 aagagaatgg acctatgaac tgatggttgg tgaagaaaac aatattttgg tgctgggatt 11940 cttttttttt ctggatgcca gcttaaaaag cgggctccat tatatttagt ggatgccagg 12000 aataaactgt tcacccagac acctacgatg ttatatattc tgtgtaaccc gccccctatt 12060 ttgggcatgt acgggttaca gcagaattaa aaggctaatt ttttgactaa ataaagttag 12120 gaaaatcact actattaatt atttacgtat tctttgaaat ggcagtattg ataatgataa 12180 actcgaactg aaaaagcgtg ttttttattc aaaatgattc taactccctt acgtaatcaa 12240 ggaatctttt tgccttggcc tccgcgtcat taaacttctt gttgttgacg ctaacattca 12300 acgctagtat atattcgttt ttttcaggta agttcttttc aacgggtctt actgatgagg 12360 cagtcgcgtc tgaacctgtt aagaggtcaa atatgtcttc ttgaccgtac gtgtcttgca 12420 tgttattagc tttgggaatt tgcatcaagt cataggaaaa tttaaatctt ggctctcttg 12480 ggctcaaggt gacaaggtcc tcgaaaatag ggcgcgcccc accgcggtgg agctccagct 12540 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc 12600 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 12660 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 12720 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 12780 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 12840 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 12900 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 12960 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 13020 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 13080 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 13140 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 13200 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 13260 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 13320 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 13380 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 13440 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 13500 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 13560 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 13620 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 13680 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 13740 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 13800 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 13860 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 13920 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 13980 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 14040 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 14100 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 14160 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 14220 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 14280 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 14340 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 14400 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 14460 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 14520 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 14580 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 14640 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 14700 tgcccctga tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 14820 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct 14880 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga 14940 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct 15000 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat 15060 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg 15120 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa 15180 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt 15240 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg 15300 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc 15360 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc 15420 actctatgaa tagttcttac tacaattttt ttgtct 15456 <210> 68 <211> 1559 <212> DNA <213> Artificial sequence <220> <223> Template <400> 68 gcattgcgga ttacgtattc taatgttcag taccgttcgt ataatgtatg ctatacgaag 60 ttatgcagat tgtactgaga gtgcaccata ccaccttttc aattcatcat ttttttttta 120 ttcttttttt tgatttcggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa 180 ggaagaacga aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga 240 agaaacatga aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa 300 acgaagataa atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc 360 tgttgctgcc aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt 420 ggatgttcgt accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg 480 tttactaaaa acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc 540 gctaaaggca ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga 600 cattggtaat acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc 660 agacattacg aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc 720 ggcagaagaa gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa 780 gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa 840 agattttgtt atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga 900 ttggttgatt atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca 960 gtatgaacc gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg 1020 actatttgca aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg 1080 ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc 1140 atgtatacta aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc 1200 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt 1260 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1320 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1380 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1440 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagataact 1500 tcgtataatg tatgctatac gaacggtacc agtgatgata caacgagtta gccaaggtg 1559 <210> 69 <211> 1047 <212> DNA <213> Achromobacter xylosoxidans <400> 69 atgaaagctc tggtttatca cggtgaccac aagatctcgc ttgaagacaa gcccaagccc 60 acccttcaaa agcccacgga tgtagtagta cgggttttga agaccacgat ctgcggcacg 120 gatctcggca tctacaaagg caagaatcca gaggtcgccg acgggcgcat cctgggccat 180 gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca cgcagttcaa gaaaggcgac 240 aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg actactgcaa gaagcagctt 300 tactcccatt gccgcgacgg cgggtggatc ctgggttaca tgatcgatgg cgtgcaggcc 360 gaatacgtcc gcatcccgca tgccgacaac agcctctaca agatccccca gacaattgac 420 gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg gccacgaaat cggcgtccag 480 tatgggaatg tccagccggg cgatgcggtg gctattgtcg gcgcgggccc cgtcggcatg 540 tccgtactgt tgaccgccca gttctactcc ccctcgacca tcatcgtgat cgacatggac 600 gagaatcgcc tccagctcgc caaggagctc ggggcaacgc acaccatcaa ctccggcacg 660 ggaacgttg tcgaagccgt gcataggatt gcggcagagg gagtcgatgt tgcgatcgag 720 gcggtgggca taccggcgac ttgggacatc tgccaggaga tcgtcaagcc cggcgcgcac 780 atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg agattcagaa gctctggatc 840 aagaacctga cgatcaccac gggactggtg aacacgaaca cgacgcccat gctgatgaag 900 gtcgcctcga ccgacaagct tccgttgaag aagatgatta cccatcgctt cgagctggcc 960 gagatcgagc acgcctatca ggtattcctc aatggcgcca aggagaaggc gatgaagatc 1020 atcctctcga acgcaggcgc tgcctga 1047 <210> 70 <211> 348 <212> PRT <213> Achromobacter xylosoxidans <400> 70 Met Lys Ala Leu Val Tyr His Gly Asp His Lys Ile Ser Leu Glu Asp 1 5 10 15 Lys Pro Lys Pro Thr Leu Gln Lys Pro Thr Asp Val Val Arg Val             20 25 30 Leu Lys Thr Thr Ile Cys Gly Thr Asp Leu Gly Ile Tyr Lys Gly Lys         35 40 45 Asn Pro Glu Val Ala Asp Gly Arg Ile Leu Gly His Glu Gly Val Gly     50 55 60 Val Ile Glu Glu Val Gly Glu Ser Val Thr Gln Phe Lys Lys Gly Asp 65 70 75 80 Lys Val Leu Ile Ser Cys Val Thr Ser Cys Gly Ser Cys Asp Tyr Cys                 85 90 95 Lys Lys Gln Leu Tyr Ser His Cys Arg Asp Gly Gly Trp Ile Leu Gly             100 105 110 Tyr Met Ile Asp Gly Val Gln Ala Glu Tyr Val Arg Ile Pro His Ala         115 120 125 Asp Asn Ser Leu Tyr Lys Ile Pro Gln Thr Ile Asp Asp Glu Ile Ala     130 135 140 Val Leu Leu Ser Asp Ile Leu Pro Thr Gly His Glu Ile Gly Val Gln 145 150 155 160 Tyr Gly Asn Val Gln Pro Gly Asp Ala Val Ala Ile Val Gly Ala Gly                 165 170 175 Pro Val Gly Met Ser Val Leu Leu Thr Ala Gln Phe Tyr Ser Ser Ser             180 185 190 Thr Ile Ile Val Ile Asp Met Asp Glu Asn Arg Leu Gln Leu Ala Lys         195 200 205 Glu Leu Gly Ala Thr His Thr Ile Asn Ser Gly Thr Glu Asn Val Val     210 215 220 Glu Ala Val His Arg Ile Ala Ala Glu Gly Val Asp Val Ala Ile Glu 225 230 235 240 Ala Val Gly Ile Pro Ala Thr Trp Asp Ile Cys Gln Glu Ile Val Lys                 245 250 255 Pro Gly Ala His Ile Ala Asn Val Gly Val His Gly Val Lys Val Asp             260 265 270 Phe Glu Ile Gln Lys Leu Trp Ile Lys Asn Leu Thr Ile Thr Thr Gly         275 280 285 Leu Val Asn Thr Asn Thr Thr Pro Met Leu Met Lys Val Ala Ser Thr     290 295 300 Asp Lys Leu Pro Leu Lys Lys Met Ile Thr His Arg Phe Glu Leu Ala 305 310 315 320 Glu Ile Glu His Ala Tyr Gln Val Phe Leu Asn Gly Ala Lys Glu Lys                 325 330 335 Ala Met Lys Ile Ile Leu Ser Asn Ala Gly Ala Ala             340 345 <210> 71 <211> 1644 <212> DNA Artificial sequence <220> <223> codon optimized L. lactis kivD coding region for S. cerevisiae        expression <400> 71 atgtatacagag taggtgacta tctgttggac agattacacg aattaggtat agaagaaata 60 ttcggagtac caggtgacta caatttgcaa tttctagatc aaattatttc acacaaagat 120 atgaaatggg tgggaaatgc taatgagtta aatgcctcct atatggccga cgggtacgca 180 agaacgaaaa aggctgcggc attcttgact acatttggtg ttggcgaatt atccgcagtt 240 aatggcttag cgggctccta tgctgagaac ctgcctgttg ttgagatcgt gggatctcct 300 acctcgaaag tgcagaacga aggtaagttt gttcaccata cgttggctga tggtgatttc 360 aagcacttta tgaagatgca cgaaccggtt actgctgcca ggactttatt gacagccgag 420 aatgcaactg ttgaaattga tagagtgttg tctgccttac taaaggaaag aaagccggtt 480 tacatcaatt tacctgtaga tgtagctgcc gctaaggctg aaaaaccatc cttgcctctt 540 aagaaggaaa attccacgtc gaatacatct gatcaagaga ttctgaacaa aatacaggaa 600 agtctgaaga atgccaagaa accaattgta atcacaggcc atgaaattat atcgttcggc 660 ctagagaaga ctgttactca gtttatttca aagactaagt tacctattac tactttgaac 720 tttggtaaat catctgttga tgaagcattg ccctcatttt tggggattta caacggtact 780 ctgtcagagc caaacttgaa ggaatttgtg gaatctgctg attttattct tatgttgggt 840 gtaaagctta ccgattctag tacgggtgca tttactcacc atcttaatga aaataaaatg 900 atttccttga atatcgatga aggtaaaatt ttcaacgaaa gaatccaaaa tttcgacttc 960 gaatccctga tatcatctct tcttgacttg tccgaaattg aatataaagg caagtacata 1020 gataaaaagc aagaagattt tgtaccttct aacgcgctgt tgtcacaaga tagactgtgg 1080 caagctgtcg aaaatttgac ccaaagtaat gagacgatcg tggctgaaca aggcacttct 1140 ttcttcggtg cctcatctat atttctgaaa tcgaaatcac attttattgg tcaacccttg 1200 tggggatcta taggatacac tttccccgca gctctaggca gccaaattgc agataaagaa 1260 tctagacatt tattgtttat cggagatgga tcattgcaac tgactgtcca agaattagga 1320 ctagccatta gagagaagat aaacccaatc tgctttatca ttaataacga tggttacacg 1380 gttgagaggg aaattcatgg tccgaaccag agttataatg acattcctat gtggaattac 1440 tcaaaactgc cagaaagttt cggggcaacg gaagacagag ttgtgtccaa aattgtgaga 1500 acagaaaatg aattcgtatc cgtgatgaaa gaagctcaag cagatccaaa taggatgtat 1560 tggatagaac ttattctagc aaaggagggt gcacctaaag ttttgaaaaa gatgggtaag 1620 ttatttgcag aacaaaacaa gagc 1644 <210> 72 <211> 753 <212> DNA <213> Saccharomyces cerevisiae <400> 72 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300 gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360 cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420 ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480 agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540 aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600 ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660 gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720 ttaataatcc aaacaaacac acatattaca ata 753 <210> 73 <211> 316 <212> DNA <213> Saccharomyces cerevisiae <400> 73 gagtaagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata 60 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 120 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 180 cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cacccaattg 240 tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta tgtcctcaga 300 ggacaacacc tgtggt 316 <210> 74 <211> 30 <212> DNA Artificial sequence <220> <223> primer <400> 74 ggaattcaca catgaaagct ctggtttatc 30 <210> 75 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 75 gcgtccaggg cgtcaaagat caggcagc 28 <210> 76 <211> 55 <212> DNA Artificial sequence <220> <223> primer <400> 76 aaacaaacac acatattaca atagctgagg atgaaagctc tggtttatca cggtg 55 <210> 77 <211> 55 <212> DNA Artificial sequence <220> <223> primer <400> 77 atcataagaa attcgcttac tcttaattaa tcaggcagcg cctgcgttcg agagg 55 <210> 78 <211> 8994 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 78 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg 60 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 120 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa 180 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 240 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 300 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 360 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 420 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 480 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 540 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 600 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 660 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 720 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 780 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 840 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 900 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 960 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 1020 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 1080 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 1140 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1200 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 1260 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 1320 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 1380 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 1440 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 1500 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 1560 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 1620 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 1680 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 1740 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 1800 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 1860 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 1920 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 1980 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 2040 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 2100 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 2160 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 2220 ccccgaaaag tgccacctga acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa 2280 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 2340 caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa 2400 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag 2460 aaatgcaacg cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac 2520 aaaaatgcat cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc 2580 ccttgtgcgc tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt 2640 tagaagaagg ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc 2700 ccgcgtttac tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc 2760 gattatattc tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg 2820 atgattcttc attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta 2880 cgtataggaa atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac 2940 tacaattttt ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag 3000 tttagatgca agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca 3060 gagatatata gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt 3120 ttagtagctc gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc 3180 ttttggtttt caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa 3240 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca 3300 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca 3360 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat 3420 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg 3480 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt 3540 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcatactaag aaaccattat 3600 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt 3660 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 3720 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 3780 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatcga 3840 ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc accattatgg 3900 gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca ttgagtgttt 3960 tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat taggaatcgt 4020 agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc ttgtcaatat 4080 taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc aatttgctta 4140 cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt agattgcgta 4200 tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg tttctattat 4260 gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct ttttaagcaa 4320 ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg ttggaaccac 4380 ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct tcaatggcct 4440 taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac aagatagtgg 4500 cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat ggttcgtaca 4560 aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc aacaaaccca 4620 aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg ttgctggtga 4680 ttataatacc atttaggtgg gttgggttct taactaggat catggcggca gaatcaatca 4740 attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc acagtttttc 4800 tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata ggcaatggtg 4860 gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact tctggaacgg 4920 tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc ttaccaaagt 4980 aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca aattgtggct 5040 tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt aagttggcgt 5100 acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca ctaccggtac 5160 cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg gaggcttcca 5220 gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca attaaatgat 5280 tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga accttaatgg 5340 cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc ttcttagggg 5400 cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata tattgctgaa 5460 atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac 5520 aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga 5580 acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct ttttctccca 5640 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc 5700 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa 5760 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac 5820 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac 5880 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac 5940 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt 6000 tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta gaagttctcc 6060 tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 6120 aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 6180 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 6240 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 6300 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 6360 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 6420 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 6480 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 6540 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 6600 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 6660 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 6720 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 6780 ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc ctgcagcccg 6840 ggggatccgc atgcttgcat ttagtcgtgc aatgtatgac tttaagattt gtgagcagga 6900 agaaaaggga gaatcttcta acgataaacc cttgaaaaac tgggtagact acgctatgtt 6960 gagttgctac gcaggctgca caattacacg agaatgctcc cgcctaggat ttaaggctaa 7020 gggacgtgca atgcagacga cagatctaaa tgaccgtgtc ggtgaagtgt tcgccaaact 7080 tttcggttaa cacatgcagt gatgcacgcg cgatggtgct aagttacata tatatatata 7140 tatatatata tagccatagt gatgtctaag taacctttat ggtatatttc ttaatgtgga 7200 aagatactag cgcgcgcacc cacacacaag cttcgtcttt tcttgaagaa aagaggaagc 7260 tcgctaaatg ggattccact ttccgttccc tgccagctga tggaaaaagg ttagtggaac 7320 gatgaagaat aaaaagagag atccactgag gtgaaatttc agctgacagc gagtttcatg 7380 atcgtgatga acaatggtaa cgagttgtgg ctgttgccag ggagggtggt tctcaacttt 7440 taatgtatgg ccaaatcgct acttgggttt gttatataac aaagaagaaa taatgaactg 7500 attctcttcc tccttcttgt cctttcttaa ttctgttgta attaccttcc tttgtaattt 7560 tttttgtaat tattcttctt aataatccaa acaaacacac atattacaat agctagctga 7620 ggatgaaggc attagtttat catggggatc acaaaatttc gttagaagac aaaccaaaac 7680 ccactctgca gaaaccaaca gacgttgtgg ttagggtgtt gaaaacaaca atttgcggta 7740 ctgacttggg aatatacaaa ggtaagaatc ctgaagtggc agatggcaga atcctgggtc 7800 atgagggcgt tggcgtcatt gaagaagtgg gcgaatccgt gacacaattc aaaaaggggg 7860 ataaagtttt aatctcctgc gttactagct gtggatcgtg tgattattgc aagaagcaac 7920 tgtattcaca ctgtagagac ggtggctgga ttttaggtta catgatcgac ggtgtccaag 7980 ccgaatacgt cagaatacca catgctgaca attcattgta taagatcccg caaactatcg 8040 atgatgaaat tgcagtacta ctgtccgata ttttacctac tggacatgaa attggtgttc 8100 aatatggtaa cgttcaacca ggcgatgctg tagcaattgt aggagcaggt cctgttggaa 8160 tgtcagtttt gttaactgct caattttact cgcctagtac cattattgtt atcgacatgg 8220 acgaaaaccg tttacaatta gcgaaggagc ttggggccac acacactatt aactccggta 8280 ctgaaaatgt tgtcgaagct gtgcatcgta tagcagccga aggagtggat gtagcaatag 8340 aagctgttgg tatacccgca acctgggaca tctgtcagga aattgtaaaa cccggcgctc 8400 atattgccaa cgtgggagtt catggtgtta aggtggactt tgaaattcaa aagttgtgga 8460 ttaagaatct aaccatcacc actggtttgg ttaacactaa tactacccca atgttgatga 8520 aggtagcctc tactgataaa ttgcctttaa agaaaatgat tactcacagg tttgagttag 8580 ctgaaatcga acacgcatat caggttttct tgaatggcgc taaagaaaaa gctatgaaga 8640 ttattctatc taatgcaggt gccgcctaat taattaagag taagcgaatt tcttatgatt 8700 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga 8760 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt 8820 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccgag 8880 caaatgcctg caaatcgctc cccatttcac ccaattgtag atatgctaac tccagcaatg 8940 agttgatgaa tctcggtgtg tattttatgt cctcagagga caacacctgt ggta 8994 <210> 79 <211> 2145 <212> DNA Artificial sequence <220> <223> constructed chimeric gene <400> 79 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatatatata 300 tatagccata gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact 360 agcgcgcgca cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa 420 tgggattcca ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga 480 ataaaaagag agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat 540 gaacaatggt aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat 600 ggccaaatcg ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt 660 cctccttctt gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta 720 attattcttc ttaataatcc aaacaaacac acatattaca atagctagct gaggatgaag 780 gcattagttt atcatgggga tcacaaaatt tcgttagaag acaaaccaaa acccactctg 840 cagaaaccaa cagacgttgt ggttagggtg ttgaaaacaa caatttgcgg tactgacttg 900 ggaatataca aaggtaagaa tcctgaagtg gcagatggca gaatcctggg tcatgagggc 960 gttggcgtca ttgaagaagt gggcgaatcc gtgacacaat tcaaaaaggg ggataaagtt 1020 ttaatctcct gcgttactag ctgtggatcg tgtgattatt gcaagaagca actgtattca 1080 cactgtagag acggtggctg gattttaggt tacatgatcg acggtgtcca agccgaatac 1140 gtcagaatac cacatgctga caattcattg tataagatcc cgcaaactat cgatgatgaa 1200 attgcagtac tactgtccga tattttacct actggacatg aaattggtgt tcaatatggt 1260 aacgttcaac caggcgatgc tgtagcaatt gtaggagcag gtcctgttgg aatgtcagtt 1320 ttgttaactg ctcaatttta ctcgcctagt accattattg ttatcgacat ggacgaaaac 1380 cgtttacaat tagcgaagga gcttggggcc acacacacta ttaactccgg tactgaaaat 1440 gttgtcgaag ctgtgcatcg tatagcagcc gaaggagtgg atgtagcaat agaagctgtt 1500 ggtatacccg caacctggga catctgtcag gaaattgtaa aacccggcgc tcatattgcc 1560 aacgtgggag ttcatggtgt taaggtggac tttgaaattc aaaagttgtg gattaagaat 1620 ctaaccatca ccactggttt ggttaacact aatactaccc caatgttgat gaaggtagcc 1680 tctactgata aattgccttt aaagaaaatg attactcaca ggtttgagtt agctgaaatc 1740 gaacacgcat atcaggtttt cttgaatggc gctaaagaaa aagctatgaa gattattcta 1800 tctaatgcag gtgccgccta attaattaag agtaagcgaa tttcttatga tttatgattt 1860 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg 1920 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc 1980 aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg agcaaatgcc 2040 tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa tgagttgatg 2100 aatctcggtg tgtattttat gtcctcagag gacaacacct gtggt 2145 <210> 80 <211> 4280 <212> DNA Artificial sequence <220> <223> vector <400> 80 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg 60 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 120 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 180 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 240 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 300 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 360 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 420 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 480 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 540 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 600 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 660 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 720 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 780 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 840 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 900 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 960 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 1020 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 1080 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 1140 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 1200 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 1260 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 1320 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1380 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1440 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1500 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1560 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1620 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1680 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1740 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1800 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1860 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1920 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1980 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 2040 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 2100 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 2160 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 2220 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 2280 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 2340 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 2400 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 2460 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca 2520 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 2580 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 2640 tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccccgg ctctgagaca 2700 gtagtaggtt agtcatcgct ctaccgacgc gcaggaaaag aaagaagcat tgcggattac 2760 gtattctaat gttcagcccg cggaacgcca gcaaatcacc acccatgcgc atgatactga 2820 gtcttgtaca cgctgggctt ccagtgtact gagagtgcac cataccacag cttttcaatt 2880 caattcatca tttttttttt attctttttt ttgatttcgg tttctttgaa atttttttga 2940 ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata 3000 tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca 3060 cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg 3120 tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca 3180 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga 3240 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc 3300 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt 3360 cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt 3420 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat 3480 tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat 3540 gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt 3600 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg 3660 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa 3720 gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta caggatctga 3780 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga 3840 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa 3900 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt 3960 atatcagtta ttaccctatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 4020 gcatcaggaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 4080 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ttcagcccgc ggaacgccag 4140 caaatcacca cccatgcgca tgatactgag tcttgtacac gctgggcttc cagtgatgat 4200 acaacgagtt agccaaggtg agcacggatg tctaaattag aattacgttt taatatcttt 4260 ttttccatat ctagggctag 4280 <210> 81 <211> 30 <212> DNA Artificial sequence <220> <223> primer <400> 81 gcatgcttgc atttagtcgt gcaatgtatg 30 <210> 82 <211> 54 <212> DNA Artificial sequence <220> <223> primer <400> 82 gaacattaga atacgtaatc cgcaatgcac tagtaccaca ggtgttgtcc tctg 54 <210> 83 <211> 54 <212> DNA Artificial sequence <220> <223> primer <400> 83 cagaggacaa cacctgtggt actagtgcat tgcggattac gtattctaat gttc 54 <210> 84 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 84 caccttggct aactcgttgt atcatcac 28 <210> 85 <211> 100 <212> DNA Artificial sequence <220> <223> primer <400> 85 ttttaagccg aatgagtgac agaaaaagcc cacaacttat caagtgatat tgaacaaagg 60 gcgaaacttc gcatgcttgc atttagtcgt gcaatgtatg 100 <210> 86 <211> 98 <212> DNA Artificial sequence <220> <223> primer <400> 86 cccaattggt aaatattcaa caagagacgc gcagtacgta acatgcgaat tgcgtaattc 60 acggcgataa caccttggct aactcgttgt atcatcac 98 <210> 87 <211> 29 <212> DNA Artificial sequence <220> <223> primer <400> 87 caaaagccca tgtcccacac caaaggatg 29 <210> 88 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 88 caccatcgcg cgtgcatcac tgcatg 26 <210> 89 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 89 tcggtttttg caatatgacc tgtgggcc 28 <210> 90 <211> 22 <212> DNA Artificial sequence <220> <223> primer <400> 90 gagaagatgc ggccagcaaa ac 22 <210> 91 <211> 2745 <212> DNA Artificial sequence <220> <223> constructed coding region-terminator segment <400> 91 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaatgttgtc ctggttgctg tggttaagcg 1740 gccgcgttaa ttcaaattaa ttgatatagt tttttaatga gtattgaatc tgtttagaaa 1800 taatggaata ttatttttat ttatttattt atattattgg tcggctcttt tcttctgaag 1860 gtcaatgaca aaatgatatg aaggaaataa tgatttctaa aattttacaa cgtaagatat 1920 ttttacaaaa gcctagctca tcttttgtca tgcactattt tactcacgct tgaaattaac 1980 ggccagtcca ctgcggagtc atttcaaagt catcctaatc gatctatcgt ttttgatagc 2040 tcattttgga gttcgcgatt gtcttctgtt attcacaact gttttaattt ttatttcatt 2100 ctggaactct tcgagttctt tgtaaagtct ttcatagtag cttactttat cctccaacat 2160 atttaacttc atgtcaattt cggctcttaa attttccaca tcatcaagtt caacatcatc 2220 ttttaacttg aatttattct ctagctcttc caaccaagcc tcattgctcc ttgatttact 2280 ggtgaaaagt gatacacttt gcgcgcaatc caggtcaaaa ctttcctgca aagaattcac 2340 caatttctcg acatcatagt acaatttgtt ttgttctccc atcacaattt aatatacctg 2400 atggattctt atgaagcgct gggtaatgga cgtgtcactc tacttcgcct ttttccctac 2460 tccttttagt acggaagaca atgctaataa ataagagggt aataataata ttattaatcg 2520 gcaaaaaaga ttaaacgcca agcgtttaat tatcagaaag caaacgtcgt accaatcctt 2580 gaatgcttcc caattgtata ttaagagtca tcacagcaac atattcttgt tattaaatta 2640 attattattg atttttgata ttgtataaaa aaaccaaata tgtataaaaa aagtgaataa 2700 gt; <210> 92 <211> 27 <212> DNA Artificial sequence <220> <223> primer <400> 92 gacttttgga agcctgaaga aactggc 27 <210> 93 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 93 cttggcagca acaggactag 20 <210> 94 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 94 ccaggccaat tcaacagact gtcggc 26 <210> 95 <211> 2347 <212> DNA Artificial sequence <220> <223> constructed URA3 marker with flanking homologous repeat sequences        for HIS gene replacement and marker excision <400> 95 gcattgcgga ttacgtattc taatgttcag gtgctggaag aagagctgct taaccgccgc 60 gcccagggtg aagatccacg ctactttacc ctgcgtcgtc tggatttcgg cggctgtcgt 120 ctttcgctgg caacgccggt tgatgaagcc tgggacggtc cgctctcctt aaacggtaaa 180 cgtatcgcca cctcttatcc tcacctgctc aagcgttatc tcgaccagaa aggcatctct 240 tttaaatcct gcttactgaa cggttctgtt gaagtcgccc cgcgtgccgg actggcggat 300 gcgatttgcg atctggtttc caccggtgcc acgctggaag ctaacggcct gcgcgaagtc 360 gaagttatct atcgctcgaa agcctgcctg attcaacgcg atggcgaaat ggaagaatcc 420 aaacagcaac tgatcgacaa actgctgacc cgtattcagg gtgtgatcca ggcgcgcgaa 480 tcaaaataca tcatgatgca cgcaccgacc gaacgtctgg atgaagtcat ggtacctact 540 gagagtgcac cataccacag cttttcaatt caattcatca tttttttttt attctttttt 600 ttgatttcgg tttctttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg 660 aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg 720 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata 780 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc 840 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg 900 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa 960 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc 1020 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa 1080 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac 1140 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga 1200 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct 1260 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt 1320 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat 1380 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac 1440 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc 1500 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata 1560 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact 1620 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa 1680 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg ttaatatttt 1740 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 1800 cggcaaaatc tctagagtgc tggaagaaga gctgcttaac cgccgcgccc agggtgaaga 1860 tccacgctac tttaccctgc gtcgtctgga tttcggcggc tgtcgtcttt cgctggcaac 1920 gccggttgat gaagcctggg acggtccgct ctccttaaac ggtaaacgta tcgccacctc 1980 ttatcctcac ctgctcaagc gttatctcga ccagaaaggc atctctttta aatcctgctt 2040 actgaacggt tctgttgaag tcgccccgcg tgccggactg gcggatgcga tttgcgatct 2100 ggtttccacc ggtgccacgc tggaagctaa cggcctgcgc gaagtcgaag ttatctatcg 2160 ctcgaaagcc tgcctgattc aacgcgatgg cgaaatggaa gaatccaaac agcaactgat 2220 cgacaaactg ctgacccgta ttcagggtgt gatccaggcg cgcgaatcaa aatacatcat 2280 gatgcacgca ccgaccgaac gtctggatga agtcatccag tgatgataca acgagttagc 2340 caaggtg 2347 <210> 96 <211> 80 <212> DNA Artificial sequence <220> <223> primer <400> 96 cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 97 <211> 81 <212> DNA Artificial sequence <220> <223> primer <400> 97 tatacacatg tatatatatc gtatgctgca gctttaaata atcggtgtca caccttggct 60 aactcgttgt atcatcactg g 81 <210> 98 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 98 gacttgaata atgcagcggc gcttgc 26 <210> 99 <211> 30 <212> DNA Artificial sequence <220> <223> primer <400> 99 ccaccctctt caattagcta agatcatagc 30 <210> 100 <211> 25 <212> DNA Artificial sequence <220> <223> primer <400> 100 aaaaattgat tctcatcgta aatgc 25 <210> 101 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 101 ctgcagcgag gagccgtaat 20 <210> 102 <211> 90 <212> DNA Artificial sequence <220> <223> primer <400> 102 atggttcatt taggtccaaa aaaaccacaa gccagaaagg gttccatggc cgatgtgcca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 103 <211> 91 <212> DNA Artificial sequence <220> <223> primer <400> 103 ttaagcaccg atgataccaa cggacttacc ttcagcaatt cttttttggg ccaaagcagc 60 caccttggct aactcgttgt atcatcactg g 91 <210> 104 <211> 24 <212> DNA Artificial sequence <220> <223> primer <400> 104 ctaggatgag tagcagcacg ttcc 24 <210> 105 <211> 26 <212> DNA Artificial sequence <220> <223> primer <400> 105 ccaattccgt gatgtctctt tgttgc 26 <210> 106 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 106 gtgaacgagt tcacaaccgc 20 <210> 107 <211> 22 <212> DNA Artificial sequence <220> <223> primer <400> 107 gttcgttcca gaattatcac gc 22 <210> 108 <211> 28 <212> DNA Artificial sequence <220> <223> primer <400> 108 ggatccgcat gcttgcattt agtcgtgc 28 <210> 109 <211> 20 <212> DNA Artificial sequence <220> <223> primer <400> 109 gggatgcgga cgtattcggc 20 <210> 110 <211> 4506 <212> DNA <213> Saccharomyces cerevisiae <400> 110 atgtcgaata ccccttataa ttcatctgtg ccttccattg catccatgac ccagtcttcg 60 gtctcaagaa gtcctaacat gcatacagca actacgcccg gtgccaacac cagctctaac 120 tctccaccct tgcacatgtc ttcagattcg tccaagatca agaggaagcg taacagaatt 180 ccgctcagtt gcaccatttg tcggaaaagg aaagtcaaat gtgacaaact cagaccacac 240 tgccagcagt gcactaaaac tggggtagcc catctctgcc actacatgga acagacctgg 300 gcagaagagg cagagaaaga attgctgaag gacaacgaat taaagaagct tagggagcgc 360 gtaaaatctt tagaaaagac tctttctaag gtgcactctt ctccttcgtc taactccttg 420 aaaagttaca acactcccga gagcagcaac ctgtttatgg gtagcgatga acacaccacc 480 cttgttaatg caaatacagg ctctgcttcc tctgcctcgc atatgcatca gcaacaacag 540 caacagcagc aacaggaaca acaacaagac ttttccagaa gtgcgaacgc caacgcgaat 600 tcctcgtccc tttctatctc aaataaatat gacaacgatg agctggactt aactaaggac 660 tttgatcttt tgcatatcaa aagtaacgga accatccact taggtgccac ccactggttg 720 tctatcatga aaggtgaccc gtacctaaaa cttttgtggg gtcatatctt cgctatgagg 780 gaaaagttaa atgaatggta ctaccaaaaa aattcgtact ctaagctgaa gtcaagcaaa 840 tgtcccatca atcacgcgca agcgccgcct tctgccgctg ccgccgctac cagaaaatgt 900 cctgttgatc actccgcgtt ttcgtctggc atggtggccc caaaggagga gactcctctt 960 cctaggaaat gtccagttga ccacaccatg ttctcttcgg gaatgattcc tcccagagag 1020 gacacttcgt cccagaagag gtgtcccgtt gaccacacca tgtattccgc aggaatgatg 1080 ccgcccaagg acgagacacc ttccccattt tccactaaag ctatgataga ccataacaag 1140 catacaatga atccgcctca gtcaaaatgt cctgtggacc atagaaacta tatgaaggat 1200 tatccctctg acatggcaaa ttcttcttcg aacccggcaa gtcgttgccc cattgaccat 1260 tcaagcatga aaaatacagc ggccttacca gcttcaacgc acaataccat cccacaccac 1320 caaccacagt ccggatctca tgctcgttcg catcccgcac aaagcaggaa acatgattcc 1380 tacatgacag aatctgaagt cctcgcaaca ctttgtgaga tgttgccacc aaagcgcgtc 1440 atcgcattat tcatcgagaa attcttcaaa catttatacc ctgccattcc aatcttagat 1500 gaacagaatt tcaaaaatca cgtgaatcaa atgctttcgt tgtcttcgat gaatcccaca 1560 gttaacaact ttggtatgag catgccatct tcatctacac tagagaacca acccataaca 1620 caaatcaatc ttccaaaact ttccgattct tgtaacttag gtattctgat aataatcttg 1680 agattgacat ggctatccat accttctaat tcctgcgaag tcgacctggg agaagaaagt 1740 ggctcatttt tagtgcccaa cgaatctagc aatatgtctg catctgcatt gacctcgatg 1800 gctaaagaag aatcacttct gctaaagcat gagacaccgg tcgaggcact ggagctatgt 1860 caaaaatact tgattaaatt cgatgaactt tctagtattt ccaataacaa cgttaattta 1920 accacggtgc agtttgccat tttttacaac ttctatatga aaagtgcctc taatgatttg 1980 actaccttga caaataccaa caacactggc atggccaatc ctggtcacga ttccgagtct 2040 caccagatcc tattgtccaa tattactcaa atggccttta gttgtgggtt acacagagac 2100 cctgataatt ttcctcaatt aaacgctacc attccagcaa ccagccagga cgtgtctaac 2160 aacgggagca aaaaggcaaa ccctagcacc aatccaactt tgaataacaa catgtctgct 2220 gccactacca acagcagtag cagatctggc agtgctgatt caagaagtgg ttctaaccct 2280 gtgaacaaga aggaaaatca ggttagtatc gaaagattta aacacacttg gaggaaaatt 2340 tggtattaca ttgttagcat ggatgttaac caatctcttt ccctggggag ccctcgacta 2400 ctaagaaatc tgagggattt cagcgataca aagctaccaa gtgcgtcaag gattgattat 2460 gttcgcgata tcaaagagtt aatcattgtg aagaatttta ctcttttttt ccaaattgat 2520 ttgtgtatta ttgctgtatt aaatcacatt ttgaatgttt ctttagcaag aagcgtgaga 2580 aaatttgaac tggattcatt gattaattta ttgaaaaatc tgacctatgg tactgagaat 2640 gtcaatgatg tagtgagctc cttgatcaac aaagggttat taccaacttc ggaaggtggt 2700 tctgtagatt caaataatga tgaaatttac ggtctaccga aactacccga tattctaaac 2760 catggtcaac ataaccaaaa cttgtatgct gatggaagaa atacttctag tagtgatata 2820 gataagaaat tggaccttcc tcacgaatct acaacgagag ctctattctt ttccaagcat 2880 atgacaatta gaatgttgct gtacttattg aactacattt tgtttactca ttatgaacca 2940 atgggcagtg aagatcctgg tactaatatc ctagctaagg agtacgctca agaggcatta 3000 aattttgcca tggatggcta cagaaactgc atgattttct tcaacaatat cagaaacacc 3060 aattcactat tcgattacat gaatgttatc ttgtcttacc cttgtttgga cattggacat 3120 cgttctttac aatttatcgt ttgtttgatc ctgagagcta aatgtggccc attgactggt 3180 atgcgtgaat catcgatcat tactaatggt acatcaagtg gatttaatag ttcggtagaa 3240 gatgaggacg tcaaagttaa acaagaatct tctgatgaat tgaaaaaaga cgatttcatg 3300 aaagatgtaa atttggattc aggcgattca ttagcagaga ttctaatgtc aagaatgctg 3360 ctatttcaaa aactaacaaa acaactatca aagaagtaca actacgctat tcgtatgaac 3420 aaatccactg gattctttgt ctctttacta gatacacctt caaagaaatc agactcgaaa 3480 tcgggtggta gttcattcat gttgggtaat tggaaacatc caaaggtttc aaacatgagc 3540 ggatttcttg ctggtgacaa agaccaatta cagaaatgcc ccgtgtacca agatgcgctg 3600 gggtttgtta gtccaaccgg tgctaatgaa ggttctgctc cgatgcaagg catgtcctta 3660 cagggctcta ctgctaggat gggagggacc cagttgccac caattagatc atacaaacct 3720 atcacgtaca caagtagtaa tctacgtcgt atgaatgaaa cgggtgaggc agaagctaag 3780 agaagaagat ttaatgatgg ctatattgat aataatagta acaacgatat acctagagga 3840 atcagcccaa aaccttcaaa tgggctatca tcggtgcagc cactattatc gtcattttcc 3900 atgaaccagc taaacggggg taccattcca acggttccat cgttaaccaa cattacttca 3960 caaatgggag ctttaccatc tttagatagg atcaccacta atcaaataaa tttgccagac 4020 ccatctagag atgaagcatt tgacaactcc atcaagcaaa tgacgcctat gacaagtgca 4080 ttcatgaatg ctaatactac aattccaagt tcaactttaa acgggaatat gaacatgaat 4140 ggagctggaa ctgcgaatac agatacaagt gccaacggca gtgctttatc gacactgaca 4200 agcccacaag gctcagactt agcatccaat tctgctacac agtataaacc tgacttagaa 4260 gactttttga tgcaaaattc taactttaat gggctaatga taaatccttc cagtctggta 4320 gaagttgttg gtggatacaa cgatcctaat aaccttggaa gaaatgacgc ggttgatttt 4380 ctacccgttg ataatgttga aattgatggt gttggaataa aaatcaacta tcatctacta 4440 actagtattt acgttactag tatattatca tatacggtgt tagaagatga cgcaaatgat 4500 gagaaa 4506 <210> 111 <211> 99 <212> DNA Artificial sequence <220> <223> primer <400> 111 tcctttctca attattattt tctactcata acctcacgca aaataacaca gtcaaatcaa 60 tcaaagtatg actgacaaaa aaactcttaa agacttaag 99 <210> 112 <211> 77 <212> DNA Artificial sequence <220> <223> primer <400> 112 gaacattaga atacgtaatc cgcaatgctt ctttcttttc cgtttaacgt atagacttct 60 aatatatttc tccatac 77 <210> 113 <211> 45 <212> DNA Artificial sequence <220> <223> primer <400> 113 aaacggaaaa gaaagaagca ttgcggatta cgtattctaa tgttc 45 <210> 114 <211> 88 <212> DNA Artificial sequence <220> <223> primer <400> 114 tatttttcgt tacataaaaa tgcttataaa actttaacta ataattagag attaaatcgc 60 caccttggct aactcgttgt atcatcac 88 <210> 115 <211> 1713 <212> DNA <213> Streptococcus mutans <400> 115 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaa 1713 <210> 116 <211> 571 <212> PRT <213> Streptococcus mutans <400> 116 Met Thr Asp Lys Lys Thr Leu Lys Asp Leu Arg Asn Arg Ser Ser Val 1 5 10 15 Tyr Asp Ser Met Val Lys Ser Pro Asn Arg Ala Met Leu Arg Ala Thr             20 25 30 Gly Met Gln Asp Glu Asp Phe Glu Lys Pro Ile Val Gly Val Ile Ser         35 40 45 Thr Trp Ala Glu Asn Thr Pro Cys Asn Ile His Leu His Asp Phe Gly     50 55 60 Lys Leu Ala Lys Val Gly Val Lys Glu Ala Gly Ala Trp Pro Val Gln 65 70 75 80 Phe Gly Thr Ile Thr Val Ser Asp Gly Ile Ala Met Gly Thr Gln Gly                 85 90 95 Met Arg Phe Ser Leu Thr Ser Arg Asp Ile Ile Ala Asp Ser Ile Glu             100 105 110 Ala Ala Met Gly Gly His Asn Ala Asp Ala Phe Val Ala Ile Gly Gly         115 120 125 Cys Asp Lys Asn Met Pro Gly Ser Val Ile Ala Met Ala Asn Met Asp     130 135 140 Ile Pro Ala Ile Phe Ala Tyr Gly Gly Thr Ile Ala Pro Gly Asn Leu 145 150 155 160 Asp Gly Lys Asp Ile Asp Leu Val Ser Val Phe Glu Gly Val Gly His                 165 170 175 Trp Asn His Gly Asp Met Thr Lys Glu Glu Val Lys Ala Leu Glu Cys             180 185 190 Asn Ala Cys Pro Gly Pro Gly Gly Cys Gly Gly Met Tyr Thr Ala Asn         195 200 205 Thr Met Ala Thr Ala Ile Glu Val Leu Gly Leu Ser Leu Pro Gly Ser     210 215 220 Ser Ser His Pro Ala Glu Ser Ala Glu Lys Lys Ala Asp Ile Glu Glu 225 230 235 240 Ala Gly Arg Ala Val Val Lys Met Leu Glu Met Gly Leu Lys Pro Ser                 245 250 255 Asp Ile Leu Thr Arg Glu Ala Phe Glu Asp Ala Ile Thr Val Thr Met             260 265 270 Ala Leu Gly Gly Ser Thr Asn Ser Thr Leu His Leu Leu Ala Ile Ala         275 280 285 His Ala Ala Asn Val Glu Leu Thr Leu Asp Asp Phe Asn Thr Phe Gln     290 295 300 Glu Lys Val Pro His Leu Ala Asp Leu Lys Pro Ser Gly Gln Tyr Val 305 310 315 320 Phe Gln Asp Leu Tyr Lys Val Gly Gly Val Pro Ala Val Met Lys Tyr                 325 330 335 Leu Leu Lys Asn Gly Phe Leu His Gly Asp Arg Ile Thr Cys Thr Gly             340 345 350 Lys Thr Val Ala Glu Asn Leu Lys Ala Phe Asp Asp Leu Thr Pro Gly         355 360 365 Gln Lys Val Ile Met Pro Leu Glu Asn Pro Lys Arg Glu Asp Gly Pro     370 375 380 Leu Ile Ile Leu His Gly Asn Leu Ala Pro Asp Gly Ala Val Ala Lys 385 390 395 400 Val Ser Gly Val Lys Val Arg Arg His Val Gly Pro Ala Lys Val Phe                 405 410 415 Asn Ser Glu Glu Glu Ala Ile Glu Ala Val Leu Asn Asp Asp Ile Val             420 425 430 Asp Gly Asp Val Val Val Val Arg Phe Val Gly Pro Lys Gly Gly Pro         435 440 445 Gly Met Pro Glu Met Leu Ser Leu Ser Ser Met Ile Val Gly Lys Gly     450 455 460 Gln Gly Glu Lys Val Ala Leu Leu Thr Asp Gly Arg Phe Ser Gly Gly 465 470 475 480 Thr Tyr Gly Leu Val Val Gly His Ile Ala Pro Glu Ala Gln Asp Gly                 485 490 495 Gly Pro Ile Ala Tyr Leu Gln Thr Gly Asp Ile Val Thr Ile Asp Gln             500 505 510 Asp Thr Lys Glu Leu His Phe Asp Ile Ser Asp Glu Glu Leu Lys His         515 520 525 Arg Gln Glu Thr Ile Glu Leu Pro Pro Leu Tyr Ser Arg Gly Ile Leu     530 535 540 Gly Lys Tyr Ala His Ile Val Ser Ser Ala Ser Arg Gly Ala Val Thr 545 550 555 560 Asp Phe Trp Lys Pro Glu Glu Thr Gly Lys Lys                 565 570 <210> 117 <211> 548 <212> PRT <213> Lactococcus lactis <400> 117 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu             20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn         35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys     50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile                 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His             100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu         115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val     130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro                 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln             180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro         195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr     210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile                 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser             260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr         275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn     290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys                 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala             340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln         355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala     370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile                 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu             420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn         435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu     450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser                 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala             500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys         515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu     530 535 540 Gln asn lys ser 545 <210> 118 <211> 1125 <212> DNA Artificial sequence <220> <223> horse ADH coding region codon optimized for S. cerevisiae        expression <400> 118 atgtcaacag ccggtaaagt tattaagtgt aaagcggcag ttttgtggga agagaaaaag 60 ccgtttagca tagaagaagt agaagtagcg ccaccaaaag cacacgaggt tagaatcaag 120 atggttgcca ccggaatctg tagatccgac gaccatgtgg tgagtggcac tctagttact 180 cctttgccag taatcgcggg acacgaggct gccggaatcg ttgaatccat aggtgaaggt 240 gttaccactg ttcgtcctgg tgataaagtg atcccactgt tcactcctca atgtggtaag 300 tgtagagtct gcaaacatcc tgagggtaat ttctgcctta aaaatgattt gtctatgcct 360 agaggtacta tgcaggatgg tacaagcaga tttacatgca gagggaaacc tatacaccat 420 ttccttggta cttctacatt ttcccaatac acagtggtgg acgagatatc tgtcgctaaa 480 atcgatgcag cttcaccact ggaaaaagtt tgcttgatag ggtgcggatt ttccaccggt 540 tacggttccg cagttaaagt tgcaaaggtt acacagggtt cgacttgtgc agtattcggt 600 ttaggaggag taggactaag cgttattatg gggtgtaaag ctgcaggcgc agcgaggatt 660 ataggtgtag acatcaataa ggacaaattt gcaaaagcta aggaggtcgg ggctactgaa 720 tgtgttaacc ctcaagatta taagaaacca atacaagaag tccttactga aatgtcaaac 780 ggtggagttg atttctcttt tgaagttata ggccgtcttg atactatggt aactgcgttg 840 tcctgctgtc aagaggcata tggagtcagt gtgatcgtag gtgttcctcc tgattcacaa 900 aatttgtcga tgaatcctat gctgttgcta agcggtcgta catggaaggg agctatattt 960 ggcggtttta agagcaagga tagtgttcca aaacttgttg ccgactttat ggcgaagaag 1020 tttgctcttg atcctttaat tacacatgta ttgccattcg agaaaatcaa tgaagggttt 1080 gatttgttaa gaagtggtga atctattcgt acaattttaa ctttt 1125 <210> 119 <211> 375 <212> PRT <213> Equus caballus <400> 119 Met Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro             20 25 30 Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg         35 40 45 Ser Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val     50 55 60 Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly 65 70 75 80 Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro                 85 90 95 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys             100 105 110 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr         115 120 125 Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr     130 135 140 Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys 145 150 155 160 Ile Asp Ala Ser Ser Leu Glu Lys Val Cys Leu Ile Gly Cys Gly                 165 170 175 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln             180 185 190 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val         195 200 205 Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp     210 215 220 Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 240 Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr                 245 250 255 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg             260 265 270 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly         275 280 285 Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met     290 295 300 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe 305 310 315 320 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe                 325 330 335 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro             340 345 350 Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser         355 360 365 Ile Arg Thr Ile Leu Thr Phe     370 375 <210> 120 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 120 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 121 <211> 9089 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 121 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac tggccattaa 2040 tctttcccat attagatttc gccaagccat gaaagttcaa gaaaggtctt tagacgaatt 2100 acccttcatt tctcaaactg gcgtcaaggg atcctggtat ggttttatcg ttttatttct 2160 ggttcttata gcatcgtttt ggacttctct gttcccatta ggcggttcag gagccagcgc 2220 agaatcattc tttgaaggat acttatcctt tccaattttg attgtctgtt acgttggaca 2280 taaactgtat actagaaatt ggactttgat ggtgaaacta gaagatatgg atcttgatac 2340 cggcagaaaa caagtagatt tgactcttcg tagggaagaa atgaggattg agcgagaaac 2400 attagcaaaa agatccttcg taacaagatt tttacatttc tggtgttgaa gggaaagata 2460 tgagctatac agcggaattt ccatatcact cagattttgt tatctaattt tttccttccc 2520 acgtccgcgg gaatctgtgt atattactgc atctagatat atgttatctt atcttggcgc 2580 gtacatttaa ttttcaacgt attctataag aaattgcggg agtttttttc atgtagatga 2640 tactgactgc acgcaaatat aggcatgatt tataggcatg atttgatggc tgtaccgata 2700 ggaacgctaa gagtaacttc agaatcgtta tcctggcgga aaaaattcat ttgtaaactt 2760 taaaaaaaaa agccaatatc cccaaaatta ttaagagcgc ctccattatt aactaaaatt 2820 tcactcagca tccacaatgt atcaggtatc tactacagat attacatgtg gcgaaaaaga 2880 caagaacaat gcaatagcgc atcaagaaaa aacacaaagc tttcaatcaa tgaatcgaaa 2940 atgtcattaa aatagtatat aaattgaaac taagtcataa agctataaaa agaaaattta 3000 tttaaatgca agatttaaag taaattcacg gccctgcagg ccctaacctg ctaggacaca 3060 acgtctttgc ctggtaaagt ttctagctga cgtgattcct tcacctgtgg atccggcaat 3120 tgtaaaggtt gtgaaaccct cagcttcata accgacacct gcaaatgact ttgcattctt 3180 aacaaagata gttgtatcaa tttcacgttc gaatctatta aggttatcga tgttcttaga 3240 ataaatgtag gcggaatgtt ttctattctg ctcagctatc ttggcgtatt taatggcttc 3300 atcaatgtcc ttcactctaa ctataggcaa aattggcatc atcaactccg tcataacgaa 3360 cggatggttt gcgttgactt cacaaataat acactttaca ttacttggtg actctacatc 3420 tatttcatcc aaaaacagtt tagcgtcctt accaacccac ttcttattaa tgaaatattc 3480 ttgagtttca ttgttctttt gaagaacaag gtctatcagc ttggatactt ggtcttcatt 3540 gataatgacg gcgttgtttt tcaacatgtt agagatcaga tcatctgcaa cgttttcaaa 3600 cacgaacact tctttttccg cgatacaagg aagattgttg tcaaacgaac aaccttcaat 3660 aatgcttctg ccggccttct cgatatctgc tgtatcgtct acaataaccg gaggattacc 3720 cgcgccagct ccgatggcct ttttaccaga attaagaagg gtttttacca tacccgggcc 3780 acccgtaccg cacaacaatt ttatggatgg atgtttgata atagcgtcta aactttccat 3840 agttgggttc tttatagtag tgacaaggtt ttcaggtcca ccacagctaa ttatggcttt 3900 gtttatcatt tctactgcga aagcgacaca ctttttggcg catgggtgac cattaaatac 3960 aactgcattc cccgcagcta tcatacctat agaattgcag ataacggttt ctgttggatt 4020 cgtgcttgga gttatagcgc cgataactcc gtatggactc atttcaacca ctgttagtcc 4080 attatcgccg gaccatgctg ttgttgtcag atcttcagtg cctggggtat acttggccac 4140 taattcatgt ttcaagattt tatcctcata ccttcccatg tgggtttcct ccaggatcat 4200 tgtggctaag acctctttat tctgtaatgc ggcttttctt atttcggtga ttattttctc 4260 tctttgttcc tttgtgtagt gtagggaaag aatcttttgt gcatgtactg cagaagaaat 4320 ggcattctca acattttcaa atactccaaa acatgaagag ttatctttgt aattctttaa 4380 gttgatgttt tcaccattag tcttcacttt caagtctttg gtggttggga ttaaggtatc 4440 tttatccatg gtgtttgttt atgtgtgttt attcgaaact aagttcttgg tgttttaaaa 4500 ctaaaaaaaa gactaactat aaaagtagaa tttaagaagt ttaagaaata gatttacaga 4560 attacaatca atacctaccg tctttatata cttattagtc aagtagggga ataatttcag 4620 ggaactggtt tcaacctttt ttttcagctt tttccaaatc agagagagca gaaggtaata 4680 gaaggtgtaa gaaaatgaga tagatacatg cgtgggtcaa ttgccttgtg tcatcattta 4740 ctccaggcag gttgcatcac tccattgagg ttgtgcccgt tttttgcctg tttgtgcccc 4800 tgttctctgt agttgcgcta agagaatgga cctatgaact gatggttggt gaagaaaaca 4860 atattttggt gctgggattc tttttttttc tggatgccag cttaaaaagc gggctccatt 4920 atatttagtg gatgccagga ataaactgtt cacccagaca cctacgatgt tatatattct 4980 gtgtaacccg ccccctattt tgggcatgta cgggttacag cagaattaaa aggctaattt 5040 tttgactaaa taaagttagg aaaatcacta ctattaatta tttacgtatt ctttgaaatg 5100 gcagtattga taatgataaa ctcgaactga aaaagcgtgt tttttattca aaatgattct 5160 aactccctta cgtaatcaag gaatcttttt gccttggcct ccgcgtcatt aaacttcttg 5220 ttgttgacgc taacattcaa cgctagtata tattcgtttt tttcaggtaa gttcttttca 5280 acgggtctta ctgatgaggc agtcgcgtct gaacctgtta agaggtcaaa tatgtcttct 5340 tgaccgtacg tgtcttgcat gttattagct ttgggaattt gcatcaagtc ataggaaaat 5400 ttaaatcttg gctctcttgg gctcaaggtg acaaggtcct cgaaaatagg gcgcgcccca 5460 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 5520 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5580 ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta 5640 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5700 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5760 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5820 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5880 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5940 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6000 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6060 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6120 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6180 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6240 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6300 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6360 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6420 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6480 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6540 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6600 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6660 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6720 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6780 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6840 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6900 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6960 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7020 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7080 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7140 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7200 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7260 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7320 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7380 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7440 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7500 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7560 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7620 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa 7680 cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca 7740 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta 7800 ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt 7860 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta 7920 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc 7980 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca 8040 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg 8100 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc 8160 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt 8220 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa 8280 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt 8340 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga 8400 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag 8460 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata 8520 cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg 8580 gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc 8640 tgaagttcct atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc 8700 gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt 8760 cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg 8820 tgtttatgct taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag 8880 tacctcctgt gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc 8940 ctttagctgt tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat 9000 catttccttt gatattggat catactaaga aaccattatt atcatgacat taacctataa 9060 aaataggcgt atcacgaggc cctttcgtc 9089 <210> 122 <211> 1023 <212> DNA <213> Saccharomyces cerevisiae <400> 122 caccgcggtg gggcgcgccc tattttcgag gaccttgtca ccttgagccc aagagagcca 60 agatttaaat tttcctatga cttgatgcaa attcccaaag ctaataacat gcaagacacg 120 tacggtcaag aagacatatt tgacctctta acaggttcag acgcgactgc ctcatcagta 180 agacccgttg aaaagaactt acctgaaaaa aacgaatata tactagcgtt gaatgttagc 240 gtcaacaaca agaagtttaa tgacgcggag gccaaggcaa aaagattcct tgattacgta 300 agggagttag aatcattttg aataaaaaac acgctttttc agttcgagtt tatcattatc 360 aatactgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc taactttatt 420 tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa atagggggcg 480 ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca 540 ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga atcccagcac 600 caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac 660 agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct 720 gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt 780 acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa 840 ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg gtaggattattg 900 attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt 960 ttttttagtt ttaaaacacc aagaacttag tttcgaataa acacacataa actagtaaac 1020 aaa 1023 <210> 123 <211> 21 <212> DNA Artificial sequence <220> <223> primer <400> 123 caaaagctga gctccaccgc g 21 <210> 124 <211> 44 <212> DNA Artificial sequence <220> <223> primer <400> 124 gtttactagt ttatgtgtgt ttattcgaaa ctaagttctt ggtg 44 <210> 125 <400> 125 000 <210> 126 <211> 39 <212> DNA Artificial sequence <220> <223> primer <400> 126 cacacatatt acaatagcta gctgaggatg aaagctctg 39 <210> 127 <211> 39 <212> DNA Artificial sequence <220> <223> primer <400> 127 cagagctttc atcctcagct agctattgta atatgtgtg 39 <210> 128 <211> 9491 <212> DNA Artificial sequence <220> &Lt; 223 > constructed plasmid <400> 128 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt 720 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca 780 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag 840 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag 900 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag 960 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta 1020 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca 1080 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct 1140 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat 1200 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat 1260 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt 1320 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt 1380 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata 1440 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 1500 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 1560 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 1620 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 1680 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 1740 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 1800 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 1860 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 1980 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2040 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcg 2100 acggcgcgcc actggtagag agcgactttg tatgccccaa ttgcgaaacc cgcgatatcc 2160 ttctcgattc tttagtaccc gaccaggaca aggaaaagga ggtcgaaacg tttttgaaga 2220 aacaagagga actacacgga agctctaaag atggcaacca gccagaaact aagaaaatga 2280 agttgatgga tccaactggc accgctggct tgaacaacaa taccagcctt ccaacttctg 2340 taaataacgg cggtacgcca gtgccaccag taccgttacc tttcggtata cctcctttcc 2400 ccatgtttcc aatgcccttc atgcctccaa cggctactat cacaaatcct catcaagctg 2460 acgcaagccc taagaaatga ataacaatac tgacagtact aaataattgc ctacttggct 2520 tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg attatcgtaa 2580 tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg 2640 ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt ggcatcctct 2700 ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat atctaacaac 2760 tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag tattggatgg 2820 ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa aatctacaat 2880 caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt cgcccacgtt 2940 aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat aaaaagacaa 3000 agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat tcttctgttc 3060 ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa ctagtatgac 3120 tgacaaaaaa actcttaaag acttaagaaa tcgtagttct gtttacgatt caatggttaa 3180 atcacctaat cgtgctatgt tgcgtgcaac tggtatgcaa gatgaagact ttgaaaaacc 3240 tatcgtcggt gtcatttcaa cttgggctga aaacacacct tgtaatatcc acttacatga 3300 ctttggtaaa ctagccaaag tcggtgttaa ggaagctggt gcttggccag ttcagttcgg 3360 aacaatcacg gtttctgatg gaatcgccat gggaacccaa ggaatgcgtt tctccttgac 3420 atctcgtgat attattgcag attctattga agcagccatg ggaggtcata atgcggatgc 3480 ttttgtagcc attggcggtt gtgataaaaa catgcccggt tctgttatcg ctatggctaa 3540 catggatatc ccagccattt ttgcttacgg cggaacaatt gcacctggta atttagacgg 3600 caaagatatc gatttagtct ctgtctttga aggtgtcggc cattggaacc acggcgatat 3660 gaccaaagaa gaagttaaag ctttggaatg taatgcttgt cccggtcctg gaggctgcgg 3720 tggtatgtat actgctaaca caatggcgac agctattgaa gttttgggac ttagccttcc 3780 gggttcatct tctcacccgg ctgaatccgc agaaaagaaa gcagatattg aagaagctgg 3840 tcgcgctgtt gtcaaaatgc tcgaaatggg cttaaaacct tctgacattt taacgcgtga 3900 agcttttgaa gatgctatta ctgtaactat ggctctggga ggttcaacca actcaaccct 3960 tcacctctta gctattgccc atgctgctaa tgtggaattg acacttgatg atttcaatac 4020 tttccaagaa aaagttcctc atttggctga tttgaaacct tctggtcaat atgtattcca 4080 agacctttac aaggtcggag gggtaccagc agttatgaaa tatctcctta aaaatggctt 4140 ccttcatggt gaccgtatca cttgtactgg caaaacagtc gctgaaaatt tgaaggcttt 4200 tgatgattta acacctggtc aaaaggttat tatgccgctt gaaaatccta aacgtgaaga 4260 tggtccgctc attattctcc atggtaactt ggctccagac ggtgccgttg ccaaagtttc 4320 tggtgtaaaa gtgcgtcgtc atgtcggtcc tgctaaggtc tttaattctg aagaagaagc 4380 cattgaagct gtcttgaatg atgatattgt tgatggtgat gttgttgtcg tacgttttgt 4440 aggaccaaag ggcggtcctg gtatgcctga aatgctttcc ctttcatcaa tgattgttgg 4500 taaagggcaa ggtgaaaaag ttgcccttct gacagatggc cgcttctcag gtggtactta 4560 tggtcttgtc gtgggtcata tcgctcctga agcacaagat ggcggtccaa tcgcctacct 4620 gcaaacagga gacatagtca ctattgacca agacactaag gaattacact ttgatatctc 4680 cgatgaagag ttaaaacatc gtcaagagac cattgaattg ccaccgctct attcacgcgg 4740 tatccttggt aaatatgctc acatcgtttc gtctgcttct aggggagccg taacagactt 4800 ttggaagcct gaagaaactg gcaaaaaatg ttgtcctggt tgctgtggtt aagcggccgc 4860 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 4920 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 4980 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 5040 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 5100 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 5160 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 5220 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 5280 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 5340 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 5400 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 5460 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 5520 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 5580 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 5640 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 5700 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 5760 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 5820 accaagtatg gagaaatata ttagaagtct atacgttaaa ccaccgcggt ggagctccag 5880 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt 5940 tcctgtgtga aattgttatc cgctcacaat tccacacaac ataggagccg gaagcataaa 6000 gtgtaaagcc tggggtgcct aatgagtgag gtaactcaca ttaattgcgt tgcgctcact 6060 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 6120 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 6180 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 6240 cilgaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 6300 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 6360 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 6420 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 6480 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 6540 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 6600 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 6660 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 6720 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 6780 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6840 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6900 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6960 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 7020 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 7080 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 7140 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 7200 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 7260 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 7320 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 7380 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 7440 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 7500 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 7560 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 7620 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 7680 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7740 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7800 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7860 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7920 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7980 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 8040 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc 8100 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct 8160 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg 8220 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 8280 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat 8340 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc 8400 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt 8460 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat 8520 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt 8580 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg 8640 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct 8700 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat 8760 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa 8820 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta 8880 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg 8940 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt 9000 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt 9060 ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg 9120 aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt 9180 gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc 9240 gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta 9300 tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat 9360 gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg 9420 gatcatctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 9480 gccctttcgt c 9491 <210> 129 <211> 1000 <212> DNA <213> Saccharymoces cerevisiae <400> 129 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 420 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 480 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 540 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 600 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 660 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 720 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 780 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 840 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 900 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 960 accaagtatg gagaaatata ttagaagtct atacgttaaa 1000 <210> 130 <211> 16387 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 130 gatcctctag tttctcggta ctatgcatat gatccaatat caaaggaaat gatagcattg 60 aaggatgaga ctaatccaat tgaggagtgg cagcatatag aacagctaaa gggtagtgct 120 gaaggaagca tacgataccc cgcatggaat gggataatat cacaggaggt actagactac 180 ctttcatcct acataaatag acgcatataa gtacgcattt aagcataaac acgcactatg 240 ccgttcttct catgtatata tatatacagg caacacgcag atataggtgc gacgtgaaca 300 gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct 360 ttgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc agagcgcttt 420 tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac caaaaacgca ccggactgta 480 acgagctact aaaatattgc gaataccgct tccacaaaca ttgctcaaaa gtatctcttt 540 gctatatatc tctgtgctat atccctatat aacctaccca tccacctttc gctccttgaa 600 cttgcatcta aactcgacct ctacattttt tatgtttatc tctagtatta ctctttagac 660 aaaaaaattg tagtaagaac tattcataga gtgaatcgaa aacaatacga aaatgtaaac 720 atttcctata cgtagtatat agagacaaaa tagaagaaac cgttcataat tttctgacca 780 atgaagaatc atcaacgcta tcactttctg ttcacaaagt atgcgcaatc cacatcggta 840 tagaatataa tcggggatgc ctttatcttg aaaaaatgca cccgcagctt cgctagtaat 900 cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa gagaaaatag acaccaaagt 960 agccttcttc taaccttaac ggacctacag tgcaaaaagt tatcaagaga ctgcattata 1020 gagcgcacaa aggagaaaaa aagtaatcta agatgctttg ttagaaaaat agcgctctcg 1080 ggatgcattt ttgtagaaca aaaaagaagt atagattctt tgttggtaaa atagcgctct 1140 cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc 1200 tctcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc 1260 gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 1320 agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc gttaacaaag 1380 atatgctatt gaagtgcaag atggaaacgc agaaaatgaa ccggggatgc gacgtgcaag 1440 attacctatg caatagatgc aatagtttct ccaggaaccg aaatacatac attgtcttcc 1500 gtaaagcgct agactatata ttattataca ggttcaaata tactatctgt ttcagggaaa 1560 actcccaggt tcggatgttc aaaattcaat gatgggtaac aagtacgatc gtaaatctgt 1620 aaaacagttt gtcggatatt aggctgtatc tcctcaaagc gtattcgaat atcattgaga 1680 agctgcattt tttttttttt tttttttttt tttttttata tatatttcaa ggatatacca 1740 ttgtaatgtc tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa 1800 tcacagccga agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt 1860 tcgatttcga aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc 1920 cagatgaggc gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg 1980 gtcctaaatg gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag 2040 aacttcaatt gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact 2100 tatctccaat caagccacaa tttgctaaag gtactgactt cgttgttgtt agagaattag 2160 tgggaggtat ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata 2220 gtgaacaata caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc 2280 tacaacatga gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt 2340 caagattatg gagaaaaact gtggaggaaa ccatcaagaa cgaattccct acattgaaag 2400 ttcaacatca attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa 2460 atggtattat aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta 2520 tcccaggctc cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca 2580 ccgcatttgg tttgtacgaa ccatgccatg gttccgctcc agatttgcca aagaataagg 2640 tcaaccctat cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc 2700 ctgaagaagg taaagccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa 2760 ctggtgattt aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag 2820 ttaagaaaat ccttgcttaa aaagattctc tttttttatg atatttgtac aaaaaaaaaa 2880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aatgcagcgt cacatcggat 2940 aataatgatg gcagccattg tagaagtgcc ttttgcattt ctagtctctt tctcggtcta 3000 gctagtttta ctacatcgcg aagatagaat cttagatcac actgcctttg ctgagctgga 3060 tcaatagagt aacaaaagag tggtaaggcc tcgttaaagg acaaggacct gagcggaagt 3120 gtatcgtaca gtagacggag tatactagag tcgacctgca ggcatgcaag cttttcaatt 3180 catcattttt tttttattct tttttttgat ttcggtttcc ttgaaatttt tttgattcgg 3240 taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 3300 gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 3360 caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 3420 ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 3480 acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 3540 taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 3600 agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag 3660 acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 3720 gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 3780 gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 3840 cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 3900 ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 3960 gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 4020 acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta 4080 ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 4140 acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 4200 tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 4260 agttattacc cgggaatctc ggtcgtaatg atttttataa tgacgaaaaa aaaaaaattg 4320 gaaagaaaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 4380 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 4440 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 4500 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 4560 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 4620 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 4680 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 4740 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 4800 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 4860 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 4920 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 4980 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 5040 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 5100 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 5160 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 5220 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 5280 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 5340 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 5400 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 5460 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 5520 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 5580 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 5640 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 5700 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 5760 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 5820 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 5880 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 5940 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 6000 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 6060 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 6120 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 6180 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 6240 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 6300 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 6360 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 6420 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 6480 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 6540 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 6600 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 6660 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 6720 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta 6780 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac cgaaatcggc 6840 aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt tccagtttgg 6900 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 6960 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc 7020 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 7080 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc taaggcgctg 7140 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta 7200 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa 7260 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 7320 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 7380 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 7440 aattcgagct ccaccgcgga tagatctgaa atgaataaca atactgacag tactaaataa 7500 ttgcctactt ggcttcacat acgttgcata cgtcgatata gataataatg ataatgacag 7560 caggattatc gtaatacgta atagttgaaa atctcaaaaa tgtgtgggtc attacgtaaa 7620 taatgatagg aatgggattc ttctattttt cctttttcca ttctagcagc cgtcgggaaa 7680 acgtggcatc ctctctttcg ggctcaattg gagtcacgct gccgtgagca tcctctcttt 7740 ccatatctaa caactgagca cgtaaccaat ggaaaagcat gagcttagcg ttgctccaaa 7800 aaagtattgg atggttaata ccatttgtct gttctcttct gactttgact cctcaaaaaa 7860 aaaaaatcta caatcaacag atcgcttcaa ttacgccctc acaaaaactt ttttccttct 7920 tcttcgccca cgttaaattt tatccctcat gttgtctaac ggatttctgc acttgattta 7980 ttataaaaag acaaagacat aatacttctc tatcaatttc agttattgtt cttccttgcg 8040 ttattcttct gttcttcttt ttcttttgtc atatataacc ataaccaagt aatacatatt 8100 caaatctaga gctgaggatg ttgaagcaaa tcaacttcgg tggtactgtt gaaaccgtct 8160 acgaaagagc tgactggcca agagaaaagt tgttggacta cttcaagaac gacacttttg 8220 ctttgatcgg ttacggttcc caaggttacg gtcaaggttt gaacttgaga gacaacggtt 8280 tgaacgttat cattggtgtc cgtaaagatg gtgcttcttg gaaggctgcc atcgaagacg 8340 gttgggttcc aggcaagaac ttgttcactg ttgaagatgc tatcaagaga ggtagttacg 8400 ttatgaactt gttgtccgat gccgctcaat cagaaacctg gcctgctatc aagccattgt 8460 tgaccaaggg taagactttg tacttctccc acggtttctc cccagtcttc aaggacttga 8520 ctcacgttga accaccaaag gacttagatg ttatcttggt tgctccaaag ggttccggta 8580 gaactgtcag atctttgttc aaggaaggtc gtggtattaa ctcttcttac gccgtctgga 8640 acgatgtcac cggtaaggct cacgaaaagg cccaagcttt ggccgttgcc attggttccg 8700 gttacgttta ccaaaccact ttcgaaagag aagtcaactc tgacttgtac ggtgaaagag 8760 gttgtttaat gggtggtatc cacggtatgt tcttggctca atacgacgtc ttgagagaaa 8820 acggtcactc cccatctgaa gctttcaacg aaaccgtcga agaagctacc caatctctat 8880 acccattgat cggtaagtac ggtatggatt acatgtacga tgcttgttcc accaccgcca 8940 gaagaggtgc tttggactgg tacccaatct tcaagaatgc tttgaagcct gttttccaag 9000 acttgtacga atctaccaag aacggtaccg aaaccaagag atctttggaa ttcaactctc 9060 aacctgacta cagagaaaag ctagaaaagg aattagacac catcagaaac atggaaatct 9120 ggaaggttgg taaggaagtc agaaagttga gaccagaaaa ccaataatta attaatcatg 9180 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag 9240 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 9300 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 9360 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 9420 tgcgggcggc cgctctagag agttgttagc aaccttttgt ttcttttgag ctggttcaga 9480 cattatgtac acgtatatgt gacgagttcg agaagtattt tactatcgta ctaaatttta 9540 cctgaaaaat tatatactcg agaaagagga agccaagaat tgagaaaaaa gaaaaacccg 9600 cgagtaagga aattaaatac aggtgtacac atacacgcac acatatatat atatatatat 9660 atgtatatgt gtatatagga agcgcgcgca tgttagtata tacgattcgt tggaaagggg 9720 ccgtccacca aacgtgactt gacgagttga caaattgacc tcaatatggc tcagtcagta 9780 atttttagtt ccgctttatt cccgccatct ttcaggccac gagggtagct cataacgccg 9840 cgctaatgcc gctgcgtcac agcaaccagt agctcagcca aaaccgaaag agaaatcgta 9900 gctgtcccga tgaggactta tacacttgtc accatctaaa taaattattt attcgcgttt 9960 cggttcttgt tttcgattta attagattgt tcattgaatc ataataaata tgtaaaaaat 10020 atatatattt gaagctgctt cagaaaaaca gggcttccta gtgtacagat gtatgtcgga 10080 tgaaaaaaaa aaaatcttaa atgtgaaatt gggtcaattc aattgactat gacttgatgt 10140 tgcaaaaatt ccaagagaaa aagtttccag cacttgatat tattttcctc tttaattttt 10200 cgccttgtct acgatcttat tagcaccgat ccagggcatc atagacctta actgttcacc 10260 aataatttcg ataccatgtg ctgcattgtt tcttctttta gcagtcatac tcgggtaacc 10320 cgtagcgcct tcacttatga acatcttagc gtattcaccg tcctggatac gtttcaaggc 10380 atttctcatg gcttgtcttg attctgcgtt aatgacttca ggtccggtga catactcacc 10440 atattctgca ttatttgaaa tggaatagtt catattagct ataccacctt catacattaa 10500 gtctactatc aacttcaatt catgtagaca ttcgaagtat gccatttcgg gagcgtaccc 10560 tgcttcgaca agcgtctcaa agcctgcttt aaccaattca acagttcctc cgcacagaac 10620 cgcttgttct ccaaataaat ctgtctcagt ctcgtcttta aaagtggttt ctattatacc 10680 cgttctcccg ccaccaactc ctgctgcgta gcttaaagct acattcttag cgtttccgct 10740 tgcgtcttgg tatatagcga tcaaatctgg aataccacca cccttaacaa attcgctcct 10800 aacagtatgc cccggagcct taggtgcaat cataataacg tccaaatctg ccctggggac 10860 tacttgattg taatgaatgg caaatccatg actgaaggcc aaggtagcgc ccttcttaat 10920 gtttggttct atttcatttt tgtacaattg cgattgaaat tcatctggcg ttaaaatcat 10980 gactaaatca gcgccggcaa cagccgctgc aacatctgtg actttcaagc catgtgcttc 11040 agcctttgca acggtagcac taccttttct cagacctact gtcacgtcga ccccagaatc 11100 tttcaagtta caggcttgtg cgtgtccttg ggaaccatat cctataatag caaccttctt 11160 tccctggatg atgctcagat cgcagtcttt atcgtaaaac accttcatgt tttatttttt 11220 acttatattg ctggtagggt aaaaaaatat aactcctagg aataggttgt ctatatgttt 11280 ttgtcttgct tctataattg taacaaacaa ggaaagggaa aatactgggt gtaaaagcca 11340 ttgagtcaag ttaggtcatc ccttttatac aaaatttttc aatttttttt ccaagattct 11400 tgtacgatta attatttttt ttttgcgtcc tacagcgtga tgaaaatttc cgcctgctgc 11460 aagatgagcg ggaacgggcg aaatgtgcac gcgcacaact tacgaaacgc ggatgagtca 11520 ctgacagcca ccgcagaggt tctgactcct actgagctct attggaggtg gcagaaccgg 11580 taccggagga gaccgctata accggtttga atttattgtc acagtgtcac atcagcggca 11640 actcagaagt ttgacagcaa gcaagttcat cattcgaact agccttattg ttttagttca 11700 gtgacagcga actgccgtac tcgatgcttt atttctcacg gtagagcgga agaacagata 11760 ggggcagcgt gagaagagtt agaaagtaaa tttttatcac gtctgaagta ttcttattca 11820 taggaaattt tgcaaggttt tttagctcaa taacgggcta agttatataa ggtgttcacg 11880 cgattttctt gttatgtata cctcttctct gaggaatggt actactgtcc tgatgtaggc 11940 tccttaaatt ggtgggcaag aataacttat cgatattttg tatattggtc ttggagttca 12000 ccacgtaatg cctgtttaag accatcagtt aactctagta ttatttggtc ttggctactg 12060 gccgtttgct attattcaag tcttttgtgc cttcccgtcg ggtaagggag ttatttaggg 12120 atacagaatc taacgaaaac taaatctcaa tgattaactc catttaatcc ttttttgaaa 12180 ggcaaaagag gtcccttgtt cacttacaac gttcttagcc aaattcgctt atcacttact 12240 acttcacgat atacagaagt aaaaacatat aaaaagatgt ctgtttgttt agccatcaca 12300 aaaggtatcg cagtttcttc tataggcctc tactctggtc ttttggcttc cgcttcattg 12360 attacatcta ctactccact agaggtttta acaggatctc taaaaacatc gatatcgtct 12420 ctgcgttcca atcctacggt gaatatattt ccaagcaatt cactgaagaa gaaagagaag 12480 atgttgtgga acatgcatgc ccaggtcctg gttcttgtgg tggtatgtat actgccaaca 12540 caatggcttc tgccgctgaa gtgctaggtt tgaccattcc aaactcctct tccttcccag 12600 ccgtttccaa ggagaagtta gctgagtgtg acaacattgg tgaatacatc aagaagacaa 12660 tggaattggg tattttacct cgtgatatcc tcacaaaaga ggcttttgaa aacgccatta 12720 cttatgtcgt tgcaaccggt gggtccacta atgctgtttt gcatttggtg gctgttgctc 12780 actctgcggg tgtcaagttg tcaccagatg atttccaaag aatcagtgat actacaccat 12840 tgatcggtga cttcaaacct tctggtaaat acgtcatggc cgatttgatt aacgttggtg 12900 gtacccaatc tgtgattaag tatctatatg aaaacaacat gttgcacggt aacacaatga 12960 ctgttaccgg tgacactttg gcagaacgtg caaagaaagc accaagccta cctgaaggac 13020 aagagattat taagccactc tcccacccaa tcaaggccaa cggtcacttg caaattctgt 13080 acggttcatt ggcaccaggt ggagctgtgg gtaaaattac cggtaaggaa ggtacttact 13140 tcaagggtag agcacgtgtg ttcgaagagg aaggtgcctt tattgaagcc ttggaaagag 13200 gtgaaatcaa gaagggtgaa aaaaccgttg ttgttatcag atatgaaggt ccaagaggtg 13260 caccaggtat gcctgaaatg ctaaagcctt cctctgctct gatgggttac ggtttgggta 13320 aagatgttgc attgttgact gatggtagat tctctggtgg ttctcacggg ttcttaatcg 13380 gccacattgt tcccgaagcc gctgaaggtg gtcctatcgg gttggtcaga gacggcgatg 13440 agattatcat tgatgctgat aataacaaga ttgacctatt agtctctgat aaggaaatgg 13500 ctcaacgtaa acaaagttgg gttgcacctc cacctcgtta cacaagaggt actctatcca 13560 agtatgctaa gttggtttcc aacgcttcca acggttgtgt tttagatgct tgattaatta 13620 agagtaagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat 13680 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag 13740 taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga 13800 ccacacctct accggcatgc cgagcaaatg cctgcaaatc gctccccatt tcacccaatt 13860 gtagatatgc taactccagc aatgagttga tgaatctcgg tgtgtatttt atgtcctcag 13920 aggacaacac ctgtggtact agttctagag cggccgcccg caaattaaag ccttcgagcg 13980 tcccaaaacc ttctcaagca aggttttcag tataatgtta catgcgtaca cgcgtctgta 14040 cagaaaaaaa agaaaaattt gaaatataaa taacgttctt aatactaaca taactataaa 14100 aaaataaata gggacctaga cttcaggttg tctaactcct tccttttcgg ttagagcgga 14160 tgtgggggga gggcgtgaat gtaagcgtga cataactaat tacatgatta attaactaga 14220 gagctttcgt tttcatgagt tccccgaatt ctttcggaag cttgtcactt gctaaattaa 14280 tgttatcact gtagtcaacc gggacatcga tgatgacagg accttcagcg ttcatgcctt 14340 gacgcagaac atctgccagc tggtctggtg attctacgcg caagccagtt gctccgaagc 14400 tttccgcata tttcacgata tcgatatttc cgaaatcgac cgcagatgta cggttatatt 14460 ttttcaattg ctggaatgca accatgtcat atgtgctgtc gttccataca atgtgtacaa 14520 ttggtgcttt tagtcgaact gctgtctcta attccattgc tgagaataag aaaccgccgt 14580 caccagagac agaaaccact ttttctcccg gtttcaccaa tgaagcgccg attgcccaag 14640 gaagcgcaac gccgagtgtt tgcataccgt tactgatcat taatgttaac ggctcgtagc 14700 tgcggaaata acgtgacatc caaatggcgt gcgaaccgat atcgcaagtt actgtaacat 14760 gatcatcgac tgcattacgc aactctttaa cgatttcaag agggtgcgct ctgtctgatt 14820 tccaatctgc aggcacctgc tcaccttcat gcatatattg ttttaaatca gaaaggattt 14880 tctgctcacg ctctgcaaat tccactttca cagcatcgtg ttcgatatga ttgatcgtgg 14940 acggaatgtc accgatcaat tcaagatcag gctggtaagc atgatcaatg tcagcgataa 15000 tctcgtctaa atggataatt gtccggtctc cattgatatt ccagaatttc ggatcatatt 15060 caatcgggtc atagccgatc gtcagaacaa catctgcctg ctctagcagt aaatcgccag 15120 gctggttgcg gaacaaaccg atacggccaa aatattgatc ctctaaatct ctagaaaggg 15180 taccggcagc ttgatatgtt tcaacaaatg gaagctgaac ctttttcaaa agcttgcgaa 15240 ccgctttaat tgcttccggt cttccgcctt tcatgccgac caaaacgaca ggaagttttg 15300 ctgtttggat ttttgctatg gccgcactga ttgcatcatc tgctgcagga ccgagttttg 15360 gcgctgcaac agcacgcacg tttttcgtat ttgtgacttc attcacaaca tcttgcggaa 15420 agctcacaaa agcggcccca gcctgccctg ctgacgctat cctaaatgca tttgtaacag 15480 cttccggtat attttttaca tcttgaactt ctacactgta ttttgtaatc ggctggaata 15540 gcgccgcatt atccaaagat tgatgtgtcc gttttaaacg atctgcacgg atcacgtttc 15600 cagcaagcgc aacgacaggg tctccttcag tgttcgctgt cagcaggcct gttgccaagt 15660 tagaggcacc cggtcctgat gtgactaaca cgactcccgg ttttccagtt aaacggccga 15720 ctgcttgggc catgaatgct gcgttttgtt cgtgccgggc aacgataatt tcaggtcctt 15780 tatcttgtaa agcgtcaaat accgcatcaa tttttgcacc tggaatgcca aatacatgtg 15840 tgacaccttg ctccactaag caatcaacaa caagctccgc ccctctgttt ttcacaaggg 15900 atttttgttc ttttgttgct tttgtcaaca tcctcagcga tgattgattg attgattgta 15960 cagtttgttt ttcttaatat ctatttcgat gacttctata tgatattgca ctaacaagaa 16020 gatattataa tgcaattgat acaagacaag gagttatttg cttctctttt atatgattct 16080 gacaatccat attgcgttgg tagtcttttt tgctggaacg gttcagcgga aaagacgcat 16140 cgctcttttt gcttctagaa gaaatgccag caaaagaatc tcttgacagt gactgacagc 16200 aaaaatgtct ttttctaact agtaacaagg ctaagatatc agcctgaaat aaagggtggt 16260 gaagtaataa ttaaatcatc cgtataaacc tatacacata tatgaggaaa aataatacaa 16320 aagtgtttta aatacagata catacatgaa catatgcacg tatagcgccc aaatgtcggt 16380 aatggga 16387 <210> 131 <211> 1188 <212> DNA <213> Saccharomyces cerevisiae <400> 131 atgttgagaa ctcaagccgc cagattgatc tgcaactccc gtgtcatcac tgctaagaga 60 acctttgctt tggccacccg tgctgctgct tacagcagac cagctgcccg tttcgttaag 120 ccaatgatca ctacccgtgg tttgaagcaa atcaacttcg gtggtactgt tgaaaccgtc 180 tacgaaagag ctgactggcc aagagaaaag ttgttggact acttcaagaa cgacactttt 240 gctttgatcg gttacggttc ccaaggttac ggtcaaggtt tgaacttgag agacaacggt 300 ttgaacgtta tcattggtgt ccgtaaagat ggtgcttctt ggaaggctgc catcgaagac 360 ggttgggttc caggcaagaa cttgttcact gttgaagatg ctatcaagag aggtagttac 420 gttatgaact tgttgtccga tgccgctcaa tcagaaacct ggcctgctat caagccattg 480 ttgaccaagg gtaagacttt gtacttctcc cacggtttct ccccagtctt caaggacttg 540 actcacgttg aaccaccaaa ggacttagat gttatcttgg ttgctccaaa gggttccggt 600 agaactgtca gatctttgtt caaggaaggt cgtggtatta actcttctta cgccgtctgg 660 aacgatgtca ccggtaaggc tcacgaaaag gcccaagctt tggccgttgc cattggttcc 720 ggttacgttt accaaaccac tttcgaaaga gaagtcaact ctgacttgta cggtgaaaga 780 ggttgtttaa tgggtggtat ccacggtatg ttcttggctc aatacgacgt cttgagagaa 840 aacggtcact ccccatctga agctttcaac gaaaccgtcg aagaagctac ccaatctcta 900 tacccattga tcggtaagta cggtatggat tacatgtacg atgcttgttc caccaccgcc 960 agaagaggtg ctttggactg gtacccaatc ttcaagaatg ctttgaagcc tgttttccaa 1020 gacttgtacg aatctaccaa gaacggtacc gaaaccaaga gatctttgga attcaactct 1080 caacctgact acagagaaaa gctagaaaag gaattagaca ccatcagaaa catggaaatc 1140 tggaaggttg gtaaggaagt cagaaagttg agaccagaaa accaataa 1188 <210> 132 <211> 1014 <212> DNA <213> Pseudomonas fluorescens <400> 132 atgaaagttt tctacgataa agactgcgac ctgtcgatca tccaaggtaa gaaagttgcc 60 atcatcggct acggttccca gggccacgct caagcatgca acctgaagga ttccggcgta 120 gacgtgactg ttggcctgcg taaaggctcg gctaccgttg ccaaggctga agcccacggc 180 ttgaaagtga ccgacgttgc tgcagccgtt gccggtgccg acttggtcat gatcctgacc 240 ccggacgagt tccagtccca gctgtacaag aacgaaatcg agccgaacat caagaagggc 300 gccactctgg ccttctccca cggcttcgcg atccactaca accaggttgt gcctcgtgcc 360 gacctcgacg tgatcatgat cgcgccgaag gctccaggcc acaccgtacg ttccgagttc 420 gtcaagggcg gtggtattcc tgacctgatc gcgatctacc aggacgcttc cggcaacgcc 480 aagaacgttg ccctgtccta cgccgcaggc gtgggcggcg gccgtaccgg catcatcgaa 540 accaccttca aggacgagac tgaaaccgac ctgttcggtg agcaggctgt tctgtgtggc 600 ggtaccgtcg agctggtcaa agccggtttc gaaaccctgg ttgaagctgg ctacgctcca 660 gaaatggcct acttcgagtg cctgcacgaa ctgaagctga tcgttgacct catgtacgaa 720 ggcggtatcg ccaacatgaa ctactcgatc tccaacaacg ctgaatacgg cgagtacgtg 780 actggtccag aagtcatcaa cgccgaatcc cgtcaggcca tgcgcaatgc tctgaagcgc 840 atccaggacg gcgaatacgc gaagatgttc atcagcgaag gcgctaccgg ctacccatcg 900 atgaccgcca agcgtcgtaa caacgctgct cacggtatcg aaatcatcgg cgagcaactg 960 cgctcgatga tgccttggat cggtgccaac aaaatcgtcg acaaagccaa gaac 1014 <210> 133 <211> 250 <212> DNA <213> Saccharomyces cerevisiae <400> 133 ccgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 60 ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 120 cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 180 ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 240 aattacatga 250 <210> 134 <211> 1181 <212> DNA <213> Saccharomyces cerevisiae <400> 134 taaaacctct agtggagtag tagatgtaat caatgaagcg gaagccaaaa gaccagagta 60 gaggcctata gaagaaactg cgataccttt tgtgatggct aaacaaacag acatcttttt 120 atatgttttt acttctgtat atcgtgaagt agtaagtgat aagcgaattt ggctaagaac 180 gttgtaagtg aacaagggac ctcttttgcc tttcaaaaaa ggattaaatg gagttaatca 240 ttgagattta gttttcgtta gattctgtat ccctaaataa ctcccttacc cgacgggaag 300 gcacaaaaga cttgaataat agcaaacggc cagtagccaa gaccaaataa tactagagtt 360 aactgatggt cttaaacagg cattacgtgg tgaactccaa gaccaatata caaaatatcg 420 ataagttatt cttgcccacc aatttaagga gcctacatca ggacagtagt accattcctc 480 agagaagagg tatacataac aagaaaatcg cgtgaacacc ttatataact tagcccgtta 540 ttgagctaaa aaaccttgca aaatttccta tgaataagaa tacttcagac gtgataaaaa 600 tttactttct aactcttctc acgctgcccc tatctgttct tccgctctac cgtgagaaat 660 aaagcatcga gtacggcagt tcgctgtcac tgaactaaaa caataaggct agttcgaatg 720 atgaacttgc ttgctgtcaa acttctgagt tgccgctgat gtgacactgt gacaataaat 780 tcaaaccggt tatagcggtc tcctccggta ccggttctgc cacctccaat agagctcagt 840 aggagtcaga acctctgcgg tggctgtcag tgactcatcc gcgtttcgta agttgtgcgc 900 gtgcacattt cgcccgttcc cgctcatctt gcagcaggcg gaaattttca tcacgctgta 960 ggacgcaaaa aaaaaataat taatcgtaca agaatcttgg aaaaaaaatt gaaaaatttt 1020 gtataaaagg gatgacctaa cttgactcaa tggcttttac acccagtatt ttccctttcc 1080 ttgtttgtta caattataga agcaagacaa aaacatatag acaacctatt cctaggagtt 1140 atattttttt accctaccag caatataagt aaaaaactag t 1181 <210> 135 <211> 759 <212> DNA <213> Saccharomyces cerevisiae <400> 135 ggccctgcag gcctatcaag tgctggaaac tttttctctt ggaatttttg caacatcaag 60 tcatagtcaa ttgaattgac ccaatttcac atttaagatt tttttttttt catccgacat 120 acatctgtac actaggaagc cctgtttttc tgaagcagct tcaaatatat atatttttta 180 catatttatt atgattcaat gaacaatcta attaaatcga aaacaagaac cgaaacgcga 240 ataaataatt tatttagatg gtgacaagtg tataagtcct catcgggaca gctacgattt 300 ctctttcggt tttggctgag ctactggttg ctgtgacgca gcggcattag cgcggcgtta 360 tgagctaccc tcgtggcctg aaagatggcg ggaataaagc ggaactaaaa attactgact 420 gagccatatt gaggtcaatt tgtcaactcg tcaagtcacg tttggtggac ggcccctttc 480 caacgaatcg tatatactaa catgcgcgcg cttcctatat acacatatac atatatatat 540 atatatatat gtgtgcgtgt atgtgtacac ctgtatttaa tttccttact cgcgggtttt 600 ttttttttct caattcttgg cttcctcttt ctcgagtata taatttttca ggtaaaattt 660 agtacgatag taaaatactt ctcgaactcg tcacatatac gtgtacataa tgtctgaacc 720 agctcaaaag aaacaaaagg ttgctaacaa ctctctaga 759 <210> 136 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 136 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 137 <211> 1716 <212> DNA <213> Bacillus subtilis <400> 137 atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60 gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120 attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180 gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240 gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300 actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360 cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420 gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480 gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540 aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600 atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660 aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720 ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780 tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840 gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900 ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960 cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020 gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080 catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140 gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200 cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260 aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320 ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380 ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440 tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500 ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560 tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620 atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680 gaattcgggg aactcatgaa aacgaaagct ctctag 1716 <210> 138 <211> 571 <212> PRT <213> Bacillus subtilis <400> 138 Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg 1 5 10 15 Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His             20 25 30 Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu         35 40 45 Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala     50 55 60 Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val 65 70 75 80 Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu                 85 90 95 Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn             100 105 110 Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn         115 120 125 Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp     130 135 140 Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser 145 150 155 160 Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val                 165 170 175 Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys             180 185 190 Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile         195 200 205 Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg     210 215 220 Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu 225 230 235 240 Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu                 245 250 255 Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly             260 265 270 Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp         275 280 285 Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr     290 295 300 Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln 305 310 315 320 Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile                 325 330 335 Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile             340 345 350 Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala         355 360 365 Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu     370 375 380 Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser 385 390 395 400 His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr                 405 410 415 Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp             420 425 430 Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val         435 440 445 Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala     450 455 460 Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr 465 470 475 480 Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser                 485 490 495 Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe             500 505 510 Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val         515 520 525 Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro     530 535 540 Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys 545 550 555 560 Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu                 565 570 <210> 139 <211> 448 <212> DNA <213> Saccharomyces cerevisiae <400> 139 cccattaccg acatttgggc gctatacgtg catatgttca tgtatgtatc tgtatttaaa 60 acacttttgt attatttttc ctcatatatg tgtataggtt tatacggatg atttaattat 120 tacttcacca ccctttattt caggctgata tcttagcctt gttactagtt agaaaaagac 180 atttttgctg tcagtcactg tcaagagatt cttttgctgg catttcttct agaagcaaaa 240 agagcgatgc gtcttttccg ctgaaccgtt ccagcaaaaa agactaccaa cgcaatatgg 300 attgtcagaa tcatataaaa gagaagcaaa taactccttg tcttgtatca attgcattat 360 aatatcttct tgttagtgca atatcatata gaagtcatcg aaatagatat taagaaaaac 420 aaactgtaca atcaatcaat caatcatc 448 <210> 140 <211> 16387 <212> DNA Artificial sequence <220> <223> plasmid construct <400> 140 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt cagcttccat ttgttgaaac atatcaagct 1200 gccggtaccc tttctagaga tttagaggat caatattttg gccgtatcgg tttgttccgc 1260 aaccagcctg gcgatttact gctagagcag gcagatgttg ttctgacgat cggctatgac 1320 ccgattgaat atgatccgaa attctggaat atcaatggag accggacaat tatccattta 1380 gacgagatta tcgctgacat tgatcatgct taccagcctg atcttgaatt gatcggtgac 1440 attccgtcca cgatcaatca tatcgaacac gatgctgtga aagtggaatt tgcagagcgt 1500 gagcagaaaa tcctttctga tttaaaacaa tatatgcatg aaggtgagca ggtgcctgca 1560 gattggaaat cagacagagc gcaccctctt gaaatcgtta aagagttgcg taatgcagtc 1620 gatgatcatg ttacagtaac ttgcgatatc ggttcgcacg ccatttggat gtcacgttat 1680 ttccgcagct acgagccgtt aacattaatg atcagtaacg gtatgcaaac actcggcgtt 1740 gcgcttcctt gggcaatcgg cgcttcattg gtgaaaccgg gagaaaaagt ggtttctgtc 1800 tctggtgacg gcggtttctt attctcagca atggaattag agacagcagt tcgactaaaa 1860 gcaccaattg tacacattgt atggaacgac agcacatatg acatggttgc attccagcaa 1920 ttgaaaaaat ataaccgtac atctgcggtc gatttcggaa atatcgatat cgtgaaatat 1980 gcggaaagct tcggagcaac tggcttgcgc gtagaatcac cagaccagct ggcagatgtt 2040 ctgcgtcaag gcatgaacgc tgaaggtcct gtcatcatcg atgtcccggt tgactacagt 2100 gataacatta atttagcaag tgacaagctt ccgaaagaat tcggggaact catgaaaacg 2160 aaagctctct agttaattaa tcatgtaatt agttatgtca cgcttacatt cacgccctcc 2220 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 2280 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt 2340 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt 2400 tttgggacgc tcgaaggctt taatttgcgg gcggccgctc tagaactagt accacaggtg 2460 ttgtcctctg aggacataaa atacacaccg agattcatca actcattgct ggagttagca 2520 tatctacaat tgggtgaaat ggggagcgat ttgcaggcat ttgctcggca tgccggtaga 2580 ggtgtggtca ataagagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa 2640 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta 2700 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc 2760 ttactcttaa ttaatcaagc atctaaaaca caaccgttgg aagcgttgga aaccaactta 2820 gcatacttgg atagagtacc tcttgtgtaa cgaggtggag gtgcaaccca actttgttta 2880 cgttgagcca tttccttatc agagactaat aggtcaatct tgttattatc agcatcaatg 2940 ataatctcat cgccgtctct gaccaacccg ataggaccac cttcagcggc ttcgggaaca 3000 atgtggccga ttaagaaccc gtgagaacca ccagagaatc taccatcagt caacaatgca 3060 acatctttac ccaaaccgta acccatcaga gcagaggaag gctttagcat ttcaggcata 3120 cctggtgcac ctcttggacc ttcatatctg ataacaacaa cggttttttc acccttcttg 3180 atttcacctc tttccaaggc ttcaataaag gcaccttcct cttcgaacac acgtgctcta 3240 cccttgaagt aagtaccttc cttaccggta attttaccca cagctccacc tggtgccaat 3300 gaaccgtaca gaatttgcaa gtgaccgttg gccttgattg ggtgggagag tggcttaata 3360 atctcttgtc cttcaggtag gcttggtgct ttctttgcac gttctgccaa agtgtcaccg 3420 gtaacagtca ttgtgttacc gtgcaacatg ttgttttcat atagatactt aatcacagat 3480 tgggtaccac caacgttaat caaatcggcc atgacgtatt taccagaagg tttgaagtca 3540 ccgatcaatg gtgtagtatc actgattctt tggaaatcat ctggtgacaa cttgacaccc 3600 gcagagtgag caacagccac caaatgcaaa acagcattag tggacccacc ggttgcaacg 3660 acataagtaa tggcgttttc aaaagcctct tttgtgagga tatcacgagg taaaataccc 3720 aattccattg tcttcttgat gtattcacca atgttgtcac actcagctaa cttctccttg 3780 gaaacggctg ggaaggaaga ggagtttgga atggtcaaac ctagcacttc agcggcagaa 3840 gccattgtgt tggcagtata cataccacca caagaaccag gacctgggca tgcatgttcc 3900 acaacatctt ctctttcttc ttcagtgaat tgcttggaaa tatattcacc gtaggattgg 3960 aacgcagaga cgatatcgat gtttttagag atcctgttaa aacctctagt ggagtagtag 4020 atgtaatcaa tgaagcggaa gccaaaagac cagagtagag gcctatagaa gaaactgcga 4080 taccttttgt gatggctaaa caaacagaca tctttttata tgtttttact tctgtatatc 4140 gtgaagtagt aagtgataag cgaatttggc taagaacgtt gtaagtgaac aagggacctc 4200 ttttgccttt caaaaaagga ttaaatggag ttaatcattg agatttagtt ttcgttagat 4260 tctgtatccc taaataactc ccttacccga cgggaaggca caaaagactt gaataatagc 4320 aaacggccag tagccaagac caaataatac tagagttaac tgatggtctt aaacaggcat 4380 tacgtggtga actccaagac caatatacaa aatatcgata agttattctt gcccaccaat 4440 ttaaggagcc tacatcagga cagtagtacc attcctcaga gaagaggtat acataacaag 4500 aaaatcgcgt gaacacctta tataacttag cccgttattg agctaaaaaa ccttgcaaaa 4560 tttcctatga ataagaatac ttcagacgtg ataaaaattt actttctaac tcttctcacg 4620 ctgcccctat ctgttcttcc gctctaccgt gagaaataaa gcatcgagta cggcagttcg 4680 ctgtcactga actaaaacaa taaggctagt tcgaatgatg aacttgcttg ctgtcaaact 4740 tctgagttgc cgctgatgtg acactgtgac aataaattca aaccggttat agcggtctcc 4800 tccggtaccg gttctgccac ctccaataga gctcagtagg agtcagaacc tctgcggtgg 4860 ctgtcagtga ctcatccgcg tttcgtaagt tgtgcgcgtg cacatttcgc ccgttcccgc 4920 tcatcttgca gcaggcggaa attttcatca cgctgtagga cgcaaaaaaa aaataattaa 4980 tcgtacaaga atcttggaaa aaaaattgaa aaattttgta taaaagggat gacctaactt 5040 gactcaatgg cttttacacc cagtattttc cctttccttg tttgttacaa ttatagaagc 5100 aagacaaaaa catatagaca acctattcct aggagttata tttttttacc ctaccagcaa 5160 tataagtaaa aaactagtat gaaggtgttt tacgataaag actgcgatct gagcatcatc 5220 cagggaaaga aggttgctat tataggatat ggttcccaag gacacgcaca agccttgaac 5280 ttgaaagatt ctggggtcga cgtgacagta ggtctgtata aaggtgctgc tgatgcagca 5340 aaggctgaag cacatggctt taaagtcaca gatgttgcag cggctgttgc tggcgctgat 5400 ttagtcatga ttttaattcc agatgaattt caatcgcaat tgtacaaaaa tgaaatagaa 5460 ccaaacatta agaagggcgc taccttggcc ttcagtcatg gatttgccat tcattacaat 5520 caagtagtcc ccagggcaga tttggacgtt attatgattg cacctaaggc tccggggcat 5580 actgttagga gcgaatttgt taagggtggt ggtattccag atttgatcgc tatataccaa 5640 gacgttagcg gaaacgctaa gaatgtagct ttaagctacg cagcaggagt tggtggcggg 5700 agaacgggta taatagaaac cacttttaaa gacgagactg agacagattt atttggagaa 5760 caagcggttc tgtgcggagg aactgttgaa ttggttaaag caggctttga gacgcttgtc 5820 gaagcagggt acgctcccga aatggcatac ttcgaatgtc tacatgaatt gaagttgata 5880 gtagacttaa tgtatgaagg tggtatagct aatatgaact attccatttc aaataatgca 5940 gaatatggtg agtatgtcac cggacctgaa gtcattaacg cagaatcaag acaagccatg 6000 agaaatgcct tgaaacgtat ccaggacggt gaatacgcta agatgttcat aagtgaaggc 6060 gctacgggtt acccgagtat gactgctaaa agaagaaaca atgcagcaca tggtatcgaa 6120 attattggtg aacagttaag gtctatgatg ccctggatcg gtgctaataa gatcgtagac 6180 aaggcgaaaa attaaggccc tgcaggccta tcaagtgctg gaaacttttt ctcttggaat 6240 ttttgcaaca tcaagtcata gtcaattgaa ttgacccaat ttcacattta agattttttt 6300 tttttcatcc gacatacatc tgtacactag gaagccctgt ttttctgaag cagcttcaaa 6360 tatatatatt ttttacatat ttattatgat tcaatgaaca atctaattaa atcgaaaaca 6420 agaaccgaaa cgcgaataaa taatttattt agatggtgac aagtgtataa gtcctcatcg 6480 ggacagctac gatttctctt tcggttttgg ctgagctact ggttgctgtg acgcagcggc 6540 attagcgcgg cgttatgagc taccctcgtg gcctgaaaga tggcgggaat aaagcggaac 6600 taaaaattac tgactgagcc atattgaggt caatttgtca actcgtcaag tcacgtttgg 6660 tggacggccc ctttccaacg aatcgtatat actaacatgc gcgcgcttcc tatatacaca 6720 tatacatata tatatatata tatatgtgtg cgtgtatgtg tacacctgta tttaatttcc 6780 ttactcgcgg gtttttcttt tttctcaatt cttggcttcc tctttctcga gtatataatt 6840 tttcaggtaa aatttagtac gatagtaaaa tacttctcga actcgtcaca tatacgtgta 6900 cataatgtct gaaccagctc aaaagaaaca aaaggttgct aacaactctc tagagcggcc 6960 gcccgcaaat taaagccttc gagcgtccca aaaccttctc aagcaaggtt ttcagtataa 7020 tgttacatgc gtacacgcgt ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg 7080 ttcttaatac taacataact ataaaaaaat aaatagggac ctagacttca ggttgtctaa 7140 ctccttcctt ttcggttaga gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa 7200 ctaattacat gattaattaa ttattggttt tctggtctca actttctgac ttccttacca 7260 accttccaga tttccatgtt tctgatggtg tctaattcct tttctagctt ttctctgtag 7320 tcaggttgag agttgaattc caaagatctc ttggtttcgg taccgttctt ggtagattcg 7380 tacaagtctt ggaaaacagg cttcaaagca ttcttgaaga ttgggtacca gtccaaagca 7440 cctcttctgg cggtggtgga acaagcatcg tacatgtaat ccataccgta cttaccgatc 7500 aatgggtata gagattgggt agcttcttcg acggtttcgt tgaaagcttc agatggggag 7560 tgaccgtttt ctctcaagac gtcgtattga gccaagaaca taccgtggat accacccatt 7620 aaacaacctc tttcaccgta caagtcagag ttgacttctc tttcgaaagt ggtttggtaa 7680 acgtaaccgg aaccaatggc aacggccaaa gcttgggcct tttcgtgagc cttaccggtg 7740 acatcgttcc agacggcgta agaagagtta ataccacgac cttccttgaa caaagatctg 7800 acagttctac cggaaccctt tggagcaacc aagataacat ctaagtcctt tggtggttca 7860 acgtgagtca agtccttgaa gactggggag aaaccgtggg agaagtacaa agtcttaccc 7920 ttggtcaaca atggcttgat agcaggccag gtttctgatt gagcggcatc ggacaacaag 7980 ttcataacgt aactacctct cttgatagca tcttcaacag tgaacaagtt cttgcctgga 8040 acccaaccgt cttcgatggc agccttccaa gaagcaccat ctttacggac accaatgata 8100 acgttcaaac cgttgtctct caagttcaaa ccttgaccgt aaccttggga accgtaaccg 8160 atcaaagcaa aagtgtcgtt cttgaagtag tccaacaact tttctcttgg ccagtcagct 8220 ctttcgtaga cggtttcaac agtaccaccg aagttgattt gcttcaacat cctcagctct 8280 agatttgaat atgtattact tggttatggt tatatatgac aaaagaaaaa gaagaacaga 8340 agaataacgc aaggaagaac aataactgaa attgatagag aagtattatg tctttgtctt 8400 tttataataa atcaagtgca gaaatccgtt agacaacatg agggataaaa tttaacgtgg 8460 gcgaagaaga aggaaaaaag tttttgtgag ggcgtaattg aagcgatctg ttgattgtag 8520 attttttttt tttgaggagt caaagtcaga agagaacaga caaatggtat taaccatcca 8580 atactttttt ggagcaacgc taagctcatg cttttccatt ggttacgtgc tcagttgtta 8640 gatatggaaa gagaggatgc tcacggcagc gtgactccaa ttgagcccga aagagaggat 8700 gccacgtttt cccgacggct gctagaatgg aaaaaggaaa aatagaagaa tcccattcct 8760 atcattattt acgtaatgac ccacacattt ttgagatttt caactattac gtattacgat 8820 aatcctgctg tcattatcat tattatctat atcgacgtat gcaacgtatg tgaagccaag 8880 taggcaatta tttagtactg tcagtattgt tattcatttc agatctatcc gcggtggagc 8940 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 9000 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 9060 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat 9120 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 9180 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 9240 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 9300 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 9360 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 9420 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 9480 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag 9540 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg 9600 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 9660 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 9720 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 9780 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 9840 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9900 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9960 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 10020 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 10080 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 10140 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 10200 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 10260 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 10320 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 10380 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 10440 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 10500 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 10560 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 10620 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 10680 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 10740 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 10800 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 10860 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 10920 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 10980 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 11040 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 11100 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 11160 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 11220 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 11280 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 11340 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 11400 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 11460 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 11520 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 11580 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 11640 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 11700 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 11760 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 11820 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 11880 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 11940 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 12000 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt 12060 ttctttccaa tttttttttt ttcgtcatta taaaaatcat tacgaccgag attcccgggt 12120 aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact 12180 tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc ccagcctgct 12240 tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag tcctcttcca 12300 acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata ctgttgaccc 12360 aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca atcgtaacct 12420 tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc tttgtcgctc 12480 ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc cttgcatgac 12540 aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc cgcctgcttc 12600 aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct 12660 gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat gtcagcaaat 12720 tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag cggcttaact 12780 gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa acaaattttg 12840 ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc caatgaagca 12900 cacaagtttg tttgcttttc gtgcatgata ttaaatagct tggcagcaac aggactagga 12960 tgagtagcag cacgttcctt atatgtagct ttcgacatga tttatcttcg tttcctgcag 13020 gtttttgttc tgtgcagttg ggttaagaat actgggcaat ttcatgtttc ttcaacacta 13080 catatgcgta tatataccaa tctaagtctg tgctccttcc ttcgttcttc cttctgttcg 13140 gagattaccg aatcaaaaaa atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa 13200 aatgatgaat tgaaaagctt gcatgcctgc aggtcgactc tagtatactc cgtctactgt 13260 acgatacact tccgctcagg tccttgtcct ttaacgaggc cttaccactc ttttgttact 13320 ctattgatcc agctcagcaa aggcagtgtg atctaagatt ctatcttcgc gatgtagtaa 13380 aactagctag accgagaaag agactagaaa tgcaaaaggc acttctacaa tggctgccat 13440 cattattatc cgatgtgacg ctgcattttt tttttttttt tttttttttt tttttttttt 13500 tttttttttt tttttttgta caaatatcat aaaaaaagag aatcttttta agcaaggatt 13560 ttcttaactt cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa 13620 tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat ggctttacct 13680 tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata 13740 gggttgacct tattctttgg caaatctgga gcggaaccat ggcatggttc gtacaaacca 13800 aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggag 13860 cctgggataa cggaggcttc atcggagatg atatcaccaa acatgttgct ggtgattata 13920 ataccattta ggtgggttgg gttcttaact aggatcatgg cggcagaatc aatcaattga 13980 tgttgaactt tcaatgtagg gaattcgttc ttgatggttt cctccacagt ttttctccat 14040 aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca 14100 tgttgtaggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat 14160 tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata 14220 cctcccacta attctctaac aacaacgaag tcagtacctt tagcaaattg tggcttgatt 14280 ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat 14340 tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc ggtaccccat 14400 ttaggaccac ccacagcacc taacaaaacg gcatcagcct tcttggaggc ttccagcgcc 14460 tcatctggaa gtggaacacc tgtagcatcg atagcagcac caccaattaa atgattttcg 14520 aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg 14580 gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac 14640 attacaatgg tatatccttg aaatatatat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 14700 tgcagcttct caatgatatt cgaatacgct ttgaggagat acagcctaat atccgacaaa 14760 ctgttttaca gatttacgat cgtacttgtt acccatcatt gaattttgaa catccgaacc 14820 tgggagtttt ccctgaaaca gatagtatat ttgaacctgt ataataatat atagtctagc 14880 gctttacgga agacaatgta tgtatttcgg ttcctggaga aactattgca tctattgcat 14940 aggtaatctt gcacgtcgca tccccggttc attttctgcg tttccatctt gcacttcaat 15000 agcatatctt tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga 15060 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg 15120 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa 15180 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 15240 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa 15300 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg 15360 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa 15420 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg 15480 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta 15540 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat 15600 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat 15660 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa 15720 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag 15780 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat 15840 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt 15900 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg 15960 gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga 16020 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac 16080 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga 16140 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg 16200 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc 16260 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct 16320 catccttcaa tgctatcatt tcctttgata ttggatcata tgcatagtac cgagaaacta 16380 gaggatc 16387 <210> 141 <211> 2237 <212> DNA <213> Artificial Sequence <220> <223> PDC1 Fragment A-ilvDSm <400> 141 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acctttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattatt 2237 <210> 142 <211> 4420 <212> DNA <213> Artificial Sequence <220> <223> PDC1A-ilVDSm-BUC cassette <400> 142 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acctttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattattagt taaagtttta taagcatttt tatgtaacga aaaataaatt 2280 ggttcatatt attactgcac tgtcacttac catggaaaga ccagacaaga agttgccgac 2340 agtctgttga attggcctgg ttaggcttaa gtctgggtcc gcttctttac aaatttggag 2400 aatttctctt aaacgatatg tatattcttt tcgttggaaa agatgtcttc caaaaaaaaa 2460 accgatgaat tagtggaacc aaggaaaaaa aaagaggtat ccttgattaa ggaacactgt 2520 ttaaacagtg tggtttccaa aaccctgaaa ctgcattagt gtaatagaag actagacacc 2580 tcgatacaaa taatggttac tcaattcaaa actgccagcg aattcgactc tgcaattgct 2640 caagacaagc tagttgtcgt agatttctac gccacttggt gcggtccatg taaaatgatt 2700 gctccaatga ttgaaaaatg tggctgtggt ttcagggtcc ataaagcttt tcaattcatc 2760 tttttttttt ttgttctttt ttttgattcc ggtttctttg aaattttttt gattcggtaa 2820 tctccgagca gaaggaagaa cgaaggaagg agcacagact tagattggta tatatacgca 2880 tatgtggtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg cacagaacaa 2940 aaacctgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa cgtgctgcta 3000 ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag caaacaaact 3060 tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt gaagcattag 3120 gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt tccatggagg 3180 gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc ttcgaagaca 3240 gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt gtatacagaa 3300 tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt attgttagcg 3360 gtttgaagca ggcggcggaa gaagtaacaa aggaacctag aggccttttg atgttagcag 3420 aattgtcatg caagggctcc ctagctactg gagaatatac taagggtact gttgacattg 3480 cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg ggtggaagag 3540 atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac aagggagacg 3600 cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct gacattatta 3660 ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt gaacgttaca 3720 gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa aaaactgtat 3780 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt 3840 tattacccgg gaatctcggt cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa 3900 agaaaaagct tcatggcctt ccactttccc aaacaacacc tacggtatct ctcaagtctt 3960 atggggttcc attggtttca ccactggtgc taccttgggt gctgctttcg ctgctgaaga 4020 aattgatcca aagaagagag ttatcttatt cattggtgac ggttctttgc aattgactgt 4080 tcaagaaatc tccaccatga tcagatgggg cttgaagcca tacttgttcg tcttgaacaa 4140 cgatggttac accattgaaa agttgattca cggtccaaag gctcaataca acgaaattca 4200 aggttgggac cacctatcct tgttgccaac tttcggtgct aaggactatg aaacccacag 4260 agtcgctacc accggtgaat gggacaagtt gacccaagac aagtctttca acgacaactc 4320 taagatcaga atgattgaaa tcatgttgcc agtcttcgat gctccacaaa acttggttga 4380 acaagctaag ttgactgctg ctaccaacgc taagcaataa 4420 <210> 143 <211> 3521 <212> DNA <213> Artificial Sequence <220> <223> PDC5A-sADB-BUC cassette <400> 143 aaggaaataa agcaaataac aataacacca ttattttaat tttttttcta ttactgtcgc 60 taacacctgt atggttgcaa ccaggtgaga atccttctga tgcatacttt atgcgtttat 120 gcgttttgcg ccccttggaa aaaaattgat tctcatcgta aatgcatact acatgcgttt 180 atgggaaaag cctccatatc caaaggtcgc gtttctttta gaaaaactaa tacgtaaacc 240 tgcattaagg taagattata tcagaaaatg tgttgcaaga aatgcattat gcaatttttt 300 gattatgaca atctctcgaa agaaatttca tatgatgaga cttgaataat gcagcggcgc 360 ttgctaaaag aacttgtata taagagctgc cattctcgat caatatactg tagtaagtcc 420 tttcctctct ttcttattac acttatttca cataatcaat ctcaaagaga acaacacaat 480 acaataacaa gaagaacaaa atgaaagctc tggtttatca cggtgaccac aagatctcgc 540 ttgaagacaa gcccaagccc acccttcaaa agcccacgga tgtagtagta cgggttttga 600 agaccacgat ctgcggcacg gatctcggca tctacaaagg caagaatcca gaggtcgccg 660 acgggcgcat cctgggccat gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca 720 cgcagttcaa gaaaggcgac aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg 780 actactgcaa gaagcagctt tactcccatt gccgcgacgg cgggtggatc ctgggttaca 840 tgatcgatgg cgtgcaggcc gaatacgtcc gcatcccgca tgccgacaac agcctctaca 900 agatccccca gacaattgac gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg 960 gccacgaaat cggcgtccag tatgggaatg tccagccggg cgatgcggtg gctattgtcg 1020 gcgcgggccc cgtcggcatg tccgtactgt tgaccgccca gttctactcc ccctcgacca 1080 tcatcgtgat cgacatggac gagaatcgcc tccagctcgc caaggagctc ggggcaacgc 1140 acaccatcaa ctccggcacg gagaacgttg tcgaagccgt gcataggatt gcggcagagg 1200 gagtcgatgt tgcgatcgag gcggtgggca taccggcgac ttgggacatc tgccaggaga 1260 tcgtcaagcc cggcgcgcac atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg 1320 agattcagaa gctctggatc aagaacctga cgatcaccac gggactggtg aacacgaaca 1380 cgacgcccat gctgatgaag gtcgcctcga ccgacaagct tccgttgaag aagatgatta 1440 cccatcgctt cgagctggcc gagatcgagc acgcctatca ggtattcctc aatggcgcca 1500 aggagaaggc gatgaagatc atcctctcga acgcaggcgc tgcctgagct aattaacata 1560 aaactcatga ttcaacgttt gtgtattttt ttacttttga aggttataga tgtttaggta 1620 aataattggc atagatatag ttttagtata ataaatttct gatttggttt aaaatatcaa 1680 ctattttttt tcacatatgt tcttgtaatt acttttctgt cctgtcttcc aggttaaaga 1740 ttagcttcta atattttagg tggtttatta tttaatttta tgctgattaa tttatttact 1800 tgtttaaacg gccggccaat gtggctgtgg tttcagggtc cataaagctt ttcaattcat 1860 cttttttttt tttgttcttt tttttgattc cggtttcttt gaaatttttt tgattcggta 1920 atctccgagc agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc 1980 atatgtggtg ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca 2040 aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct 2100 actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac 2160 ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta 2220 ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag 2280 ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaagac 2340 agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga 2400 atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc 2460 ggtttgaagc aggcggcgga agaagtaaca aaggaaccta gaggcctttt gatgttagca 2520 gaattgtcat gcaagggctc cctagctact ggagaatata ctaagggtac tgttgacatt 2580 gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga 2640 gattgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagac 2700 gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc tgacattatt 2760 attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac 2820 agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta 2880 ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag 2940 ttattacccg ggaatctcgg tcgtaatgat ttctataatg acgaaaaaaa aaaaattgga 3000 aagaaaaagc ttcatggcct tctactttcc caacagatgt atacgctatc gtccaagtct 3060 tgtggggttc cattggtttc acagtcggcg ctctattggg tgctactatg gccgctgaag 3120 aactggcc aaagaagaga gttattttat tcattggtga cggttctcta caattgactg 3180 ttcaagaaat ctctaccatg attagatggg gtttgaagcc atacattttt gtcttgaata 3240 acaacggtta caccattgaa aaattgattc acggtcctca tgccgaatat aatgaaattc 3300 aaggttggga ccacttggcc ttattgccaa cttttggtgc tagaaactac gaaacccaca 3360 gagttgctac cactggtgaa tgggaaaagt tgactcaaga caaggacttc caagacaact 3420 ctaagattag aatgattgaa gttatgttgc cagtctttga tgctccacaa aacttggtta 3480 aacaagctca attgactgcc gctactaacg ctaaacaata a 3521 <210> 144 <211> 2686 <212> DNA <213> Artificial Sequence <220> <223> pUC19 <400> 144 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686 <210> 145 <211> 1685 <212> DNA <213> Artificial Sequence <220> <223> gpd2 :: loxP-URA3-loxP cassette <400> 145 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag ataacttcgt atagcataca ttatacgaag 120 ttatgcagat tgtactgaga gtgcaccata ccacagcttt tcaattcaat tcatcatttt 180 ttttttattc ttttttttga tttcggtttc tttgaaattt ttttgattcg gtaatctccg 240 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta 300 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct 360 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc 420 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg 480 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca 540 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag 600 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat 660 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag 720 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga 780 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt 840 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga 900 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag 960 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg 1020 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg 1080 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag 1140 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag 1200 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac 1260 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg 1320 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 1380 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 1440 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 1500 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 1560 gataacttcg tatagcatac attatacgaa gttatccagt gatgatacaa cgagttagcc 1620 aaggtgacac tctccccccc cctccccctc tgatctttcc tgttgcctct ttttccccca 1680 accaa 1685

Claims (32)

(a) 발효성 탄소 공급원으로부터 생성물 알코올을 생성하는 재조합 미생물을 포함하는 발효 브로쓰를 제공하는 단계 - 상기 재조합 미생물은 피루베이트 데카르복실라아제(pyruvate decarboxylase; PDC) 활성의 감소 또는 제거를 포함함 - 와;
(b) 발효 브로쓰를 발효성 탄소 공급원과 접촉시키고, 이에 의해 재조합 미생물이 발효성 탄소 공급원을 소비하고 생성물 알코올을 생성하게 하는 단계와;
(c) 발효 브로쓰를, 상기 발효 과정에서의 단계에서 바이오매스로부터 유래된 지방산과 접촉시키는 단계를 포함하며,
재조합 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 큰 방법.(a) providing a fermentation broth comprising a recombinant microorganism that produces a product alcohol from a fermentable carbon source, the recombinant microorganism comprising a reduction or elimination of pyruvate decarboxylase (PDC) activity - Wow;
(b) contacting the fermentation broth with a fermentable carbon source, thereby causing the recombinant microorganism to consume the fermentable carbon source and produce product alcohol;
(c) contacting the fermentation broth with fatty acids derived from biomass in the step of the fermentation process,
Wherein at least one of the (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater than the growth rate and / or fermentable carbon consumption of the recombinant microorganism in the presence of the fatty acid, in the absence of the fatty acid. 제1항에 있어서, 지방산은 올레산, 팔미트산, 미리스트산 및 그 혼합물로부터 선택되는 방법.The method of claim 1 wherein the fatty acid is selected from oleic acid, palmitic acid, myristic acid and mixtures thereof. 제1항에 있어서, 바이오매스는 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀(grasses), 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스(switchgrass), 폐지, 사탕수수 바가스(bagasse), 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물로부터 유래되는 방법.The biomass of claim 1, wherein the biomass is corn grain, corncob, crop residues, for example corn husk, corncobs, grasses, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass ), Waste paper, sugar cane bagasse, sorghum, sugar cane, soybean, ingredients obtained by milling of grains, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, wood chips, sawdust , Shrubs and bushes, vegetables, fruits, flowers, animal manure and mixtures thereof. 제1항에 있어서, 생성물 알코올은 부탄올인 방법.The method of claim 1 wherein the product alcohol is butanol. 제1항에 있어서, 단계 (b) 및 단계 (c)는 실질적으로 동시에 일어나는 방법.The method of claim 1, wherein step (b) and step (c) occur substantially simultaneously. 제1항에 있어서, 발효성 탄소 공급원은 바이오매스로부터 유래되는 방법.The method of claim 1, wherein the fermentable carbon source is derived from biomass. 제1항에 있어서, 발효 브로쓰는 에탄올을 추가로 포함하는 방법.The method of claim 1, wherein the fermentation broth further comprises ethanol. 제1항에 있어서, 재조합 미생물은 하나 이상의 피루베이트 데카르복실라아제(PDC) 유전자 결실을 갖는 방법.The method of claim 1, wherein the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions. (a) 발효성 탄소 공급원 및 오일을 포함하는 바이오매스를 제공하는 단계와;
(b) 오일의 적어도 일부분을 지방산으로 전환시켜 지방산을 포함하는 바이오매스를 형성하는 단계와;
(c) 바이오매스를, 발효성 탄소 공급원으로부터 생성물 알코올을 생성할 수 있는 재조합 미생물을 포함하는 발효 브로쓰와 접촉시키는 단계 - 상기 재조합 미생물은 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함함 - 와;
(d) 지방산과 발효 브로쓰를 접촉시키는 단계를 포함하며,
재조합 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 큰, 생성물 알코올을 생성하는 방법.(a) providing a biomass comprising a fermentable carbon source and an oil;
(b) converting at least a portion of the oil to fatty acids to form a biomass comprising fatty acids;
(c) contacting the biomass with a fermentation broth comprising recombinant microorganisms capable of producing product alcohols from the fermentable carbon source, wherein the recombinant microorganism comprises a reduction or elimination of pyruvate decarboxylase activity. - Wow;
(d) contacting the fatty acid with the fermentation broth,
At least one of the (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater than the growth rate and / or fermentable carbon consumption of the recombinant microorganism in the presence of the fatty acid, in the absence of the fatty acid to produce a product alcohol. Way. 제9항에 있어서, 오일의 적어도 일부분을 지방산으로 전환시키는 단계 (b)는 오일을, 오일의 일부를 지방산으로 가수분해시킬 수 있는 하나 이상의 물질과 접촉시키는 것을 포함하는 방법.10. The method of claim 9, wherein converting at least a portion of the oil to a fatty acid comprises contacting the oil with one or more substances capable of hydrolyzing a portion of the oil to the fatty acid. 제10항에 있어서, 상기 하나 이상의 물질은 하나 이상의 효소를 포함하는 방법.The method of claim 10, wherein the one or more substances comprise one or more enzymes. 제11항에 있어서, 상기 하나 이상의 효소는 리파아제 효소를 포함하는 방법.The method of claim 11, wherein the one or more enzymes comprise a lipase enzyme. 제11항에 있어서,
단계 (c) 전에, 오일의 적어도 일부분이 가수분해된 후에 하나 이상의 효소를 불활성화시키는 단계를 추가로 포함하는 방법.12. The method of claim 11,
Prior to step (c), further comprising inactivating one or more enzymes after at least a portion of the oil has been hydrolyzed. 제9항에 있어서, 단계 (b), 단계 (c), 및 단계 (d) 중 하나 이상은 발효 용기 내에서 일어나는 방법.The method of claim 9, wherein one or more of steps (b), (c), and (d) occur in the fermentation vessel. 제9항에 있어서, 단계 (b), 단계 (c), 및 단계 (d) 중 하나 이상은 실질적으로 동시에 일어나는 방법.The method of claim 9, wherein at least one of step (b), step (c), and step (d) occur substantially simultaneously. 제9항에 있어서, 바이오매스는 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀, 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스, 폐지, 사탕수수 바가스, 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물로부터 유래되는 방법.10. The biomass of claim 9, wherein the biomass is corn grain, corncob, crop residues, for example corn husk, corncobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane. Vargas, sorghum, sugar cane, soybeans, ingredients obtained by milling grains, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, chips, sawdust, shrubs and bushes, vegetables, fruits , Flowers, animal manure and mixtures thereof. 제9항에 있어서, 지방산은 올레산, 팔미트산, 미리스트산 및 그 혼합물로부터 선택되는 방법.The method of claim 9, wherein the fatty acid is selected from oleic acid, palmitic acid, myristic acid and mixtures thereof. 제9항에 있어서,
발효성 탄소 공급원을 발효시켜 생성물 알코올을 생성하는 단계를 추가로 포함하는 방법.10. The method of claim 9,
Fermenting the fermentable carbon source to produce the product alcohol. 제18항에 있어서, 생성물 알코올은 부탄올인 방법.The method of claim 18, wherein the product alcohol is butanol. 제9항에 있어서, 발효 브로쓰는 에탄올을 추가로 포함하는 방법.The method of claim 9, wherein the fermentation broth further comprises ethanol. 제9항에 있어서, 재조합 미생물은 하나 이상의 피루베이트 데카르복실라아제 (PDC) 유전자 결실을 갖는 방법.The method of claim 9, wherein the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions. 제9항에 있어서,
오일의 적어도 일부분을 지방산으로 전환시키는 단계 (b) 전에 오일을 바이오매스로부터 분리시키는 단계를 추가로 포함하는 방법.10. The method of claim 9,
Separating the oil from the biomass prior to (b) converting at least a portion of the oil into fatty acids. 제9항에 있어서,
바이오매스를 액화시켜 액화된 바이오매스를 생성하는 단계 - 상기 액화된 바이오매스는 올리고당류를 포함함 - 와;
액화된 바이오매스를, 올리고당류를 발효성 당으로 전환시킬 수 있는 당화 효소와 접촉시켜 당화된 바이오매스를 형성하는 단계를 추가로 포함하며,
단계 (c)는 당화된 바이오매스를, 재조합 미생물을 포함하는 발효 브로쓰와 접촉시키는 것을 포함하는 방법.10. The method of claim 9,
Liquefying the biomass to produce a liquefied biomass, wherein the liquefied biomass comprises oligosaccharides;
Contacting the liquefied biomass with a saccharifying enzyme capable of converting oligosaccharides into fermentable sugars to form a glycosylated biomass,
Step (c) comprises contacting the glycated biomass with a fermentation broth comprising recombinant microorganisms. (a) 공급재료(feedstock)를 제공하는 단계와;
(b) 상기 공급재료를 액화시켜 공급재료 슬러리를 생성하는 단계와;
(c) 공급재료 슬러리를 분리시켜 (i) 발효성 탄소 공급원을 포함하는 수성 층, (ii) 오일 층, 및 (iii) 고형물 층을 포함하는 생성물을 생성하는 단계와;
(d) 오일 층으로부터 오일을 수득하고 오일의 적어도 일부분을 지방산으로 전환시키는 단계와;
(e) (c)의 수성 층을 발효성 탄소 공급원으로부터 생성물 알코올을 생성할 수 있는 재조합 미생물을 포함하는 발효 브로쓰가 들어 있는 발효 용기에 공급하는 단계 - 상기 재조합 미생물은 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함함 - 와;
(f) 수성 층의 발효성 탄소 공급원을 발효시켜 생성물 알코올을 생성하는 단계와;
(g) 발효 브로쓰를 지방산과 접촉시키는 단계를 포함하며,
재조합 미생물의 (i) 성장 속도 및 (ii) 발효성 탄소 소비량 중 적어도 하나는 지방산의 존재 하에서, 지방산의 부재 하에서의 재조합 미생물의 성장 속도 및/또는 발효성 탄소 소비량보다 더 큰, 생성물 알코올을 생성하는 방법.(a) providing a feedstock;
(b) liquefying the feedstock to produce a feedstock slurry;
(c) separating the feed slurry to produce a product comprising (i) an aqueous layer comprising a fermentable carbon source, (ii) an oil layer, and (iii) a solids layer;
(d) obtaining an oil from the oil layer and converting at least a portion of the oil to a fatty acid;
(e) feeding the aqueous layer of (c) to a fermentation vessel containing a fermentation broth comprising recombinant microorganisms capable of producing product alcohols from the fermentable carbon source, wherein the recombinant microorganism is pyruvate decarboxylase. Including reducing or eliminating activity;
(f) fermenting the fermentable carbon source of the aqueous layer to produce product alcohol;
(g) contacting the fermentation broth with a fatty acid,
At least one of the (i) growth rate and (ii) fermentable carbon consumption of the recombinant microorganism is greater than the growth rate and / or fermentable carbon consumption of the recombinant microorganism in the presence of the fatty acid, in the absence of the fatty acid to produce a product alcohol. Way. 제24항에 있어서, 오일의 적어도 일부분을 지방산으로 전환시키는 단계 (d)는 오일을, 오일의 일부를 지방산으로 가수분해시킬 수 있는 하나 이상의 물질과 접촉시키는 것을 포함하는 방법.The method of claim 24, wherein converting at least a portion of the oil into a fatty acid comprises contacting the oil with one or more substances capable of hydrolyzing a portion of the oil to the fatty acid. 제25항에 있어서, 상기 하나 이상의 물질은 하나 이상의 효소를 포함하는 방법.The method of claim 25, wherein the one or more substances comprise one or more enzymes. 제26항에 있어서, 상기 하나 이상의 효소는 리파아제 효소를 포함하는 방법.The method of claim 26, wherein the one or more enzymes comprises a lipase enzyme. 제24항에 있어서, 공급재료는 옥수수 알곡, 옥수수 속대, 작물 잔사, 예를 들어 옥수수 껍질, 옥수수대, 풀, 밀, 호밀, 밀짚, 보리, 보릿짚, 건초, 볏짚, 스위치그래스, 폐지, 사탕수수 바가스, 수수, 사탕수수, 대두, 곡물의 제분에 의해 수득되는 성분, 셀룰로오스계 물질, 리그노셀룰로오스계 물질, 나무, 가지, 뿌리, 잎, 나뭇조각, 톱밥, 관목 및 떨기나무, 야채, 과실, 꽃, 동물 분뇨 및 그 혼합물로부터 유래되는 하나 이상의 발효성 당을 포함하는 방법.25. The feed material of claim 24, wherein the feedstock is corn grain, corncob, crop residues such as corn husk, corncobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane. Vargas, sorghum, sugar cane, soybeans, ingredients obtained by milling grains, cellulosic materials, lignocellulosic materials, trees, branches, roots, leaves, chips, sawdust, shrubs and bushes, vegetables, fruits And one or more fermentable sugars derived from flowers, animal manure, and mixtures thereof. 제24항에 있어서, 지방산은 올레산, 팔미트산, 미리스트산 및 그 혼합물로부터 선택되는 방법.The method of claim 24, wherein the fatty acid is selected from oleic acid, palmitic acid, myristic acid and mixtures thereof. 제18항에 있어서, 생성물 알코올은 부탄올인 방법.The method of claim 18, wherein the product alcohol is butanol. 제24항에 있어서, 재조합 미생물은 하나 이상의 피루베이트 데카르복실라아제 (PDC) 유전자 결실을 갖는 방법.The method of claim 24, wherein the recombinant microorganism has one or more pyruvate decarboxylase (PDC) gene deletions. 지방산 및 피루베이트 데카르복실라아제 활성의 감소 또는 제거를 포함하는 재조합 미생물을 포함하는 조성물.A composition comprising a recombinant microorganism comprising reducing or eliminating fatty acid and pyruvate decarboxylase activity.

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