KR101759673B1 - Genetically engineered yeast cell having enhanced growth rate and method of producing target materials using the same - Google Patents

Genetically engineered yeast cell having enhanced growth rate and method of producing target materials using the same Download PDF

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KR101759673B1
KR101759673B1 KR1020150187643A KR20150187643A KR101759673B1 KR 101759673 B1 KR101759673 B1 KR 101759673B1 KR 1020150187643 A KR1020150187643 A KR 1020150187643A KR 20150187643 A KR20150187643 A KR 20150187643A KR 101759673 B1 KR101759673 B1 KR 101759673B1
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한지숙
백승호
권윤정
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Abstract

생장 속도가 증대된 유전적으로 조작된 효모 세포 및 그를 사용하여 목적 물질을 생산하는 방법에 관한 것으로, 일 양상에 따른 효모 세포 및 그를 사용하여 목적 물질을 생산하는 방법에 의하면, 효모 세포의 생장 속도 및 포도당 소비량 증대에 의해 목적 물질을 고효율 및 고수율로 생산할 수 있다. The present invention relates to genetically engineered yeast cells having increased growth rate and a method for producing a target substance using the same. According to a yeast cell according to one aspect and a method for producing a target substance using the yeast cell, The target substance can be produced with high efficiency and high yield by increasing glucose consumption.

Description

생장 속도가 증대된 유전적으로 조작된 효모 세포 및 그를 사용하여 목적 물질을 생산하는 방법{Genetically engineered yeast cell having enhanced growth rate and method of producing target materials using the same}[0001] The present invention relates to a genetically engineered yeast cell having increased growth rate and a method for producing a target substance using the yeast cell,

생장 속도가 증대된 유전적으로 조작된 효모 세포 및 그를 사용하여 목적 물질을 생산하는 방법에 관한 것이다. A genetically engineered yeast cell with increased growth rate and a method for producing a target substance using the same.

화석연료의 과다한 사용으로 인해 지구온난화 및 자원고갈 문제가 대두됨에 따라 바이오매스를 비롯한 친환경적인 대체에너지 자원에 대한 관심이 증가하고 있다. 이와 함께 미생물을 활용한 바이오 화학제품과 바이오 연료 등을 생산하는 바이오리파이너리 기술에 대한 연구도 활발히 진행되고 있다. As global warming and resource depletion problems arise due to excessive use of fossil fuels, interest in environmentally friendly alternative energy resources including biomass is increasing. In addition, research on bio-refinery technology that produces bio-chemical products and bio-fuels using microorganisms is actively under way.

사카로마이세스 세레비지애(Saccharomyces cerevisiae)는 고대부터 주조 및 양조, 식품 발효에 사용되어 왔으며 진핵생물의 모델생체로서 연구되고 있다. 나아가 사카로마이세스 세레비지애는 젖산(lactate), 숙신산(succinate) 등의 유기산과 에탄올(ethanol), 아이소부탄올(isobutanol), 2,3-부탄다이올(2,3-butanediol) 등 바이오 연료 생산을 위한 기반 미생물로도 활용되고 있다. Saccharomyces cerevisiae has been used in casting and brewing food fermentation since ancient times and has been studied as a model organism of eukaryotes. Furthermore, the saccharomyces cerevisiae is produced by mixing organic acids such as lactate and succinate with biofuels such as ethanol, isobutanol, and 2,3-butanediol, It is also used as a microbial base for production.

피루브산(pyruvate) 유래의 바이오 연료 및 유기산의 생산성은 경쟁 경로로 작용하는 에탄올 또는 글리세롤 합성 경로의 저해 또는 제거, 또는 바이오 연료 및 유기산의 합성 경로의 과발현 등을 통해 향상될 수 있지만, 유전적으로 조작된 균주의 생장 속도가 감소하는 문제점이 발생한다. The productivity of biofuels and organic acids derived from pyruvate can be improved through the inhibition or elimination of the ethanol or glycerol synthesis pathway, which acts as a competitive pathway, or the overexpression of biofuel and organic acid synthesis pathways, The growth rate of the strain is decreased.

따라서, 유전적으로 조작된 균주의 생장 속도를 증대시킴으로써 목적 산물의 생산능과 생산 수율을 증대시키기 위한 요구가 존재한다. Therefore, there is a need to increase the yield of the target product and the production yield by increasing the growth rate of the genetically engineered strain.

일 양상은 모세포에 비하여 생장 속도가 증대된 유전적으로 조작된 효모 세포를 제공하는 것이다. One aspect is to provide genetically engineered yeast cells with increased growth rates relative to the parent cells.

다른 양상은 상기 효모 세포를 사용하여 목적 물질을 생산하는 방법을 제공하는 것이다. Another aspect is to provide a method for producing a target substance using the yeast cell.

일 양상은 모세포에 비하여 생장 속도가 증대된 유전적으로 조작된 효모 세포를 제공한다. One aspect provides genetically engineered yeast cells with increased growth rates relative to the parent cells.

상기 효모 세포는 모세포에 비하여 글루코오스 신호 인자(glucose signaling factor: GSF)의 활성이 감소되어 있는 것일 수 있다. The yeast cell may have decreased glucose signaling factor (GSF) activity as compared to the parent cell.

본 명세서에서 사용된 용어 "유전적 조작 (genetic engineering)" 또는 "유전적으로 조작된 (genetically engineered)"은 세포에 대하여 하나 이상의 유전적 변형 (genetic modification)을 도입하는 행위 또는 그에 의하여 만들어진 세포를 나타낸다. The term "genetic engineering" or "genetically engineered " as used herein refers to the act of introducing one or more genetic modifications to a cell or cells made thereby .

본 명세서에서 사용된 용어 "활성 증가 (increase in activity)", 또는 "증가된 활성 (increased activity)"은 주어진 유전적으로 조작되지 않은 모세포(예, 야생형)가 갖지 않는 또는 갖는 내재적 단백질 또는 효소의 활성에 비해, 동일한 타입의 단백질 또는 효소의 활성이 보다 더 높은 활성을 갖는 것을 의미할 수 있다. 단백질 또는 효소의 증가된 활성을 갖는 세포는 당업계에 공지된 임의의 방법을 사용하여 확인될 수 있다. 상기 증가된 활성을 갖는 세포 또는 미생물은, 유전적 변형을 갖지 않은 세포 또는 미생물에 비하여 하나 이상의 효소 또는 폴리펩티드의 활성을 증가시키는 유전적 변형을 갖는 것일 수 있다. As used herein, the term " increase in activity ", or "increased activity" refers to the activity of an endogenous protein or enzyme that does not have or has a given genetically untreated parental , The activity of the same type of protein or enzyme may have higher activity. Cells with increased activity of proteins or enzymes can be identified using any method known in the art. The cell or microorganism having the increased activity may be one having a genetic modification that increases the activity of one or more enzymes or polypeptides relative to a cell or microorganism that does not have a genetic modification.

본 명세서에서 사용된 용어 "활성 감소 (decrease in activity)" 또는 "감소된 활성 (decreased activity)"은 모세포 (예, 유전적으로 조작되지 않은 세포) 중에서 측정된 것보다 더 낮은 효소 또는 폴리펩티드의 활성을 갖는 세포를 나타낸다. 또한, "활성 감소 (decrease in activity)" 또는 "감소된 활성 (decreased activity)"은 본래의 (original) 또는 야생형 (wild-type)의 효소 또는 폴리펩티드보다 더 낮은 활성을 갖는 분리된 효소 또는 폴리펩티드를 나타낸다. 활성 감소 또는 감소된 활성은 활성이 없는 것, 예를 들면, 불활성화(incactivation)를 포함한다. 상기 감소된 활성을 갖는 세포는, 유전적 변형을 갖지 않은 세포에 비하여 하나 이상의 효소 또는 폴리펩티드의 활성을 감소시키는 유전적 변형 (genetic modification)을 갖는 것일 수 있다. The term " decrease in activity "or" decreased activity ", as used herein, refers to the activity of a lower enzyme or polypeptide than measured in a parent cell (e.g., Lt; / RTI > In addition, "decrease in activity" or "decreased activity" refers to an isolated enzyme or polypeptide having lower activity than the original or wild-type enzyme or polypeptide . Activity reduction or reduced activity includes those that are not active, for example, inactivation. The cell with reduced activity may be one that has a genetic modification that reduces the activity of one or more enzymes or polypeptides as compared to cells that do not have a genetic modification.

본 명세서에서 사용된 용어 "모세포 (parent cell)"는 본래 세포 (original cell), 예를 들면, 조작된 미생물, 예를 들면 상기 효모 세포에 대하여 동일 타입의 유전적으로 조작되지 않은 세포를 의미할 수 있다. 상기 모세포는 특정 유전적 변형을 갖거나 갖지 않은 세포이지만, 다른 사항에 대하여는 동일한 것일 수 있다. 따라서, 예를 들면, 상기 모세포는 글루코오스 신호 인자의 활성이 감소된 것만을 제외하고는 상기 효모 세포와 동일한 것일 수 있다. As used herein, the term "parent cell" can refer to an original cell, e. G., A manipulated microorganism, e. G., A genetically engineered cell of the same type for the yeast cell have. The parent cell is a cell with or without a particular genetic modification, but may be the same for other things. Thus, for example, the parent cell may be the same as the yeast cell except that the activity of the glucose signaling factor is reduced.

상기 모세포는 유전적으로 조작된 세포일 수 있다. 전술한 바와 같이, 상기 효모 세포의 유전적 조작은 글루코오스 신호 인자의 활성이 감소되도록 유전적 조작된 것만을 제외하고는 모세포의 유전적 조작과 동일한 것일 수 있다. The parent cell may be a genetically engineered cell. As described above, the genetic manipulation of the yeast cell may be the same as the genetic manipulation of the parental cell, except that it is genetically engineered to reduce the activity of the glucose signaling factor.

일 구체예에 있어서, 상기 모세포 또는 효모 세포는 유전적 조작 전의 세포에 비하여 목적 물질, 예를 들면, 젖산(예를 들면, D 형 또는 L 형 젖산), 숙신산, 에탄올, 이소부탄올, 2,3-부탄다이올, 아세토인, 또는 그들의 조합의 생산이 증대되도록 유전적으로 조작된 효모 세포일 수 있다. In one embodiment, the parent cell or yeast cell is a cell of a target substance, such as lactic acid (for example, D or L type lactic acid), succinic acid, ethanol, isobutanol, 2,3 -Butanediol, acetone, or a combination thereof. ≪ / RTI >

상기 모세포 또는 효모 세포에서 목적 물질의 생산이 증대되도록 유전적으로 조작되기 위하여, 상기 모세포 또는 효모 세포는 유전적 조작 전의 세포에 비하여 알코올 탈수소효소(alcohol dehydrogenase), 글리세롤-3-인산 탈수소효소(glycerol-3-phosphate dehydrogenase), 2,3-부탄다이올 탈수소효소(2,3-butanediol dehydrogenase), 젖산 생산능을 갖지 않는 균주의 D형 젖산 탈수소효소(D-lactate dehydrogenase), 모노카복실레이트/수소 동시수송단백질, 또는 피루브산 탈탄산효소(pyruvate decarboxylase)의 활성이 감소되어 있는 유전적으로 조작된 효모 세포일 수 있다. 또한, 상기 모세포는 유전적 조작 전의 세포에 비하여 아세토락테이트 신타아제(acetolactate synthase), 아세토락테이트 디카복실레이즈(acetolactate decarboxylase), 2,3-부탄다이올 탈수소효소, 젖산 생산능을 갖는 균주의 D형 젖산 탈수소효소, 또는 NADH 산화효소(NADH oxidase)의 활성이 증가되어 있는, 유전적으로 조작된 효모 세포일 수 있다. 예를 들면, 목적 물질의 합성 경로에 관련된 유전자의 발현, 또는 목적 물질의 합성 경로에 경쟁적인 대사 경로에 관련된 유전자의 제거를 통해 목적 생산능을 갖는 모세포 또는 효모 세포를 제작할 수 있다. 상세하게는 상기 모세포 또는 효모 세포는 아세토락테이트 신타아제, 아세토락테이트 디카복실레이즈 및/또는 NADH 산화효소의 활성이 증가되어 있고, 및/또는 알코올 탈수소효소, 글리세롤-3-인산 탈수소효소, 및/또는 2,3-부탄다이올 탈수소효소의 활성이 감소되어 있는, 아세토인 생산능을 갖는 유전적으로 조작된 효모 세포일 수 있다. 또한 상기 모세포 또는 효모 세포는 아세토락테이트 신타아제, 아세토락테이트 디카복실레이즈, 2,3-부탄다이올 탈수소효소 및/또는 NADH 산화효소의 활성이 증가되어 있고, 및/또는 알코올 탈수소효소, 및/또는 글리세롤-3-인산 탈수소효소의 활성이 감소되어 있는, 2,3-부탄다이올 생산능을 갖는 유전적으로 조작된 효모 세포일 수 있다. 또한 상기 모세포 또는 효모 세포는 젖산 생산능을 갖는 균주의 D형 젖산 탈수소효소, 및/또는 NADH 산화효소의 활성이 증가되어 있고, 및/또는 젖산 생산능을 갖지 않는 균주의 D형 젖산 탈수소효소, 알코올 탈수소효소, 글리세롤-3-인산 탈수소효소, 모노카복실레이트/수소 동시수송단백질, 및/또는 피루브산 탈탄산효소의 활성이 감소되어 있는 젖산 생산능을 갖는 유전적으로 조작된 효모 세포일 수 있다. 상기는 목적 물질의 생산을 증대시키기 위한 유전적 조작의 일 예시들이고, 통상의 당업자는 미생물의 알려진 대사 경로에서 특정 유전자의 발현 또는 제거를 통해 다양한 목적 물질의 생산능이 증대된 모세포 또는 효모 세포를 제작할 수 있다. 이에 추가적으로, 상기 모세포 또는 효모 세포에 글루코오스 신호 인자의 활성을 감소시킴으로써, 생장 속도가 증대되거나, 포도당 소비가 증대되거나, 목적 물질의 생산능이 더 증대된 효모 세포를 제작할 수 있다. In order to be genetically manipulated so as to increase the production of a target substance in the parent cells or yeast cells, the parent cells or yeast cells are required to have an alcohol dehydrogenase, a glycerol-3-phosphate dehydrogenase, 3-phosphate dehydrogenase, 2,3-butanediol dehydrogenase, D-lactate dehydrogenase of a strain not producing lactic acid production, monocarboxylate / hydrogen simultaneously Or a genetically engineered yeast cell in which the activity of pyruvate decarboxylase is reduced. In addition, the above-described parent cells have been found to be more effective than acetolactate synthase, acetolactate decarboxylase, 2,3-butanediol dehydrogenase, and lactic acid producing strain D-type lactate dehydrogenase, or NADH oxidase, may be a genetically engineered yeast cell. For example, a parent cell or a yeast cell having an objective production ability can be produced through expression of a gene involved in a synthesis pathway of a target substance, or removal of a gene involved in a competitive metabolic pathway in a synthesis pathway of a target substance. Specifically, the parent cell or yeast cell has increased activity of acetolactate synthase, acetolactate dicarboxylase and / or NADH oxidase, and / or alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase, and / RTI > and / or 2,3-butanediol dehydrogenase activity is reduced. In addition, the parent cells or yeast cells may have increased activity of acetolactate synthase, acetolactate dicarboxylase, 2,3-butanediol dehydrogenase and / or NADH oxidase, and / or alcohol dehydrogenase, and Or a genetically engineered yeast cell having 2,3-butanediol producing ability, wherein the activity of the glycerol-3-phosphate dehydrogenase is reduced. In addition, the parent cells or yeast cells may be selected from the group consisting of D-type lactate dehydrogenase, D-type lactate dehydrogenase, and / or NADH oxidase, which have increased lactic acid production ability and / It may be a genetically engineered yeast cell having the ability to produce lactic acid with reduced activity of alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase, monocarboxylate / hydrogen co-transport protein, and / or pyruvate decarboxylase. The above is an example of genetic manipulation for increasing the production of a target substance, and a person skilled in the art will be able to produce a parent cell or yeast cell whose productivity of various target substances is increased through the expression or elimination of a specific gene in a known metabolic pathway of the microorganism . In addition, by decreasing the activity of the glucose signaling factor in the parent cells or yeast cells, yeast cells having increased growth rate, increased glucose consumption, or increased production capacity of the target substance can be produced.

상기 언급된 효소는 이름이 상이하더라도 그와 유사한 활성을 갖는 효소(예를 들면, 동질효소(isoenzyme) 또는 동족체(homolog))를 포함할 수 있다. 예를 들면, 상기 아세토락테이트 신타아제는 아세토하이드록시산 신타아제(acetohydroxy acid synthase: AHAS)와 호환적으로 사용되고, 바실러스 서브틸리스 유래의 alsS에 의해 암호화되는 아세토락테이트 신타아제, 대장균 유래의 ilvB 또는 ilvN에 의해 암호화되는 아세토락테이트 신타아제 I, 대장균 유래의 ilvGMEDA에 의해 암호화되는 아세토락테이트 신타아제 II, 또는 대장균 유래의 ilvI 또는 ilvH에 의해 암호화되는 아세토락테이트 신타아제 III를 포함할 수 있다. 상기 아세토락테이트 디카복실레이즈는 바실러스 서브틸리스 유래의 alsD, 락토바실러스 델브루키(Lactobacillus delbrueckii subsp . lactis) 유래의 aldB, 브레비바실러스 브레비스(Brevibacillus brevis), 엔테로박터 에어로게네스(Enterobacter aerogenes), 류코노스톡 락티스(Leuconostoc lactis), 사카로마이세스 세레비지애, 스타필로코커스 아우레우스(Staphylococcus aureus) 유래의 아세토락테이트 디카복실레이즈일 수 있다. 상기 NADH 산화효소는 nox1, nox3, nox4, 락토코커스 락티스 유래의 noxE를 포함할 수 있다. 상기 알코올 탈수소효소는 ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 또는 SFA1를 포함할 수 있다. 상기 글리세롤-3-인산 탈수소효소는 GPD1 또는 GPD2를 포함할 수 있다. 상기 2,3-부탄다이올 탈수소효소는 사카로마이세스 세레비지애 유래의 BDH1, 패니바실러스 폴리믹사(Paenibacillus polymyxa) 유래의 BDH99::67, 바실러스 서브틸리스, 엔테로코커스 패시움(Enterococcus faecium) 엔테로코커스 듀란스(Enterococcus durans) 마이코박테리움 속(Mycobacterium sp .) 락토바실러스 락티스 유래의 2,3-부탄다이올 탈수소효소일 수 있다. 상기 젖산 생산능을 갖지 않는 균주의 D형 젖산 탈수소효소는 효모 균주, 예를 들면, 사카로마이세스 세레비지애 유래의 DLD1을 포함할 수 있다. 상기 모노카복실레이트/수소 동시수송단백질은 피루브산 퍼미아제(pyruvate permease), 또는 젖산 퍼미아제(lactate permease)를 포함할 수 있다. 예를 들면, 상기 모노카복실레이트/수소 동시수송단백질은 사카로마이세스 세레비지애 유래의 JEN1을 포함할 수 있다. 상기 피루브산 탈탄산효소는 효모 균주, 예를 들면, 사카로마이세스 세레비지애 유래의 PDC1, PDC5 또는 PDC6을 포함할 수 있다. 상기 젖산 생산능을 갖는 균주의 D형 젖산 탈수소효소는 예를 들면, 락토바실러스 종, 예를 들면, 락토바실러스 브레비스(Lactobacillus brevis), 락토바실러스 펜토수스(Lactobacillus pentosus), 락토바실러스 람노서스(Lactobacillus rhamnosus), 락토바실러스 젠세니(Lactobacillus jensenii), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 파라플란타럼(Lactobacillus paraplantarum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 파라카세이(Lactobacillus paracasei), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 존스니(Lactobacillus johnsonii), 락토바실러스 카세이(Lactobacillus case), 또는 류코노스톡 종, 예를 들면, 류코노스톡 메센테로이드(Leuconostoc mesenteroides), 류코노스톡 카르노섬(Leuconostoc carnosum), 류코노스톡 시트리움(Leuconostoc citreum), 류코노스톡 겔리둠(Leuconostoc gelidum), 또는 류코노스톡 수도메센테로이드(Leuconostoc pseudomesenteroides), 또는 페디오코커스 종(Pediococcus spp .) 유래의 젖산 탈수소효소를 포함할 수 있다. The above-mentioned enzymes may include an enzyme (e.g., an isoenzyme or a homolog) having similar activity even if the name is different. For example, the acetolactate synthase may be used in combination with acetohydroxy acid synthase (AHAS), acetolactate synthase encoded by alsS from Bacillus subtilis, Acetolactate synthase I encoded by ilvB or ilvN, Acetolactate synthase II encoded by ilvGMEDA from E. coli, or Acetolactate synthase III encoded by ilvI or ilvH from E. coli. have. The acetolactate dicarboxylase may be selected from the group consisting of alsD from Bacillus subtilis, Lactobacillus delbrueckii subsp . lactis- derived aldB, Brevibacillus < RTI ID = 0.0 > brevis , Enterobacter aerogenes , Leuconostoc lactis , Saccharomyces cerevisiae, Staphylococcus aureus, and the like. The NADH oxidase may include nox1, nox3, nox4, and noxE derived from lactococcus lactis. The alcohol dehydrogenase may include ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 or SFA1. The glycerol-3-phosphate dehydrogenase may comprise GPDl or GPD2. The 2,3-butanediol dehydrogenase can be obtained from BDH1 derived from Saccharomyces cerevisiae, Paenibacillus < RTI ID = 0.0 > polymyxa ), BDH99 :: 67, Bacillus subtilis, Enterococcus faecium ) Enterococcus Durans can be a 2,3-butanediol dehydrogenase derived from Mycobacterium sp . Lactobacillus lactis . The D-lactate dehydrogenase enzyme of the strain having no lactic acid producing ability may contain a yeast strain, for example, DLD1 derived from Saccharomyces cerevisiae. The monocarboxylate / hydrogen co-transport protein may comprise a pyruvate permease, or a lactate permease. For example, the monocarboxylate / hydrogen co-transport protein may comprise JEN1 from Saccharomyces cerevisiae. The pyruvic acid decarboxylase may include a yeast strain, for example, PDC1, PDC5 or PDC6 derived from Saccharomyces cerevisiae. The D-lactate dehydrogenase enzyme of the strain having lactic acid producing ability is, for example, a lactobacillus species such as Lactobacillus brevis , Lactobacillus pentosus , Lactobacillus rhamnosus , Lactobacillus jensenii , Lactobacillus plantarum , Lactobacillus paraplantarum , Lactobacillus fermentum , Lactobacillus paracasei , Lactobacillus spp ., And Lactobacillus spp . Lactobacillus acidophilus , Lactobacillus johnsonii , Lactobacillus case , or Leuconostoc sp., Such as leuconostocercenteroid ( Leuconostoc mesenteroides , Leuconostoc carnosum , Leuconostoc citreum , Leuconostoc gelidum ), or the Ryukono Stock cappuccine ( Leuconostoc pseudo mesenteroides), or the caucus Pedy five species (Pediococcus spp . ) Derived lactate dehydrogenase.

본 명세서에서 용어 "글루코오스 신호 인자(glucose signaling factor: GSF)"는 소포체에 위치하여 소포체로부터의 Hxt1(hexose transporter 1), Gal2(galactose permease)와 같은 특정 당수송 단백질의 분비에 관여하는 인자를 의미할 수 있다. 상기 글루코오스 신호 인자는 단백질의 이름이 상이하더라도, 그와 유사한 활성을 갖는 단백질을 포함할 수 있으며, 예를 들면, GSF1 또는 GSF2를 포함할 수 있다. 상기 글루코오스 신호 인자는 서열번호 2의 아미노산 서열과 약 70% 이상, 약 75% 이상, 약 80% 이상, 약 85%이상, 약 90% 이상, 약 92% 이상, 약 95% 이상, 약 97% 이상, 약 98% 이상, 또는 약 99% 이상의 서열 상동성을 갖는 폴리펩티드 일 수 있다. 상기 글루코오스 신호 인자 유전자는 서열번호 1의 뉴클레오티드 서열과 약 70% 이상, 약 75% 이상, 약 80% 이상, 약 85%이상, 약 90% 이상, 약 92% 이상, 약 95% 이상, 약 97% 이상, 약 98% 이상, 또는 약 99% 이상의 서열 상동성을 갖는 것일 수 있다.
As used herein, the term "glucose signaling factor (GSF)" refers to a factor involved in the secretion of specific sugar transport proteins such as Hxt1 (hexose transporter 1) and Gal2 (galactose permease) from the endoplasmic reticulum can do. The glucose signaling factor may include proteins having similar activities, even though the names of the proteins are different, and may include, for example, GSF1 or GSF2. The glucose signaling factor is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97% of the amino acid sequence of SEQ ID NO: , At least about 98%, or at least about 99% sequence homology. The glucose signaling factor gene is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97% of the nucleotide sequence of SEQ ID NO: , At least about 98%, or at least about 99% sequence homology.

상기 모세포 또는 효모 세포는 전술된 효소의 활성을 증가시키기 위해 그를 코딩하는 외인성 유전자 (exogenous gene)를 포함하는 것일 수 있다. 용어 "외인성 (exogenous)"은 언급된 분자 (referenced molecule) 또는 언급된 활성 (referenced activity)이 숙주 세포로 도입된 것을 의미할 수 있다. 분자는 예를 들면, 숙주 염색체 내로의 삽입에 의하는 것과 같은 코딩 핵산 (encoding nucleic acid)의 숙주 유전 물질 내로의 도입 또는 플라스미드와 같은 비염색체 유전물질로서 도입될 수 있다. 코딩 핵산의 발현과 관련하여, 상기 용어 "외인성"은 상기 코딩 핵산이 개체 내로 발현 가능한 형태로 도입된 것을 나타낸다. 생합성 활성과 관련하여, 상기 용어 "외인성"은 숙주 모세포에 도입된 활성을 나타낸다. 그 기원 (source)은 예를 들면, 숙주 모세포에 도입된 후 언급된 활성을 발현하는 동질성 (homologous) 또는 이질성 (heterologous) 코딩 핵산일 수 있다. 그러므로, 용어 "내인성 (endogenous)"은 상기 숙주 세포에 존재하는 언급된 분자 또는 활성을 나타낸다. 비슷하게, 코딩 핵산의 발현과 관련하여, 상기 용어 "내인성"은 개체 내에 포함된 코딩 핵산의 발현을 나타낸다. 용어 "이질성 (heterologous)"은 언급된 종 외의 다른 기원으로부터의 분자 또는 활성을 나타내고 용어 "동질성 (homologous)"은 숙주 모세포로부터의 분자 또는 활성을 나타낸다. 따라서, 코딩 핵산의 외인성 발현은 이질성 (heterologous) 또는 동질성 (homologous) 코딩 핵산 중 어느 하나 또는 둘 다를 이용할 수 있다. 상기 외인성 유전자는, 상기 모세포 또는 효모 세포에서 유전적 조작 전에 비하여 언급된 효소의 활성이 증가되기에 충분한 양으로 발현된 것일 수 있다. 이러한 외인성 유전자는 미생물에서 발현되기에 적합한 코돈으로 변경된 서열, 최적화된 코돈을 갖는 서열로 변경될 수 있다. 이 코돈 변경은 단백질의 아미노산 서열이 바뀌지 않는 범위 내에서 적절히 이루어질 수 있다. The parent cell or yeast cell may comprise an exogenous gene encoding the same to increase the activity of the aforementioned enzyme. The term "exogenous" may refer to the introduction of a referenced molecule or a referenced activity into a host cell. Molecules may be introduced into the host genetic material, for example, by encoding nucleic acid, such as by insertion into a host chromosome, or as a non-chromosomal genetic material, such as a plasmid. In connection with the expression of a coding nucleic acid, the term "exogenous" indicates that the coding nucleic acid has been introduced into a form capable of expression into the subject. With respect to biosynthesis activity, the term "exogenous" refers to the activity introduced into the host cell. The source may be, for example, a homologous or heterologous coding nucleic acid which is introduced into the host cell and expresses the activity mentioned. Thus, the term "endogenous" refers to the mentioned molecule or activity present in the host cell. Similarly, in connection with the expression of a coding nucleic acid, the term "endogenous" refers to the expression of a coding nucleic acid contained within an individual. The term " heterologous "refers to a molecule or activity from a different source than the species mentioned, and the term" homologous "refers to a molecule or activity from a host cell. Thus, the exogenous expression of the coding nucleic acid may utilize either or both of heterologous or homologous coding nucleic acids. The exogenous gene may be expressed in an amount sufficient to increase the activity of the enzyme mentioned before the genetic manipulation in the parental or yeast cell. Such an exogenous gene can be changed to a sequence having a codon suitable for expression in a microorganism, a sequence having an optimized codon. This codon change can be made appropriately within a range that does not change the amino acid sequence of the protein.

상기 외인성 유전자는 발현 벡터를 통하여 모세포 내로 도입된 것일 수 있다. 또한, 상기 외인성 유전자는 선형 폴리뉴클레오티드 형태로 모세포 내로 도입된 것일 수 있다. 또한, 상기 외인성 유전자는 세포 내에서 발현 벡터 (예, 플라스미드)로부터 발현되는 것일 수 있다. 또한, 상기 외인성 유전자는 안정적인 발현을 위하여 세포 내의 유전물질 (예, 염색체)에 삽입되어 발현되는 것일 수 있다. 상기 벡터는 복제개시점, 프로모터, 상기 효소를 코딩하는 폴리뉴클레오티드, 및 터미네이터를 포함할 수 있다. 상기 복제개시점은 효모 자가복제 서열 (autonomous replication sequence, ARS)을 포함할 수 있다. 상기 효모 자가복제서열은 효모 동원체 서열 (centrometric sequence, CEN)에 의해 안정화될 수 있다. 상기 프로모터는 TDH3 프로모터, TEF 프로모터, 및 FBA1 프로모터로 이루어진 군으로부터 선택된 것일 수 있다. 상기 터미네이터는 CYC1, GPM1, 및 FBA1로 이루어진 군으로부터 선택되는 것일 수 있다. 상기 벡터는 선별 마커를 더 포함할 수 있다. The exogenous gene may be introduced into the parent cell through an expression vector. In addition, the exogenous gene may be introduced into the parent cell in the form of a linear polynucleotide. In addition, the exogenous gene may be expressed from an expression vector (e.g., a plasmid) in a cell. In addition, the exogenous gene may be inserted into a genetic material (e.g., a chromosome) in a cell for stable expression. The vector may comprise a cloning start point, a promoter, a polynucleotide encoding the enzyme, and a terminator. The origin of replication may comprise an autonomous replication sequence (ARS). The yeast self-replication sequence can be stabilized by a centrometric sequence (CEN). The promoter may be selected from the group consisting of TDH3 promoter, TEF promoter, and FBA1 promoter. The terminator may be selected from the group consisting of CYC1, GPM1, and FBA1. The vector may further comprise a selection marker.

상기 모세포 또는 효모 세포는 단일 유전자, 복수의 유전자 예를 들면, 2 내지 10 카피 수를 포함할 수 있다. 상기 효모 세포는, 예를 들면, 1 내지 10, 1 내지 8, 1 내지 7, 1 내지 6, 1 내지 5, 1 내지 4, 1 내지 3, 2 내지 10, 2 내지 8, 2 내지 7, 2 내지 6, 2 내지 5, 2 내지 4, 또는 2 내지 3 카피의 상기 효소를 코딩하는 유전자를 포함할 수 있다. 상기 모세포 또는 효모 세포가 복수의 유전자를 포함하는 경우, 각각의 유전자는 동일한 유전자의 카피이거나 둘 이상의 상이한 유전자의 카피를 포함할 수 있다. 외인성 유전자의 복수의 카피는 숙주 세포의 게놈 내에 동일한 유전자 좌 (locus), 또는 여러 유전자 좌에 포함될 수 있다. The parent cell or yeast cell may contain a single gene, a plurality of genes, for example, 2 to 10 copies. The yeast cell may be cultured in a culture medium containing, for example, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 10, 2 to 8, To 6, 2 to 5, 2 to 4, or 2 to 3 copies of the enzyme. When the parent cell or yeast cell contains a plurality of genes, each gene may be a copy of the same gene or a copy of two or more different genes. Multiple copies of the exogenous gene may be contained in the same locus or multiple loci within the genome of the host cell.

본 명세서에서 사용된 폴리뉴클레오티드는 "유전자"를 포함하고, 본 명세서에서 사용된 핵산 분자는 "벡터" 또는 "플라스미드"를 포함하는 것으로 이해될 수 있다. 따라서 용어 "유전자" (일명, "구조적 유전자")는 아미노산의 특정 서열을 코딩하는 폴리뉴클레오티드를 지칭하는데, 이것은 하나 또는 그 이상의 단백질 또는 효소의 전부 또는 일부를 포함하고, 그리고 예로써, 유전자가 발현되는 조건을 결정하는 조절 (비-전사된) DNA 서열, 예를 들면, 프로모터 서열을 포함할 수 있다. 유전자의 전사된 영역은 코딩 서열뿐만 아니라 인트론, 5'-비번역 영역 (UTR), 그리고 3'-UTR을 비롯한 비번역 영역을 포함할 수 있다.
As used herein, polynucleotides include "genes" and nucleic acid molecules as used herein can be understood to include "vectors" or "plasmids". Thus, the term "gene" (aka, "structural gene") refers to a polynucleotide that encodes a particular sequence of amino acids, including all or part of one or more proteins or enzymes, (Non-transcribed) DNA sequence, e.g., a promoter sequence, that determines the conditions under which the gene is transcribed. The transcribed region of the gene may include a coding sequence as well as an untranslated region, including an intron, a 5'-untranslated region (UTR), and a 3'-UTR.

상기 모세포 또는 효모 세포는 전술된 효소의 활성을 감소시키기 위해 상기 언급된 효소 또는 단백질을 코딩하는 내인성 유전자(endogenous gene)가 제거 또는 파괴(disruption)된 것일 수 있다. 예를 들면, 상기 효모 세포는 글루코오스 신호 인자를 코딩하는 유전자가 제거 또는 파괴된 것일 수 있다. 용어 "제거(deletion)" 또는 "파괴 (disruption)"는 유전자의 발현이 감소되도록 하는 유전적 변형을 나타낸다. 상기 파괴는 유전자의 "불활성화 (inactivation)" 또는 유전자의 "감쇄 (attenuation)"를 포함할 수 있다. 상기 불활성화는 유전자의 기능적 산물 (functional product)이 발현되지 않는 것뿐만 아니라 발현은 되지만 기능적 산물이 발현되지 않는 것을 포함한다. 상기 감쇄는 유전자의 기능적 산물의 발현량 감소를 포함한다. 즉, 상기 감쇄는 유전자의 순 발현량은 증가하였더라도 기능적 산물의 발현량이 감소되는 것을 포함할 수 있다. 여기서 유전자의 기능적 산물이란 모세포 또는 야생형 세포에서 상기 유전자의 산물 (예, 효소)이 갖는 생화학적 또는 생리적 기능 (예, 효소 활성)을 보유하고 있는 것을 말한다. 따라서, 상기 제거 또는 파괴는 유전자의 기능적 제거(functional deletion) 또는 파괴(functional disruption)를 포함한다.The parent cell or yeast cell may be one in which the endogenous gene coding for the above-mentioned enzyme or protein has been removed or disrupted to reduce the activity of the aforementioned enzyme. For example, the yeast cell may be one in which the gene encoding the glucose signaling factor is deleted or destroyed. The term " deletion "or" disruption "refers to a genetic modification that results in decreased expression of the gene. Such destruction can include "inactivation" of a gene or "attenuation" of a gene. The inactivation includes not only the expression of the functional product of the gene but also the expression of the functional product but not the functional product. The attenuation includes a reduction in the expression level of the functional product of the gene. That is, the attenuation may include a decrease in the expression level of the functional product even when the net expression amount of the gene is increased. Here, the functional product of a gene refers to a gene having a biochemical or physiological function (for example, an enzyme activity) of a product (e.g., an enzyme) of the gene in a cell or a wild-type cell. Thus, the removal or destruction involves functional deletion or functional disruption of the gene.

상기 제거 또는 파괴하는 단계는 1) 상기 단백질을 암호화하는 유전자의 일부 또는 전체의 결실, 2) 상기 유전자의 발현이 감소하도록 발현조절 서열의 변형, 3) 상기 단백질의 활성이 약화되도록 염색체 상의 상기 유전자 서열의 변형 또는 4) 이의 조합 등을 사용하여 수행될 수 있다. 상기 단백질을 암호화하는 폴리뉴클레오티드의 일부 또는 전체를 결실하는 방법은 예를 들면, Cre/loxP 재조합 시스템을 사용하여 유전자 결손을 위한 카세트를 모세포에 형질전환함으로써 수행될 수 있고, 효모 내 염색체 삽입용 벡터를 통해 염색체 내 내재적 목적 단백질을 암호화하는 폴리뉴클레오티드를 일부 핵산 서열이 결실된 폴리뉴클레오티드 또는 마커 유전자로 교체함으로써 수행될 수 있다. 상기 "일부"란 폴리뉴클레오티드의 종류에 따라서 상이하지만, 예를 들면, 1 내지 700개, 1 내지 500개, 1 내지 300개, 1 내지 100개, 또는 1 내지 50개일 수 있다. 또한, 상기 뉴클레오티드의 발현이 감소하도록 발현조절 서열을 변형하는 방법은 상기 발현조절 서열의 활성을 더욱 약화하도록 핵산 서열을 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합으로 발현조절 서열상의 변이를 유도하여 수행하거나, 더욱 약한 활성을 갖는 핵산 서열로 교체함으로써 수행할 수 있다. 상기 발현조절 서열에는 프로모터, 오퍼레이터 서열, 리보좀 결합 부위를 코딩하는 서열, 및 전사와 해독의 종결을 조절하는 서열을 포함한다. 아울러, 상기 단백질을 암호화하는, 염색체 상의 폴리뉴클레오티드 서열을 변형하는 방법은 상기 단백질의 활성을 더욱 약화하도록 폴리뉴클레오티드 서열을 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합으로 서열상의 변이를 유도하여 수행하거나, 더욱 약한 활성을 갖도록 개량된 폴리뉴클레오티드 서열로 교체함으로써 수행할 수 있다.The step of removing or destroying may include 1) deletion of part or all of the gene encoding the protein, 2) modification of the expression control sequence so that the expression of the gene is decreased, 3) A modification of the sequence, or 4) a combination thereof. A method of deleting a part or all of the polynucleotides encoding the protein can be performed, for example, by transforming a cassette for gene deletion into a parent cell using a Cre / loxP recombination system, Into a polynucleotide encoding an intrinsic target protein in the chromosome with a polynucleotide or marker gene in which some nucleic acid sequences have been deleted. The "part" may be, for example, 1 to 700, 1 to 500, 1 to 300, 1 to 100, or 1 to 50 depending on the kind of the polynucleotide. In addition, the method of modifying the expression control sequence so that the expression of the nucleotide is decreased may include a step of mutating the nucleic acid sequence, deletion, insertion, non-conservative or conservative substitution or a combination thereof in the expression control sequence to further weaken the activity of the expression control sequence Or by replacing it with a nucleic acid sequence having a weaker activity. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating termination of transcription and translation. In addition, a method of modifying a polynucleotide sequence on a chromosome, which encodes the protein, may be performed by deletion, insertion, non-conservative or conservative substitution of a polynucleotide sequence or a combination thereof to induce a mutation in the sequence to further weaken the activity of the protein , Or by replacing with an improved polynucleotide sequence to have weaker activity.

상기 효모 세포는 사카로마이세스(Saccharomyces) 속에 속하는 것일 수 있다. 예를 들면, 상기 효모 세포는 사카로마이세스(Saccharomyces), 클루베로마이세스(Kluyveromyces), 피키아(Pichia), 한세눌라(Hansenula), 자이고사카로마이세스(Zygosaccharomyces) 또는 캔디다(Candida)속에 속하는 균주인 것일 수 있다. 또한, 사카로마이세스(Saccharomyces)속에서 사카로마이세스 센수 스트릭토(Saccharomyces sensustricto) 집합체에 속하는 균주인 것일 수 있다. 사카로마이세스 센수 스트릭토(Saccharomyces sensustricto) 집합체에 속하는 균주는 예를 들면, 사카로마이세스 세레비지애(S. cerevisiae), 사카로마이세스 바야누스(S. bayanus), 사카로마이세스 파라독서스 (S. paradoxus), 사카로마이세스 미카테(S. mikatae), 또는 사카로마이세스 쿠드리아브제비(S. kudriavzevii)일 수 있다. The yeast cell may be one belonging to the genus Saccharomyces . For example, the yeast cell may be selected from the group consisting of Saccharomyces , Kluyveromyces , Pichia , Hansenula , Zygosaccharomyces or Candida , Which is a strain belonging to the genus. In addition, the saccharide with my process (Saccharomyces) in a Saccharomyces sensu My process streak soil (Saccharomyces sensustricto ) cluster. Saccharomyces The strains belonging to the sensustricto cluster include, for example, S. cerevisiae , S. bayanus , S. paradoxus , romayi process may be non-catheter (S. mikatae), or a saccharide as MY-ku laundry process Havre lots (S. kudriavzevii).

일 구체예에 있어서, 모세포에 비하여 글루코오스 신호 인자의 활성이 감소되어 있는, 유전적으로 조작된 효모 세포는 모세포에 비하여 포도당 소비량이 증대된 것일 수 있다. 상기 효모 세포의 생장 속도 증대, 및 포도당 소비량 증대에 따라, 목적 물질의 생산이 증대될 수 있다. 따라서, 상기 효모 세포는 모세포에 비하여 목적 물질의 생산이 증대된 것일 수 있다.
In one embodiment, the genetically engineered yeast cell, in which the activity of the glucose signaling factor is reduced relative to the parent cell, may have increased glucose consumption relative to the parent cell. As the growth rate of the yeast cells is increased and the glucose consumption is increased, the production of the target substance can be increased. Therefore, the yeast cell may have increased production of the target substance as compared with the parent cell.

다른 양상은 상기 효모 세포를 사용하여 목적 물질을 생산하는 방법을 제공한다. Another aspect provides a method for producing a target substance using the yeast cell.

상기 방법은 모세포에 비하여 글루코오스 신호 인자(glucose signaling factor: GSF)의 활성이 감소되어 있는, 상기 세포에 비하여 생장 속도가 증대된 유전적으로 조작된 효모 세포를 배지에서 배양하는 단계; 및 상기 배양물로부터 목적 물질을 분리하는 단계를 포함할 수 있다. Culturing the genetically engineered yeast cells having a reduced glucose signaling factor (GSF) activity relative to the parent cells, wherein the growth rate is higher than that of the cells, in a culture medium; And separating the target substance from the culture.

상기 "모세포", 및 "생장 속도가 증대된 유전적으로 조작된 효모 세포"에 대해서는 상기한 바와 같다. 상기 목적 물질은 젖산(예를 들면, D형 젖산, 또는 L형 젖산), 숙신산, 에탄올, 이소부탄올, 2,3-부탄다이올, 또는 아세토인일 수 있다. The above "parent cells" and "genetically engineered yeast cells with increased growth rate" are as described above. The target material may be lactic acid (for example, D-lactic acid or L-lactic acid), succinic acid, ethanol, isobutanol, 2,3-butanediol or acetone.

본 발명의 용어 "배양"이란, 상기 효모 세포로부터 목적 물질을 생산하기 위하여, 상기 세포를 적당히 인공적으로 조절한 환경조건에서 생육시키는 일련의 행위를 의미할 수 있다. 본 발명에서 상기 세포를 배양하는 방법은 당업계에 널리 알려져 있는 방법을 이용하여 수행할 수 있다. 구체적으로 상기 배양은 배치 공정 또는 주입 배치 또는 반복 주입 배치 공정(fed batch or repeated fed batch process)에서 연속식으로 배양할 수 있다. 배양에 사용되는 배지는 목적 물질로 대사될 수 있는 하나 이상의 기질을 포함하는 것일 수 있으며, 예를 들면, 적당한 탄소원, 질소원, 아미노산, 비타민 등을 함유한 통상의 배지 내에서 호기성 조건 하에서 온도, pH 등을 조절하면서 적절한 방식으로 특정 균주의 요건을 충족해야 한다. 사용될 수 있는 탄소원으로는 글루코즈를 주탄소원으로 사용하며, 이외에 자일로즈, 수크로즈, 락토즈, 프락토즈, 말토즈, 전분, 셀룰로즈와 같은 당 및 탄수화물, 대두유, 해바라기유, 피마자유, 코코넛유 등과 같은 오일 및 지방, 팔미트산, 스테아린산, 리놀레산과 같은 지방산, 글리세롤, 에탄올과 같은 알코올, 아세트산과 같은 유기산이 포함될 수 있다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 사용될 수 있는 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 및 질산암모늄과 같은 무기질소원; 글루탐산, 메티오닌, 글루타민과 같은 아미노산 및 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해생성물 등 유기질소원이 사용될 수 있다. 이들 질소원은 단독 또는 조합되어 사용될 수 있다. 상기 배지에는 인원으로서 인산 제1칼륨, 인산 제2칼륨 및 대응되는 소듐-함유 염이 포함될 수 있다. 사용될 수 있는 인원으로는 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨-함유 염이 포함된다. 또한, 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간 및 탄산칼슘 등이 사용될 수 있다. 마지막으로, 상기 물질에 더하여 아미노산 및 비타민과 같은 필수 성장 물질이 사용될 수 있다.The term "cultivation" of the present invention may mean a series of actions in which the cells are grown under appropriately artificially controlled environmental conditions in order to produce a target substance from the yeast cells. The method for culturing the cells in the present invention can be carried out by using methods well known in the art. Specifically, the culture may be continuously cultured in a batch process, an injection batch, or a repeated batch or batch fed batch process. The medium used for the culture may be one containing one or more substrates that can be metabolized to the target substance and may be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, And the like, while meeting the requirements of a particular strain. The carbon source that can be used is glucose as the main carbon source and may also be used in combination with sugars and carbohydrates such as xylose, sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, The same oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.

통상적으로 세포는 약 20℃ 내지 약 37℃ 범위의 온도에서 적절한 배지 내에 성장시킬 수 있다. 본 발명에서 성장 배지는, 예를 들면, 효모 질소 베이스(yeast nitrogen base), 암모늄 설페이트, 및 탄소/에너지 공급원으로서의 덱스트로스를 포함하는 브로스(broth) 또는 대부분의 사카로마이세스 세레비지애 균주의 성장을 위한 최적 비율로 펩톤, 효모 추출물 및 덱스트로스를 블렌딩한 YPD 배지와 같이 상업적으로 제조된 통상적인 배지일 수 있다. 그밖에 정의되거나 합성된 성장 배지도 사용할 수 있으며, 특정 미생물의 성장에 적절한 배지는 미생물학 또는 발효과학 분야의 당업자에게 공지되어 있다. Typically, the cells can be grown in a suitable medium at a temperature ranging from about 20 < 0 > C to about 37 < 0 > C. In the present invention, the growth medium may comprise, for example, a yeast nitrogen base, ammonium sulfate, and dextrose as a carbon / energy source, such as broth or most of the Saccharomyces cerevisiae strain For example, a YPD medium in which peptone, yeast extract and dextrose are blended in an optimal ratio for growth. Other defined or synthetic growth media may also be used, and suitable media for growth of particular microorganisms are known to those skilled in the art of microbiology or fermentation.

또한, 상기 분리는 배양물, 예를 들면, 배양한 미생물, 세포, 배지, 배양 매질, 또는 그들의 조합으로부터 분리하는 것일 수 있다. In addition, the separation may be to separate from the culture, for example, the cultured microorganism, the cell, the culture medium, the culture medium, or a combination thereof.

생물 생산된 목적 물질은 당업계에 공지된 방법을 사용하여 배양 배지로부터 분리할 수 있다. 이러한 분리 방법은 원심분리, 여과, 이온교환크로마토그래피 또는 결정화일 수 있다. 예를 들면, 배양물을 저속 원심분리하여 바이오매스를 제거하고 얻어진 상등액을, 이온교환크로마토그래피를 통하여 분리할 수 있다. The biologically produced target substance can be isolated from the culture medium using methods known in the art. Such a separation method may be centrifugation, filtration, ion exchange chromatography or crystallization. For example, the culture can be centrifuged at low speed to remove the biomass, and the resulting supernatant can be separated through ion exchange chromatography.

일 양상에 따른 효모 세포 및 그를 사용하여 목적 물질을 생산하는 방법에 의하면, 효모 세포의 생장 속도 및 포도당 소비량 증대에 의해 목적 물질을 고효율 및 고수율로 생산할 수 있다. According to one aspect of the present invention, a yeast cell and a method for producing a target substance using the same can produce a target substance with high efficiency and high yield by increasing the growth rate of yeast cells and the consumption of glucose.

도 1은 일 구체예에 따른 생장 속도가 증대된 S. cerevisiae 균주의 생장 속도를 나타낸 그래프이다.
도 2는 일 구체예에 따른 생장 속도가 증대된 S. cerevisiae 균주의 생장 속도를 나타낸 그래프이다.
도 3은 일 구체예에 따른 생장 속도가 증대된 S. cerevisiae 균주의 생장 속도를 나타낸 그래프이다.
도 4는 비교예에 따른 젖산 생산능이 증가된 S. cerevisiae 균주의 대사 산물의 생산량을 나타낸 그래프이다.
도 5는 일 구체예에 따른 젖산 생산능이 증가된 S. cerevisiae 균주의 대사 산물의 생산량을 나타낸 그래프이다. FIG. 1 is a graph showing the growth rate of S. cerevisiae strain with increased growth rate according to one embodiment.
FIG. 2 is a graph showing the growth rate of S. cerevisiae strain with increased growth rate according to one embodiment.
FIG. 3 is a graph showing the growth rate of S. cerevisiae strain with an increased growth rate according to one embodiment.
FIG. 4 is a graph showing the production yield of metabolites of S. cerevisiae strain with increased lactic acid production capacity according to a comparative example.
FIG. 5 is a graph showing production yields of metabolites of S. cerevisiae strains with increased lactic acid production capacity according to one embodiment.

이하 본 발명을 실시예에 의해 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

실시예Example 1. 생장 속도가 증대된  1. Increased growth rate S. S. cerevisiaecerevisiae 균주의 제작 Production of strain

본 실시예에서는 특정 목적 물질의 생산능이 증대되도록 유전적으로 조작된 효모 세포에 추가적으로 글루코오스 신호 인자 2(glucose signaling factor 2: GSF2) 유전자 (서열번호 1의 뉴클레오티드 서열, 서열번호 2의 아미노산 서열)를 결손시켜, 생장 속도가 증대된 S. cerevisiae 균주를 제작하였다.
In this embodiment, glucose signaling factor 2 (GSF2) gene (nucleotide sequence of SEQ ID NO: 1, amino acid sequence of SEQ ID NO: 2) is deleted in addition to genetically engineered yeast cells so that the production capability of a specific target substance is increased To produce S. cerevisiae strain with increased growth rate.

1. One. ADHADH 1 내지 5 유전자 결손,  1 to 5 gene deletion, GPDGPD 1 및 2 유전자 결손 및/또는  1 and 2 gene defects and / or GSF2GSF2 유전자 결손  Gene defect S. S. cerevisiaecerevisiae 균주의 제작 Production of strain

사카로마이세스 세레비지애에는 보조인자로 NADH를 사용하는 6종의 알코올 탈수소효소 (ADH1, ADH2, ADH3, ADH4, ADH5, 및 SFA1) 및 NADPH를 사용하는 알코올 탈수소효소 (ADH6, 및 ADH7)가 존재한다. 또한, NADH를 보조인자로 사용하여 디히드록시아세톤인산 (dihydroxyacetone phosphate, DHAP) 을 글리세롤-3-인산으로 전환시키는 글리세롤-3-인산 탈수소 효소 (GPD1 및 GPD2)가 존재한다. (ADH1, ADH2, ADH3, ADH4, ADH5, and SFA1) using NADH and alcohol dehydrogenase (ADH6 and ADH7) using NADPH are present in the Saccharomyces cerevisiae do. There are also glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) that convert dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate using NADH as a cofactor.

이에, 본 실시예에서는 상기 ADH1 (서열번호 3의 뉴클레오티드 서열, 서열번호 4의 아미노산 서열), ADH2 (서열번호 5의 뉴클레오티드 서열, 서열번호 6의 아미노산 서열), ADH3 (서열번호 7의 뉴클레오티드 서열, 서열번호 8의 아미노산 서열), ADH4 (서열번호 9의 뉴클레오티드 서열, 서열번호 10의 아미노산 서열), 및ADH5 (서열번호 11의 뉴클레오티드 서열, 서열번호 12의 아미노산 서열)를 결손시킨 균주와, ADH1, GPD1 (서열번호 13의 뉴클레오티드 서열, 서열번호 14의 아미노산 서열), 및 GPD2 (서열번호 15의 뉴클레오티드 서열, 서열번호 16의 아미노산 서열)를 결손시킨 균주를 제작하고, 상기 균주에 추가적으로 GSF2를 결손시킨 균주를 제작하였다.ADH2 (the nucleotide sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6), ADH3 (the nucleotide sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: (Amino acid sequence of SEQ ID NO: 8), ADH4 (SEQ ID NO: 9, amino acid sequence of SEQ ID NO: 10), and ADH5 (nucleotide sequence of SEQ ID NO: 11, amino acid sequence of SEQ ID NO: 12) A strain lacking GPD1 (nucleotide sequence of SEQ ID NO: 13, amino acid sequence of SEQ ID NO: 14), and GPD2 (nucleotide sequence of SEQ ID NO: 15, amino acid sequence of SEQ ID NO: 16) was prepared and GSF2 was additionally deleted .

(1.1) 유전자 결손 카세트의 제작(1.1) Production of gene-deficient cassette

ADH1 내지 ADH5, GPD1 및 GPD2, 및/또는 GSF2 유전자가 결손된 돌연변이는 Cre/loxP 재조합 시스템을 이용하였다. 유전자 결손을 위한 카세트는 pUG27(loxP-his5 +-loxP 결손 카세트를 포함하는 플라스미드, Euroscarf, 독일) 또는 pUG72 (loxP - URA3 - loxP 결손 카세트를 포함하는 플라스미드, Euroscarf, 독일) 플라스미드를 주형으로 사용하여 PCR 증폭을 통해 획득하였다. 유전자 결손 카세트 제작을 위한 프라이머 세트로는, 서열번호 17 및 18 (ADH1), 서열번호 19 및 20 (ADH2), 서열번호 21 및 22 (ADH3), 서열번호 23 및 24 (ADH4), 서열번호 25 및 26 (ADH5), 서열번호 27 및 28 (GPD1), 서열번호 29 및 30 (GPD2), 및 서열번호 31 및 32(GSF2)의 조합을 각각 해당 유전자에 대해 사용하였다.Mutants lacking ADH1 to ADH5, GPD1 and GPD2, and / or GSF2 genes used the Cre / loxP recombination system. Cassette for the gene defect is pUG27 using (loxP plasmid containing the deletion cassette, Euroscarf, Germany loxP - - URA3) plasmid as a template (loxP - - his5 + loxP deletion plasmid, Euroscarf containing the cassette, Germany) or pUG72 PCR amplification. SEQ ID NOs: 17 and 18 (ADH1), SEQ ID NOs: 19 and 20 (ADH2), SEQ ID NOs: 21 and 22 (ADH3), SEQ ID NOs: 23 and 24 (ADH4), SEQ ID NO: And 26 (ADH5), SEQ ID NOs: 27 and 28 (GPD1), SEQ ID NOs: 29 and 30 (GPD2), and SEQ ID NOs: 31 and 32 (GSF2), respectively.

(1.2) 균주의 제작(1.2) Production of strain

S. cerevisiae 균주 CEN.PK2-1C(MATa ura3 -52 trp1 -289 leu2 -3,112 his3 △1 MAL2-8C SUC2) (Euroscarf, 독일)에 상기 (1.1)에서 제작한 ADH1 내지 ADH5, GPD1 및 GPD2, 및/또는 GSF2 유전자 결손용 카세트를 리튬 아세테이트를 이용한 화학적 형질전환 방법에 의하여 도입하였다. 상기 형질전환 균주를 SC 배지 (20 g/L 포도당, 6.7 g/L YNB, 적당한 아미노산 첨가물)에서 배양하여, 상기 유전자가 형질전환된 균주를 선별하였다. 상기 유전자의 결손 확인용 프라이머로서, 각각 서열번호 33 및 34 (ADH1), 서열번호 35 및 36 (ADH2), 서열번호 37 및 38 (ADH3), 서열번호 39 및 40 (ADH4), 서열번호 41 및 42 (ADH5), 서열번호 43 및 44 (GPD1), 서열번호 45 및 46 (GPD2), 및 서열번호 47 및 48 (GSF2)의 조합을 사용하여 유전자 결손 여부를 확인하였다. 유전자가 결손된 균주가 지니고 있는 선별마커를 제거하기 위하여 Cre recombinase를 발현시키는 pSH63(TRP1, Cre recombinase under the control of GAL1 promoter, Euroscarf, 독일)를 형질전환 하였고 선별마커 유전자가 제거된 결손 균주를 제작하였다. 최종적으로 수득된 비교예 균주를 S. cerevisiae adh1-5△, 및 S. cerevisiae adh1△gpd1△gpd2△로 명명하고, GSF2 유전자가 추가적으로 결손된 균주를 S. cerevisiae adh1-5△gsf2△ 및 S. cerevisiae adh1△gpd1△gpd2△gsf2△로 명명하였다.
S. cerevisiae strain CEN.PK2-1C (MATa ura3 leu2 -3,112 -289 -52 trp1 his3 △ 1 MAL2-8C SUC2) (Euroscarf, Germany) ADH1 prepared in the above step (1.1) to the ADH5, GPD1 and GPD2, and / / Cassette for GSF2 gene deletion was introduced by a chemical transformation method using lithium acetate. The transformant was cultured in SC medium (20 g / L glucose, 6.7 g / L YNB, a suitable amino acid additive) to select strains transformed with the gene. (ADH1), SEQ ID NOs: 35 and 36 (ADH2), SEQ ID NOs: 37 and 38 (ADH3), SEQ ID NOs: 39 and 40 (ADH4), SEQ ID NO: 41 and SEQ ID NO: A combination of SEQ ID NOs: 42 and 42 (ADH5), SEQ ID NOs: 43 and 44 (GPD1), SEQ ID NOs: 45 and 46 (GPD2), and SEQ ID NOs: 47 and 48 (GSF2) In order to remove the selectable markers of the gene-deficient strain, pSH63 ( TRP1 , Cre recombinase under the control of GAL1 promoter, Euroscarf, Germany) expressing Cre recombinase was transformed and a deletion strain in which a selective marker gene was deleted was prepared Respectively. The finally obtained comparative strains were designated as S. cerevisiae adh1-5Δ and S. cerevisiae adh1Δgpd1Δgpd2Δ, and strains in which the GSF2 gene was additionally deficient were named S. cerevisiae adh1-5Δgsf2Δ and S. cerevisiae adh1 . cerevisiae adh1? gpd1? gpd2? gsf2 ?.

2. 2. ADHADH 1 유전자 결손,  1 gene deficiency, GPDGPD 1 및 2 유전자 결손,  1 and 2 gene deletion, DLD1DLD1 유전자 결손,  Gene deletion, JEN1JEN1 유전자 결손, 및  Gene deletion, and GSF2GSF2 유전자 결손  Gene defect S. S. cerevisiaecerevisiae 균주의 제작 Production of strain

사카로마이세스 세레비지애 균주는 대부분의 피루브산을 에탄올 발효에 사용하기 때문에 본 균주로부터 젖산 생산량을 증대시키기 위해서는 에탄올과 글리세롤 합성 대사경로를 차단하는 것이 필요하다. 따라서, 상기 ADH1, GPD1, GPD2, S. cerevisiae의 D형 젖산 탈수소효소 (D-lactate dehydrogenase, DLD1) 유전자(서열번호 49의 뉴클레오티드 서열, 서열번호 50의 아미노산 서열), 및 모노카복실레이트/수소 동시수송단백질(Monocarboxylate/proton symporter, JEN1) 유전자(서열번호 51의 뉴클레오티드 서열, 서열번호 52의 아미노산 서열)를 결손시킨 균주를 제작하였고, 상기 균주에 추가적으로 GSF2를 결손시킨 균주를 제작하였다. In order to increase the production of lactic acid from this strain, it is necessary to block the ethanol and glycerol synthesis metabolic pathway, since most pyruvic acid is used for ethanol fermentation of Saccharomyces cerevisiae strain. Thus, the D-lactate dehydrogenase (DLD1) gene (the nucleotide sequence of SEQ ID NO: 49, the amino acid sequence of SEQ ID NO: 50) of the ADH1, GPD1, GPD2 and S. cerevisiae , and the monocarboxylate / A strain in which a transporter protein (monocarboxylate / proton symporter, JEN1) gene (nucleotide sequence of SEQ ID NO: 51 and amino acid sequence of SEQ ID NO: 52) was deleted and a strain in which GSF2 was deleted in addition to the above strain was prepared.

(2.1) 유전자 결손 카세트의 제작 (2.1) Production of gene-deficient cassette

유전자 결손 카세트 제작을 위한 프라이머 세트로서, 서열번호 53 및 54 (DLD1), 서열번호 55 및 56 (JEN1)을 사용한 것만을 제외하고는 상기 실시예 1의 (1.1)과 동일한 방법으로 DLD1 및 JEN1 유전자 결손을 위한 카세트를 제작하였다. (1.1) of Example 1, except that SEQ ID NOS: 53 and 54 (DLD1), SEQ ID NOS: 55 and 56 (JEN1) were used as a primer set for producing a gene-deficient cassette, and DLD1 and JEN1 genes A cassette for defects was prepared.

(2.2) 균주의 제작(2.2) Production of strain

유전자의 결손 확인용 프라이머로서, 각각 서열번호 57 및 58 (DLD1), 서열번호 59 및 60 (JEN1)의 조합을 사용하여 유전자 결손 여부를 확인한 것만을 제외하고는 상기 실시예 1의 (1.2)와 동일한 방법으로 균주를 제작하였다. 최종적으로 수득된 비교예 균주를 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△로 명명하고, GSF2 유전자가 추가적으로 결손된 균주를 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△gsf2△로 명명하였다.
(1.2) and (2) of Example 1, except that a gene defect was confirmed using a combination of SEQ ID NOS: 57 and 58 (DLD1), SEQ ID NOS: 59 and 60 The strain was prepared in the same manner. The finally obtained comparative strain was designated as S. cerevisiae dld1 DELTA jen1 DELTA adh1 DELTA gpd1 DELTA gpd2 DELTA and the strains in which the GSF2 gene was additionally deleted were designated as S. cerevisiae dld1 DELTA jen1 DELTA adh1 DELTA gpd1 DELTA gpd2 DELTA gsf2 DELTA Respectively.

3. 3. ldhAldhA 유전자 발현, 및  Gene expression, and ADHADH 1 유전자 결손,  1 gene deficiency, GPDGPD 1 및 2 유전자 결손,  1 and 2 gene deletion, DLD1DLD1 유전자 결손,  Gene deletion, JEN1JEN1 유전자 결손,  Gene deletion, PDC1PDC1 유전자 결손, 및  Gene deletion, and GSF2GSF2 유전자 결손  Gene defect S. cerevisiaeS. cerevisiae 균주의 제작 Production of strain

D형 젖산 생산능을 갖는 유전적으로 조작된 효모 균주에서의 GSF2의 결손 효과를 확인하기 위해, 상기 실시예 1의 (2.2)에서 제작된 균주에서 류코노스톡 메센테로이드(Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293)의 D형 젖산 탈수소효소(D-lactate dehydrogenase, ldhA)(서열번호 61의 뉴클레오티드 서열, 서열번호 62의 아미노산 서열)를 S. cerevisiae의 피루브산 탈탄산효소 1 (pyruvate decarboxylase 1, PDC1)(서열번호 63의 뉴클레오티드 서열, 서열번호 64의 아미노산 서열) 위치에 삽입시킨 균주를 제작하였고, 상기 균주에 추가적으로 GSF2를 결손시킨 균주를 제작하였다. To confirm the deletion effect of GSF2 in a genetically engineered yeast strain having D-lactic acid producing ability, leuconostoc mesenteroide ( Leuconostoc ) in the strain prepared in (2.2) mesenteroides subsp. mesenteroides ATCC 8293) D-type lactate dehydrogenase (D-lactate dehydrogenase, ldhA) (SEQ ID NO: 61 of the nucleotide sequence, the amino acid sequence of SEQ ID NO: 62), pyruvate decarboxylase enzyme of S. cerevisiae 1 (pyruvate decarboxylase 1, PDC1 a) (The nucleotide sequence of SEQ ID NO: 63 and the amino acid sequence of SEQ ID NO: 64), and a strain in which GSF2 was deleted in addition to the above strain was prepared.

(3.1) 유전자 발현 플라스미드의 제작(3.1) Production of gene expression plasmid

D형 젖산 탈수소효소 유전자를 도입하기 위해, Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293의 ldhA를 사용하였다. To introduce the D-lactate dehydrogenase gene, Leuconostoc mesenteroides subsp. ldhA of mesenteroides ATCC 8293 was used.

구체적으로, pETldhD 플라스미드로부터 Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293의 lhdA 유전자를 서열번호 65 및 66의 프라이머 세트를 사용하여 PCR을 통해 확보하였다. PCR로 확보한 ldhA 유전자는 p425TEF 플라스미드 벡터[LEU2, P TEF1 (서열번호 67), T CYC1 (서열번호 68)] (Mumberg et al., 1995)에 BamHI, PstI 제한효소를 사용하여 클로닝하고 p425TEF-Lm. ldhA로 명명하였다. 이후에, 상기 클로닝한 p425TEF-Lm.ldhA를 주형으로 서열번호 69 및 70의 프라이머 세트를 사용하여 TEF1 프로모터로부터 CYC1 터미네이터 절편을 획득하고, ApaI 및 NheI 제한효소를 이용하여 pUG72MCS 벡터에 클로닝하고 pUG72MCS_P TEF1 -Lm. ldhA -T CYC1 로 명명하였다. Specifically, from the pETldhD plasmid, Leuconostoc mesenteroides subsp. The lhdA gene of mesenteroides ATCC 8293 was obtained by PCR using the primer sets of SEQ ID NOS: 65 and 66. The ldhA gene obtained by PCR was cloned into p425TEF plasmid vector [ LEU2 , P TEF1 (SEQ ID NO: 67), T CYC1 (SEQ ID NO: 68)] (Mumberg et al., 1995) using BamHI and PstI restriction enzymes, Lm. It was named ldhA. Thereafter, the cloned p425TEF-Lm. A CYC1 terminator fragment was obtained from the TEF1 promoter using the primer set of SEQ ID NOS: 69 and 70 with ldhA as a template, cloned into pUG72MCS vector using ApaI and NheI restriction enzymes, and ligated into pUG72MCS_P TEF1 - Lm. ldhA - T CYC1 .

(3.2) 유전자 결손/삽입 카세트의 제작(3.2) Production of gene defect / insert cassette

PDC1 유전자의 위치에 상기 lhdA 유전자를 삽입하기 위해, pUG72MCS_P TEF1 -Lm.ldhA-T CYC1 를 사용하고, 서열번호 71 및 72의 프라이머 세트를 사용한 것만을 제외하고는 상기 실시예 1의 (1.1)과 동일한 방법으로 PDC1 결손/ldhA 삽입을 위한 카세트를 제작하였다. (1.1) and (2) of Example 1, except that pUG72MCS_P TEF1 - Lm.ldhA- T CYC1 was used to insert the lhdA gene at the position of the PDC1 gene and the primer set of SEQ ID NOs: 71 and 72 was used. A cassette for PDC1 deletion / ldhA insertion was constructed in the same manner.

(3.3) 균주의 제작(3.3) Production of strain

유전자의 결손/삽입 확인용 프라이머로서, 각각 서열번호 73 및 74의 조합을 사용하여 유전자 결손/삽입 여부를 확인한 것만을 제외하고는 상기 실시예 1의 (1.2)와 동일한 방법으로 균주를 제작하였다. 최종적으로 수득된 비교예 균주를 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△pdc1△::Lm. ldhA로 명명하고, GSF2 유전자가 추가적으로 결손된 균주를 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△pdc1△::Lm. ldhAgsf2△로 명명하였다.
A strain was prepared in the same manner as in (1.2) of Example 1, except that the gene deletion / insertion was confirmed using the combination of SEQ ID NOs: 73 and 74 as primers for gene deletion / insertion confirmation. The finally obtained comparative strain was designated as S. cerevisiae dld1? Jen1? Adh1? Gpd1? Gpd2? Pdc1? :: Lm. ldhA and the strain in which the GSF2 gene is additionally deleted is referred to as S. cerevisiae dld1? jen1? adh1? gpd1? gpd2? pdc1? :: Lm. ldhA gsf2 ?.

실시예Example 2. 제작된  2. Manufactured S. S. cerevisiaecerevisiae 균주의 생장 속도 및 포도당 소비량 증가, 및 그에 따른 목적 물질의 생산성 증대 확인 Increase in the growth rate and glucose consumption of the strain and increase in the productivity of the target substance

1. 생장 속도 증대 확인1. Growth rate increase confirmation

GSF2 유전자 결손 S. cerevisiae 균주의 생장 속도 증대를 확인하기 위해, 상기 실시예 1의 (1.2), 및 (2.2)에서 제작한 균주를 배지에서 배양하였다. GSF2 gene deletion To confirm the increase in the growth rate of S. cerevisiae strain, the strains prepared in (1.2) and (2.2) of Example 1 were cultured in a medium.

구체적으로, 생장 속도 증대를 확인하기 위해 상기 균주를 YPD 배지(20 g/l 포도당, 10 g/l 효모 추출물, 20 g/l 박토-펩톤)에서 배양하였다. 세포 배양은 진탕배양기를 이용하여 30 ℃에서 170 rpm으로 진행하였다. 생장속도를 비교하기 위한 배양조건은 초기접종 세포농도는 OD600=0.05로 고정하였고, 50 ml 코니컬 튜브에서 5 ml 배지로 진행하였다. 균주별 생장 속도는 Varian Cary50 UV/Vis spectrophotometer(Agilent)를 사용하여 600 nm의 파장에서 세포농도를 측정하는 방식으로 결정하였다. S. cerevisiae adh1-5△gsf2△ 및 S. cerevisiae adh1△gpd1△gpd2△gsf2△의 생장 속도를 그의 비교예 균주 S. cerevisiae adh1-5△, 및 S. cerevisiae adh1△gpd1△gpd2△와 비교한 결과를 각각 도 1 및 도 2에 나타내었고, S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△gsf2△의 생장 속도를 그의 비교예 균주 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△와 비교한 결과를 도 3에 나타내었다. Specifically, the strain was cultured in YPD medium (20 g / l glucose, 10 g / l yeast extract, 20 g / l bacto-peptone) to confirm growth rate increase. Cell culture was carried out at 30 rpm at 170 rpm using a shaking incubator. In order to compare the growth rate, the initial inoculated cell concentration was fixed to OD 600 = 0.05, and the culture was carried out in a 5 ml culture medium in a 50 ml conical tube. The growth rate of each strain was determined by measuring the cell concentration at a wavelength of 600 nm using a Varian Cary 50 UV / Vis spectrophotometer (Agilent). S. cerevisiae and S. cerevisiae adh1-5 △ △ gsf2 adh1 gpd1 △ △ △ gpd2 the growth rate of its gsf2 △ Comparative Example strain S. cerevisiae adh1-5 △, and S. cerevisiae adh1 gpd1 △ △ gpd2 compared to △ The results are shown in Figs. 1 and 2, respectively. The growth rate of S. cerevisiae dld1 DELTA jen1 DELTA adh1 DELTA gpd1 DELTA gpd2 DELTA gsf2 DELTA is compared with that of its comparative strain S. cerevisiae dld1 DELTA jen1 DELTA adh1 DELTA gpd1 DELTA gpd2 DELTA The results are shown in Fig.

도 1은 일 구체예에 따른 생장 속도가 증대된 S. cerevisiae 균주의 생장 속도를 나타낸 그래프이다. FIG. 1 is a graph showing the growth rate of S. cerevisiae strain with increased growth rate according to one embodiment.

도 2는 일 구체예에 따른 생장 속도가 증대된 S. cerevisiae 균주의 생장 속도를 나타낸 그래프이다. FIG. 2 is a graph showing the growth rate of S. cerevisiae strain with increased growth rate according to one embodiment.

도 3은 일 구체예에 따른 생장 속도가 증대된 S. cerevisiae 균주의 생장 속도를 나타낸 그래프이다. FIG. 3 is a graph showing the growth rate of S. cerevisiae strain with an increased growth rate according to one embodiment.

도 1 내지 3에 나타낸 바와 같이, GSF2 유전자의 결손을 통해 균주의 생장 속도를 현저하게 향상시킬 수 있음을 확인할 수 있다. 상기의 결과로, 특정 목적 물질의 생산을 위해 유전적으로 조작됨으로 인해 생장 속도가 감소된 세포에 상기 글루코오스 신호 인자를 결손시킴으로써 균주의 생장 속도를 향상시킬 수 있음을 알 수 있다.
As shown in Figs. 1 to 3, it can be confirmed that the growth rate of the strain can be remarkably improved through the deletion of the GSF2 gene. As a result, it can be seen that the growth rate of the strain can be improved by defeating the glucose signaling factor in a cell whose growth rate is decreased by genetically manipulating for the production of a specific target substance.

2. 포도당 소비량 증가, 및 그에 따른 목적 물질의 생산성 증대 확인2. Increased consumption of glucose and thus increased productivity of target substances

GSF2 유전자 결손 S. cerevisiae 균주의 포도당 소비량 증가, 및 그에 따른 목적 물질의 생산성 증대를 확인하기 위해, 상기 실시예 1의 (3.3)에서 제작한 젖산 생성 균주를 배지에서 배양하였다. GSF2 gene deletion The lactic acid-producing strain prepared in Example 1 (3.3) was cultivated in a medium in order to increase the glucose consumption of the S. cerevisiae strain and the resulting productivity of the target substance.

구체적으로, 젖산 생산 배지로는 YPD50(50 g/l 포도당, 10 g/l 효모 추출물, 20 g/l 박토-펩톤)를 사용하였다. 세포 배양은 진탕배양기를 이용하여 30 ℃에서 170 rpm으로 진행하였다. 초기접종 세포농도는 OD600=1로 고정하였고, 50 ml 코니컬 튜브에서 5 ml 배지로 진행하였다. 대사산물을 분석하기 위하여 배양액 800μl를 원심분리하여 상등액을 얻고, 이를 0.22 μm 필터로 여과하여 HPLC 분석을 진행하였다. UltiMate 3000 HPLC system(Thermo fishers scientific)을 이용하였고 BioRad Aminex HPX-87H 컬럼과 굴절률검출기(RI detector)를 사용하였다. 이동상은 5 mM 황산을 사용하였고 유속은 0.6 ml/분, 온도는 60 ℃로 설정하여 대사산물의 생산량을 확인하였고, 그 결과를 도 4 및 도 5에 나타내었다. Specifically, YPD50 (50 g / l glucose, 10 g / l yeast extract, 20 g / l bacto-peptone) was used as a lactic acid production medium. Cell culture was carried out at 30 rpm at 170 rpm using a shaking incubator. The initial inoculated cell concentration was fixed at OD 600 = 1 and proceeded to 5 ml medium in a 50 ml conical tube. To analyze the metabolites, 800 μl of the culture was centrifuged to obtain supernatant, which was then filtered through a 0.22 μm filter for HPLC analysis. UltiMate 3000 HPLC system (Thermo fishers scientific) was used and a BioRad Aminex HPX-87H column and a refractive index detector (RI detector) were used. The mobile phase used was 5 mM sulfuric acid, the flow rate was 0.6 ml / min, and the temperature was set at 60 ° C to produce the metabolite. The results are shown in FIGS. 4 and 5.

도 4는 비교예에 따른 젖산 생산능이 증가된 S. cerevisiae 균주의 대사 산물의 생산량을 나타낸 그래프이다.FIG. 4 is a graph showing the production yield of metabolites of S. cerevisiae strain with increased lactic acid production capacity according to a comparative example.

도 5는 일 구체예에 따른 젖산 생산능이 증가된 S. cerevisiae 균주의 대사 산물의 생산량을 나타낸 그래프이다. FIG. 5 is a graph showing production yields of metabolites of S. cerevisiae strains with increased lactic acid production capacity according to one embodiment.

도 4에 나타낸 바와 같이, 비교예 균주인 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△pdc1△::Lm. ldhA는 97시간 동안 33.5 g/l의 포도당을 소비하여 19.3 g/l의 D형 젖산을 생산하였고, 에탄올, 아세트산, 및 글리세롤을 각각 2.2 g/l, 1.3 g/l, 및 0.03 g/l를 생산하였음을 확인할 수 있다. As shown in Fig. 4, S. cerevisiae dld1? Jen1? Adh1? Gpd1? Gpd2? Pdc1? :: Lm. ldhA consumed 33.5 g / l of glucose for 97 hours to produce 19.3 g / l of D-lactic acid, while ethanol, acetic acid and glycerol were added at 2.2 g / l, 1.3 g / l and 0.03 g / l, respectively It can be confirmed that it produced.

이에 반해, 도 5에 나타낸 바와 같이, 일 구체예에 따른 젖산 생산능이 증가된 균주인 S. cerevisiae dld1△jen1△adh1△gpd1△gpd2△pdc1△::Lm. ldhA gsf2△는 포도당 소비능이 비교예 균주 대비 50% 증가된 50.3 g/l의 포도당을 소비하여 비교예 균주 대비 84% 증가된 35.6 g/l의 D형 젖산을 생산하였으며 에탄올, 아세트산, 및 글리세롤을 각각 2.5 g/l, 2.0 g/l, 및 0.02 g/l를 생산하였음을 확인할 수 있다. On the other hand, as shown in FIG. 5, the S. cerevisiae dld1? Jen1? Adh1? Gpd1? Gpd2? Pdc1? :: Lm. ldhA gsf2 △ consumed 50.3 g / l glucose, which was 50% higher than that of the comparative strain, and produced 35.6 g / l D-lactate, which was 84% higher than the strain of the comparative strain. The ethanol, acetic acid and glycerol 2.5 g / l, 2.0 g / l, and 0.02 g / l, respectively.

상기의 결과로, 글루코오스 신호 인자를 결손시킴으로써 균주의 생장 속도를 향상시킬 수 있고, 포도당 소비능을 증대시킴으로써 특정 목적 물질, 예를 들면, D 형 젖산의 생산능을 현저하게 증가시킬 수 있음을 알 수 있다. As a result, it has been found that the growth rate of the strain can be improved by eliminating the glucose signaling factor, and the ability to produce a specific target substance, for example, D-lactic acid can be remarkably increased by increasing the glucose consumption ability .

<110> SNU R&DB Foundation <120> Genetically engineered yeast cell having enhanced growth rate and method of producing target materials using the same <130> PN112274 <160> 80 <170> KopatentIn 2.0 <210> 1 <211> 1212 <212> DNA <213> Saccharomyces cerevisiae GSF2 <400> 1 atggagattt acattagact taacgcagat gtcgagcacg actatgcgtt tcaggtgtca 60 aatgaagaca ccataaataa caaaattaag aagattttcc cttccaagac gggtttggcg 120 gacttaatgg tgctgagacc atcaattttc catgagaagg agcccgtaaa gttctataag 180 tctatccatc cagggtacct gtccgaaggt ggttgtttga tgttccatta tgaagctgat 240 aacgaagaaa atctcgagga gttgaatgac tccaagccgc ttattgacca gttgtggcct 300 ggtcagctag ttgttccgga gtggaagcta tccaagaaaa acatatgggt atacaccatt 360 attatgttgg cctggctata taccgatttg cccgacgcca tttctccaac cccaggtatt 420 tgtttgacta atcaattgtc tagactattg attcctgtag ccaagcacat ggatttacct 480 gaaattgccg ctaaactcga acaagaagtt caggcaaact attctagtct cgtagctcaa 540 tggcttttct ttgtcatgca cattttcaaa gtgggcataa ttactttatt cctcaaatta 600 ggcattgcca accccatcag cttcaatcct tacaagttat ggagtttgag agatttaact 660 tctccttctg ctaacggcgc taagaattct ggcggcaata acaacactac tgacttaaag 720 acacgtttgc gctctctggg ctggattggt gctaagagag ctacttacga cgattaccag 780 actaactatt acaactatgt catagataag atgggtggtg cagttgctgc ttatagagcg 840 ggcgcgatta ggaaagctgc agcaccaggt atccaacttg tggctggaga gggtttccaa 900 agtcctctag aagataggtt cactgcttcc actttcacgg ctattaaaac cgaacggaaa 960 ttcattttaa gtgaagaata ctttgtggag ctggaaaaca atctaaagaa gatcttggaa 1020 gagtatgacg gggatattgg taagatgaat gctgaaatta ggaggtttag aaggtttggt 1080 atttatgaac cagatgaaaa actggcctcg ctagttaagc tcagaagaga aattgctgat 1140 gagaaagaga aagcatcaaa caatgatgct acttttggta taaaaaagaa tgatttgaaa 1200 aaatctaatt aa 1212 <210> 2 <211> 403 <212> PRT <213> Saccharomyces cerevisiae GSF2 <400> 2 Met Glu Ile Tyr Ile Arg Leu Asn Ala Asp Val Glu His Asp Tyr Ala 1 5 10 15 Phe Gln Val Ser Asn Glu Asp Thr Ile Asn Asn Lys Ile Lys Lys Ile 20 25 30 Phe Pro Ser Lys Thr Gly Leu Ala Asp Leu Met Val Leu Arg Pro Ser 35 40 45 Ile Phe His Glu Lys Glu Pro Val Lys Phe Tyr Lys Ser Ile His Pro 50 55 60 Gly Tyr Leu Ser Glu Gly Gly Cys Leu Met Phe His Tyr Glu Ala Asp 65 70 75 80 Asn Glu Glu Asn Leu Glu Glu Leu Asn Asp Ser Lys Pro Leu Ile Asp 85 90 95 Gln Leu Trp Pro Gly Gln Leu Val Val Pro Glu Trp Lys Leu Ser Lys 100 105 110 Lys Asn Ile Trp Val Tyr Thr Ile Ile Met Leu Ala Trp Leu Tyr Thr 115 120 125 Asp Leu Pro Asp Ala Ile Ser Pro Thr Pro Gly Ile Cys Leu Thr Asn 130 135 140 Gln Leu Ser Arg Leu Leu Ile Pro Val Ala Lys His Met Asp Leu Pro 145 150 155 160 Glu Ile Ala Ala Lys Leu Glu Gln Glu Val Gln Ala Asn Tyr Ser Ser 165 170 175 Leu Val Ala Gln Trp Leu Phe Phe Val Met His Ile Phe Lys Val Gly 180 185 190 Ile Ile Thr Leu Phe Leu Lys Leu Gly Ile Ala Asn Pro Ile Ser Phe 195 200 205 Asn Pro Tyr Lys Leu Trp Ser Leu Arg Asp Leu Thr Ser Pro Ser Ala 210 215 220 Asn Gly Ala Lys Asn Ser Gly Gly Asn Asn Asn Thr Thr Asp Leu Lys 225 230 235 240 Thr Arg Leu Arg Ser Leu Gly Trp Ile Gly Ala Lys Arg Ala Thr Tyr 245 250 255 Asp Asp Tyr Gln Thr Asn Tyr Tyr Asn Tyr Val Ile Asp Lys Met Gly 260 265 270 Gly Ala Val Ala Ala Tyr Arg Ala Gly Ala Ile Arg Lys Ala Ala Ala 275 280 285 Pro Gly Ile Gln Leu Val Ala Gly Glu Gly Phe Gln Ser Pro Leu Glu 290 295 300 Asp Arg Phe Thr Ala Ser Thr Phe Thr Ala Ile Lys Thr Glu Arg Lys 305 310 315 320 Phe Ile Leu Ser Glu Glu Tyr Phe Val Glu Leu Glu Asn Asn Leu Lys 325 330 335 Lys Ile Leu Glu Glu Tyr Asp Gly Asp Ile Gly Lys Met Asn Ala Glu 340 345 350 Ile Arg Arg Phe Arg Arg Phe Gly Ile Tyr Glu Pro Asp Glu Lys Leu 355 360 365 Ala Ser Leu Val Lys Leu Arg Arg Glu Ile Ala Asp Glu Lys Glu Lys 370 375 380 Ala Ser Asn Asn Asp Ala Thr Phe Gly Ile Lys Lys Asn Asp Leu Lys 385 390 395 400 Lys Ser Asn <210> 3 <211> 1047 <212> DNA <213> Saccharomyces cerevisiae ADH1 <400> 3 atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 4 <211> 348 <212> PRT <213> Saccharomyces cerevisiae ADH1 <400> 4 Met Ser Ile Pro Glu Thr Gln Lys Gly Val Ile Phe Tyr Glu Ser His 1 5 10 15 Gly Lys Leu Glu Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn 20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val 50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val 65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly 85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys 100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln 115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140 Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile 165 170 175 Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu 195 200 205 Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys 210 215 220 Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala Thr Asp Gly Gly Ala 225 230 235 240 His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255 Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro 260 265 270 Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser 275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly 325 330 335 Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys 340 345 <210> 5 <211> 1047 <212> DNA <213> Saccharomyces cerevisiae ADH2 <400> 5 atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60 cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120 tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180 ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480 aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540 ggtggtctag gttctttggc tgttcaatat gctaaggcga tgggttacag agtcttaggt 600 attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660 gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720 cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780 agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840 gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900 gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960 gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 6 <211> 348 <212> PRT <213> Saccharomyces cerevisiae ADH2 <400> 6 Met Ser Ile Pro Glu Thr Gln Lys Ala Ile Ile Phe Tyr Glu Ser Asn 1 5 10 15 Gly Lys Leu Glu His Lys Asp Ile Pro Val Pro Lys Pro Lys Pro Asn 20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Thr Lys Leu Pro Leu Val 50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val 65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly 85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys 100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Glu 115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140 Asp Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Arg Ala Gly His Trp Ala Ala Ile 165 170 175 Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Pro Gly Lys Glu 195 200 205 Glu Leu Phe Thr Ser Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys 210 215 220 Glu Lys Asp Ile Val Ser Ala Val Val Lys Ala Thr Asn Gly Gly Ala 225 230 235 240 His Gly Ile Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255 Thr Arg Tyr Cys Arg Ala Asn Gly Thr Val Val Leu Val Gly Leu Pro 260 265 270 Ala Gly Ala Lys Cys Ser Ser Asp Val Phe Asn His Val Val Lys Ser 275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Ser Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly 325 330 335 Gln Ile Ala Gly Arg Tyr Val Val Asp Thr Ser Lys 340 345 <210> 7 <211> 1128 <212> DNA <213> Saccharomyces cerevisiae ADH3 <400> 7 atgttgagaa cgtcaacatt gttcaccagg cgtgtccaac caagcctatt ttctagaaac 60 attcttagat tgcaatccac agctgcaatc cctaagactc aaaaaggtgt catcttttat 120 gagaataagg ggaagctgca ttacaaagat atccctgtcc ccgagcctaa gccaaatgaa 180 attttaatca acgttaaata ttctggtgta tgtcacaccg atttacatgc ttggcacggc 240 gattggccat tacctgttaa actaccatta gtaggtggtc atgaaggtgc tggtgtagtt 300 gtcaaactag gttccaatgt caagggctgg aaagtcggtg atttagcagg tatcaaatgg 360 ctgaacggtt cttgtatgac atgcgaattc tgtgaatcag gtcatgaatc aaattgtcca 420 gatgctgatt tatctggtta cactcatgat ggttctttcc aacaatttgc gaccgctgat 480 gctattcaag ccgccaaaat tcaacagggt accgacttgg ccgaagtagc cccaatatta 540 tgtgctggtg ttactgtata taaagcacta aaagaggcag acttgaaagc tggtgactgg 600 gttgccatct ctggtgctgc aggtggcttg ggttccttgg ccgttcaata tgcaactgcg 660 atgggttaca gagttctagg tattgatgca ggtgaggaaa aggaaaaact tttcaagaaa 720 ttggggggtg aagtattcat cgactttact aaaacaaaga atatggtttc tgacattcaa 780 gaagctacca aaggtggccc tcatggtgtc attaacgttt ccgtttctga agccgctatt 840 tctctatcta cggaatatgt tagaccatgt ggtaccgtcg ttttggttgg tttgcccgct 900 aacgcctacg ttaaatcaga ggtattctct catgtggtga agtccatcaa tatcaagggt 960 tcttatgttg gtaacagagc tgatacgaga gaagccttag acttctttag cagaggtttg 1020 atcaaatcac caatcaaaat tgttggatta tctgaattac caaaggttta tgacttgatg 1080 gaaaagggca agattttggg tagatacgtc gtcgatacta gtaaataa 1128 <210> 8 <211> 375 <212> PRT <213> Saccharomyces cerevisiae ADH3 <400> 8 Met Leu Arg Thr Ser Thr Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Thr Ala Ala Ile Pro Lys 20 25 30 Thr Gln Lys Gly Val Ile Phe Tyr Glu Asn Lys Gly Lys Leu His Tyr 35 40 45 Lys Asp Ile Pro Val Pro Glu Pro Lys Pro Asn Glu Ile Leu Ile Asn 50 55 60 Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu His Ala Trp His Gly 65 70 75 80 Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val Gly Gly His Glu Gly 85 90 95 Ala Gly Val Val Val Lys Leu Gly Ser Asn Val Lys Gly Trp Lys Val 100 105 110 Gly Asp Leu Ala Gly Ile Lys Trp Leu Asn Gly Ser Cys Met Thr Cys 115 120 125 Glu Phe Cys Glu Ser Gly His Glu Ser Asn Cys Pro Asp Ala Asp Leu 130 135 140 Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln Phe Ala Thr Ala Asp 145 150 155 160 Ala Ile Gln Ala Ala Lys Ile Gln Gln Gly Thr Asp Leu Ala Glu Val 165 170 175 Ala Pro Ile Leu Cys Ala Gly Val Thr Val Tyr Lys Ala Leu Lys Glu 180 185 190 Ala Asp Leu Lys Ala Gly Asp Trp Val Ala Ile Ser Gly Ala Ala Gly 195 200 205 Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Thr Ala Met Gly Tyr Arg 210 215 220 Val Leu Gly Ile Asp Ala Gly Glu Glu Lys Glu Lys Leu Phe Lys Lys 225 230 235 240 Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys Thr Lys Asn Met Val 245 250 255 Ser Asp Ile Gln Glu Ala Thr Lys Gly Gly Pro His Gly Val Ile Asn 260 265 270 Val Ser Val Ser Glu Ala Ala Ile Ser Leu Ser Thr Glu Tyr Val Arg 275 280 285 Pro Cys Gly Thr Val Val Leu Val Gly Leu Pro Ala Asn Ala Tyr Val 290 295 300 Lys Ser Glu Val Phe Ser His Val Val Lys Ser Ile Asn Ile Lys Gly 305 310 315 320 Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu Ala Leu Asp Phe Phe 325 330 335 Ser Arg Gly Leu Ile Lys Ser Pro Ile Lys Ile Val Gly Leu Ser Glu 340 345 350 Leu Pro Lys Val Tyr Asp Leu Met Glu Lys Gly Lys Ile Leu Gly Arg 355 360 365 Tyr Val Val Asp Thr Ser Lys 370 375 <210> 9 <211> 1149 <212> DNA <213> Saccharomyces cerevisiae ADH4 <400> 9 atgtcttccg ttactgggtt ttacattcca ccaatctctt tctttggtga aggtgcttta 60 gaagaaaccg ctgattacat caaaaacaag gattacaaaa aggctttgat cgttactgat 120 cctggtattg cagctattgg tctctccggt agagtccaaa agatgttgga agaacgtggc 180 ttaaacgttg ctatctatga caaaactcaa ccaaacccaa atattgccaa tgtcacagct 240 ggtttgaagg ttttgaagga agaaaactct gaaattgtcg tttccattgg tggtggttct 300 gctcacgaca atgctaaggc cattgcttta ttggctacta acggtgggga aattggagat 360 tatgaaggtg tcaaccaatc taagaaggct gctttaccgc tatttgccat caacactact 420 gctggtactg cttccgagat gaccagattc actattatct ctaatgaaga aaagaaaatc 480 aagatggcca tcattgacaa caacgtcact ccagctgttg ctgtcaacga cccatctacc 540 atgtttggtt tgccacctgc tttgactgct gctactggtc tagatgcttt gactcactgt 600 atcgaagctt acgtttccac cgcctctaac ccaatcaccg atgcttgtgc tttgaagggt 660 attgatttga tcaatgaaag cttggtcgcc gcatacaaag acggtaaaga caagaaggcc 720 agaactgata tgtgttacgc agaatacttg gcaggtatgg ctttcaacaa tgcttctcta 780 ggttatgttc atgcccttgc tcatcaactt ggtggtttct accacttgcc tcatggtgtt 840 tgtaacgctg tcttgttgcc tcatgttcaa gaggccaaca tgcaatgtcc aaaggccaag 900 aagagattag gtgaaattgc cttgcattgc ggtgcttctc aagaagatcc agaagaaacc 960 atcaaggctt tgcacgtttt aaacagaacc atgaacattc caagaaactt gaaagactta 1020 ggtgttaaaa ccgaagattt tgacattttg gctgaacacg ccatgcatga tgcctgccat 1080 ttgactaacc cagttcaatt caccaaagaa caagtggttg ccattatcaa gaaagcctat 1140 gaatattaa 1149 <210> 10 <211> 382 <212> PRT <213> Saccharomyces cerevisiae ADH4 <400> 10 Met Ser Ser Val Thr Gly Phe Tyr Ile Pro Pro Ile Ser Phe Phe Gly 1 5 10 15 Glu Gly Ala Leu Glu Glu Thr Ala Asp Tyr Ile Lys Asn Lys Asp Tyr 20 25 30 Lys Lys Ala Leu Ile Val Thr Asp Pro Gly Ile Ala Ala Ile Gly Leu 35 40 45 Ser Gly Arg Val Gln Lys Met Leu Glu Glu Arg Gly Leu Asn Val Ala 50 55 60 Ile Tyr Asp Lys Thr Gln Pro Asn Pro Asn Ile Ala Asn Val Thr Ala 65 70 75 80 Gly Leu Lys Val Leu Lys Glu Glu Asn Ser Glu Ile Val Val Ser Ile 85 90 95 Gly Gly Gly Ser Ala His Asp Asn Ala Lys Ala Ile Ala Leu Leu Ala 100 105 110 Thr Asn Gly Gly Glu Ile Gly Asp Tyr Glu Gly Val Asn Gln Ser Lys 115 120 125 Lys Ala Ala Leu Pro Leu Phe Ala Ile Asn Thr Thr Ala Gly Thr Ala 130 135 140 Ser Glu Met Thr Arg Phe Thr Ile Ile Ser Asn Glu Glu Lys Lys Ile 145 150 155 160 Lys Met Ala Ile Ile Asp Asn Asn Val Thr Pro Ala Val Ala Val Asn 165 170 175 Asp Pro Ser Thr Met Phe Gly Leu Pro Pro Ala Leu Thr Ala Ala Thr 180 185 190 Gly Leu Asp Ala Leu Thr His Cys Ile Glu Ala Tyr Val Ser Thr Ala 195 200 205 Ser Asn Pro Ile Thr Asp Ala Cys Ala Leu Lys Gly Ile Asp Leu Ile 210 215 220 Asn Glu Ser Leu Val Ala Ala Tyr Lys Asp Gly Lys Asp Lys Lys Ala 225 230 235 240 Arg Thr Asp Met Cys Tyr Ala Glu Tyr Leu Ala Gly Met Ala Phe Asn 245 250 255 Asn Ala Ser Leu Gly Tyr Val His Ala Leu Ala His Gln Leu Gly Gly 260 265 270 Phe Tyr His Leu Pro His Gly Val Cys Asn Ala Val Leu Leu Pro His 275 280 285 Val Gln Glu Ala Asn Met Gln Cys Pro Lys Ala Lys Lys Arg Leu Gly 290 295 300 Glu Ile Ala Leu His Cys Gly Ala Ser Gln Glu Asp Pro Glu Glu Thr 305 310 315 320 Ile Lys Ala Leu His Val Leu Asn Arg Thr Met Asn Ile Pro Arg Asn 325 330 335 Leu Lys Asp Leu Gly Val Lys Thr Glu Asp Phe Asp Ile Leu Ala Glu 340 345 350 His Ala Met His Asp Ala Cys His Leu Thr Asn Pro Val Gln Phe Thr 355 360 365 Lys Glu Gln Val Val Ala Ile Ile Lys Lys Ala Tyr Glu Tyr 370 375 380 <210> 11 <211> 1056 <212> DNA <213> Saccharomyces cerevisiae ADH5 <400> 11 atgccttcgc aagtcattcc tgaaaaacaa aaggctattg tcttttatga gacagatgga 60 aaattggaat ataaagacgt cacagttccg gaacctaagc ctaacgaaat tttagtccac 120 gttaaatatt ctggtgtttg tcatagtgac ttgcacgcgt ggcacggtga ttggccattt 180 caattgaaat ttccattaat cggtggtcac gaaggtgctg gtgttgttgt taagttggga 240 tctaacgtta agggctggaa agtcggtgat tttgcaggta taaaatggtt gaatgggact 300 tgcatgtcct gtgaatattg tgaagtaggt aatgaatctc aatgtcctta tttggatggt 360 actggcttca cacatgatgg tacttttcaa gaatacgcaa ctgccgatgc cgttcaagct 420 gcccatattc caccaaacgt caatcttgct gaagttgccc caatcttgtg tgcaggtatc 480 actgtttata aggcgttgaa aagagccaat gtgataccag gccaatgggt cactatatcc 540 ggtgcatgcg gtggcttggg ttctctggca atccaatacg cccttgctat gggttacagg 600 gtcattggta tcgatggtgg taatgccaag cgaaagttat ttgaacaatt aggcggagaa 660 atattcatcg atttcacgga agaaaaagac attgttggtg ctataataaa ggccactaat 720 ggcggttctc atggagttat taatgtgtct gtttctgaag cagctatcga ggcttctacg 780 aggtattgta ggcccaatgg tactgtcgtc ctggttggta tgccagctca tgcttactgc 840 aattccgatg ttttcaatca agttgtaaaa tcaatctcca tcgttggatc ttgtgttgga 900 aatagagctg atacaaggga ggctttagat ttcttcgcca gaggtttgat caaatctccg 960 atccacttag ctggcctatc ggatgttcct gaaatttttg caaagatgga gaagggtgaa 1020 attgttggta gatatgttgt tgagacttct aaatga 1056 <210> 12 <211> 351 <212> PRT <213> Saccharomyces cerevisiae ADH5 <400> 12 Met Pro Ser Gln Val Ile Pro Glu Lys Gln Lys Ala Ile Val Phe Tyr 1 5 10 15 Glu Thr Asp Gly Lys Leu Glu Tyr Lys Asp Val Thr Val Pro Glu Pro 20 25 30 Lys Pro Asn Glu Ile Leu Val His Val Lys Tyr Ser Gly Val Cys His 35 40 45 Ser Asp Leu His Ala Trp His Gly Asp Trp Pro Phe Gln Leu Lys Phe 50 55 60 Pro Leu Ile Gly Gly His Glu Gly Ala Gly Val Val Val Lys Leu Gly 65 70 75 80 Ser Asn Val Lys Gly Trp Lys Val Gly Asp Phe Ala Gly Ile Lys Trp 85 90 95 Leu Asn Gly Thr Cys Met Ser Cys Glu Tyr Cys Glu Val Gly Asn Glu 100 105 110 Ser Gln Cys Pro Tyr Leu Asp Gly Thr Gly Phe Thr His Asp Gly Thr 115 120 125 Phe Gln Glu Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro 130 135 140 Pro Asn Val Asn Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile 145 150 155 160 Thr Val Tyr Lys Ala Leu Lys Arg Ala Asn Val Ile Pro Gly Gln Trp 165 170 175 Val Thr Ile Ser Gly Ala Cys Gly Gly Leu Gly Ser Leu Ala Ile Gln 180 185 190 Tyr Ala Leu Ala Met Gly Tyr Arg Val Ile Gly Ile Asp Gly Gly Asn 195 200 205 Ala Lys Arg Lys Leu Phe Glu Gln Leu Gly Gly Glu Ile Phe Ile Asp 210 215 220 Phe Thr Glu Glu Lys Asp Ile Val Gly Ala Ile Ile Lys Ala Thr Asn 225 230 235 240 Gly Gly Ser His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile 245 250 255 Glu Ala Ser Thr Arg Tyr Cys Arg Pro Asn Gly Thr Val Val Leu Val 260 265 270 Gly Met Pro Ala His Ala Tyr Cys Asn Ser Asp Val Phe Asn Gln Val 275 280 285 Val Lys Ser Ile Ser Ile Val Gly Ser Cys Val Gly Asn Arg Ala Asp 290 295 300 Thr Arg Glu Ala Leu Asp Phe Phe Ala Arg Gly Leu Ile Lys Ser Pro 305 310 315 320 Ile His Leu Ala Gly Leu Ser Asp Val Pro Glu Ile Phe Ala Lys Met 325 330 335 Glu Lys Gly Glu Ile Val Gly Arg Tyr Val Val Glu Thr Ser Lys 340 345 350 <210> 13 <211> 1176 <212> DNA <213> Saccharomyces cerevisiae GPD1 <400> 13 atgtctgctg ctgctgatag attaaactta acttccggcc acttgaatgc tggtagaaag 60 agaagttcct cttctgtttc tttgaaggct gccgaaaagc ctttcaaggt tactgtgatt 120 ggatctggta actggggtac tactattgcc aaggtggttg ccgaaaattg taagggatac 180 ccagaagttt tcgctccaat agtacaaatg tgggtgttcg aagaagagat caatggtgaa 240 aaattgactg aaatcataaa tactagacat caaaacgtga aatacttgcc tggcatcact 300 ctacccgaca atttggttgc taatccagac ttgattgatt cagtcaagga tgtcgacatc 360 atcgttttca acattccaca tcaatttttg ccccgtatct gtagccaatt gaaaggtcat 420 gttgattcac acgtcagagc tatctcctgt ctaaagggtt ttgaagttgg tgctaaaggt 480 gtccaattgc tatcctctta catcactgag gaactaggta ttcaatgtgg tgctctatct 540 ggtgctaaca ttgccaccga agtcgctcaa gaacactggt ctgaaacaac agttgcttac 600 cacattccaa aggatttcag aggcgagggc aaggacgtcg accataaggt tctaaaggcc 660 ttgttccaca gaccttactt ccacgttagt gtcatcgaag atgttgctgg tatctccatc 720 tgtggtgctt tgaagaacgt tgttgcctta ggttgtggtt tcgtcgaagg tctaggctgg 780 ggtaacaacg cttctgctgc catccaaaga gtcggtttgg gtgagatcat cagattcggt 840 caaatgtttt tcccagaatc tagagaagaa acatactacc aagagtctgc tggtgttgct 900 gatttgatca ccacctgcgc tggtggtaga aacgtcaagg ttgctaggct aatggctact 960 tctggtaagg acgcctggga atgtgaaaag gagttgttga atggccaatc cgctcaaggt 1020 ttaattacct gcaaagaagt tcacgaatgg ttggaaacat gtggctctgt cgaagacttc 1080 ccattatttg aagccgtata ccaaatcgtt tacaacaact acccaatgaa gaacctgccg 1140 gacatgattg aagaattaga tctacatgaa gattag 1176 <210> 14 <211> 391 <212> PRT <213> Saccharomyces cerevisiae GPD1 <400> 14 Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn 1 5 10 15 Ala Gly Arg Lys Arg Ser Ser Ser Ser Val Ser Leu Lys Ala Ala Glu 20 25 30 Lys Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr 35 40 45 Ile Ala Lys Val Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe 50 55 60 Ala Pro Ile Val Gln Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu 65 70 75 80 Lys Leu Thr Glu Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr Leu 85 90 95 Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile 100 105 110 Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gln 115 120 125 Phe Leu Pro Arg Ile Cys Ser Gln Leu Lys Gly His Val Asp Ser His 130 135 140 Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly 145 150 155 160 Val Gln Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gln Cys 165 170 175 Gly Ala Leu Ser Gly Ala Asn Ile Ala Thr Glu Val Ala Gln Glu His 180 185 190 Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys Asp Phe Arg Gly 195 200 205 Glu Gly Lys Asp Val Asp His Lys Val Leu Lys Ala Leu Phe His Arg 210 215 220 Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile 225 230 235 240 Cys Gly Ala Leu Lys Asn Val Val Ala Leu Gly Cys Gly Phe Val Glu 245 250 255 Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Val Gly 260 265 270 Leu Gly Glu Ile Ile Arg Phe Gly Gln Met Phe Phe Pro Glu Ser Arg 275 280 285 Glu Glu Thr Tyr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile Thr 290 295 300 Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr 305 310 315 320 Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gln 325 330 335 Ser Ala Gln Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu 340 345 350 Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val Tyr Gln 355 360 365 Ile Val Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Asp Met Ile Glu 370 375 380 Glu Leu Asp Leu His Glu Asp 385 390 <210> 15 <211> 1323 <212> DNA <213> Saccharomyces cerevisiae GPD2 <400> 15 atgcttgctg tcagaagatt aacaagatac acattcctta agcgaacgca tccggtgtta 60 tatactcgtc gtgcatataa aattttgcct tcaagatcta ctttcctaag aagatcatta 120 ttacaaacac aactgcactc aaagatgact gctcatacta atatcaaaca gcacaaacac 180 tgtcatgagg accatcctat cagaagatcg gactctgccg tgtcaattgt acatttgaaa 240 cgtgcgccct tcaaggttac agtgattggt tctggtaact gggggaccac catcgccaaa 300 gtcattgcgg aaaacacaga attgcattcc catatcttcg agccagaggt gagaatgtgg 360 gtttttgatg aaaagatcgg cgacgaaaat ctgacggata tcataaatac aagacaccag 420 aacgttaaat atctacccaa tattgacctg ccccataatc tagtggccga tcctgatctt 480 ttacactcca tcaagggtgc tgacatcctt gttttcaaca tccctcatca atttttacca 540 aacatagtca aacaattgca aggccacgtg gcccctcatg taagggccat ctcgtgtcta 600 aaagggttcg agttgggctc caagggtgtg caattgctat cctcctatgt tactgatgag 660 ttaggaatcc aatgtggcgc actatctggt gcaaacttgg caccggaagt ggccaaggag 720 cattggtccg aaaccaccgt ggcttaccaa ctaccaaagg attatcaagg tgatggcaag 780 gatgtagatc ataagatttt gaaattgctg ttccacagac cttacttcca cgtcaatgtc 840 atcgatgatg ttgctggtat atccattgcc ggtgccttga agaacgtcgt ggcacttgca 900 tgtggtttcg tagaaggtat gggatggggt aacaatgcct ccgcagccat tcaaaggctg 960 ggtttaggtg aaattatcaa gttcggtaga atgtttttcc cagaatccaa agtcgagacc 1020 tactatcaag aatccgctgg tgttgcagat ctgatcacca cctgctcagg cggtagaaac 1080 gtcaaggttg ccacatacat ggccaagacc ggtaagtcag ccttggaagc agaaaaggaa 1140 ttgcttaacg gtcaatccgc ccaagggata atcacatgca gagaagttca cgagtggcta 1200 caaacatgtg agttgaccca agaattccca ttattcgagg cagtctacca gatagtctac 1260 aacaacgtcc gcatggaaga cctaccggag atgattgaag agctagacat cgatgacgaa 1320 tag 1323 <210> 16 <211> 440 <212> PRT <213> Saccharomyces cerevisiae GPD2 <400> 16 Met Leu Ala Val Arg Arg Leu Thr Arg Tyr Thr Phe Leu Lys Arg Thr 1 5 10 15 His Pro Val Leu Tyr Thr Arg Arg Ala Tyr Lys Ile Leu Pro Ser Arg 20 25 30 Ser Thr Phe Leu Arg Arg Ser Leu Leu Gln Thr Gln Leu His Ser Lys 35 40 45 Met Thr Ala His Thr Asn Ile Lys Gln His Lys His Cys His Glu Asp 50 55 60 His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys 65 70 75 80 Arg Ala Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr 85 90 95 Thr Ile Ala Lys Val Ile Ala Glu Asn Thr Glu Leu His Ser His Ile 100 105 110 Phe Glu Pro Glu Val Arg Met Trp Val Phe Asp Glu Lys Ile Gly Asp 115 120 125 Glu Asn Leu Thr Asp Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr 130 135 140 Leu Pro Asn Ile Asp Leu Pro His Asn Leu Val Ala Asp Pro Asp Leu 145 150 155 160 Leu His Ser Ile Lys Gly Ala Asp Ile Leu Val Phe Asn Ile Pro His 165 170 175 Gln Phe Leu Pro Asn Ile Val Lys Gln Leu Gln Gly His Val Ala Pro 180 185 190 His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys 195 200 205 Gly Val Gln Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly Ile Gln 210 215 220 Cys Gly Ala Leu Ser Gly Ala Asn Leu Ala Pro Glu Val Ala Lys Glu 225 230 235 240 His Trp Ser Glu Thr Thr Val Ala Tyr Gln Leu Pro Lys Asp Tyr Gln 245 250 255 Gly Asp Gly Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His 260 265 270 Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser 275 280 285 Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val 290 295 300 Glu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Leu 305 310 315 320 Gly Leu Gly Glu Ile Ile Lys Phe Gly Arg Met Phe Phe Pro Glu Ser 325 330 335 Lys Val Glu Thr Tyr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile 340 345 350 Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala 355 360 365 Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu Leu Leu Asn Gly 370 375 380 Gln Ser Ala Gln Gly Ile Ile Thr Cys Arg Glu Val His Glu Trp Leu 385 390 395 400 Gln Thr Cys Glu Leu Thr Gln Glu Phe Pro Leu Phe Glu Ala Val Tyr 405 410 415 Gln Ile Val Tyr Asn Asn Val Arg Met Glu Asp Leu Pro Glu Met Ile 420 425 430 Glu Glu Leu Asp Ile Asp Asp Glu 435 440 <210> 17 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH1 deletion <400> 17 ttcaagctat accaagcata caatcaacta tctcatatac acagctgaag cttcgtacgc 60 60 <210> 18 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH1 deletion <400> 18 cttatttaat aataaaaatc ataaatcata agaaattcgc gcataggcca ctagtggat 59 <210> 19 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH2 deletion <400> 19 tacaatcaac tatcaactat taactatatc gtaatacaca cagctgaagc ttcgtacgc 59 <210> 20 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH2 deletion <400> 20 ataatgaaaa ctataaatcg taaagacata agagatccgc gcataggcca ctagtggat 59 <210> 21 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH3 deletion <400> 21 gttaaaacta ggaatagtat agtcataagt taacaccatc cagctgaagc ttcgtacgc 59 <210> 22 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH3 deletion <400> 22 acaaagactt tcataaaaag tttgggtgcg taacacgcta gcataggcca ctagtggat 59 <210> 23 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH4 deletion <400> 23 caagtttaca tttgcaacaa ctaatagtca aataagaaaa cagctgaagc ttcgtacgc 59 <210> 24 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH4 deletion <400> 24 gcacacgcat aattgacgtt tatgagttcg ttcgattttt gcataggcca ctagtggat 59 <210> 25 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH5 deletion <400> 25 agaaaattat ttaactacat atctacaaaa tcaaagcatc cagctgaagc ttcgtacgc 59 <210> 26 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH5 deletion <400> 26 taaaaagtaa aaatatattc atcaaattcg ttacaaaaga gcataggcca ctagtggat 59 <210> 27 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD1 deletion <400> 27 cacccccccc ctccacaaac acaaatattg ataatataaa gcagctgaag cttcgtacgc 60 60 <210> 28 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD1 deletion <400> 28 aagtggggga aagtatgata tgttatcttt ctccaataaa tgcataggcc actagtggat 60 60 <210> 29 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD2 deletion <400> 29 tctctttccc tttccttttc cttcgctccc cttccttatc acagctgaag cttcgtacgc 60 60 <210> 30 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD2 deletion <400> 30 ggcaacagga aagatcagag ggggaggggg ggggagagtg tgcataggcc actagtggat 60 60 <210> 31 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GSF2 deletion <400> 31 gagatcggtg gactttgttt tgatagaggg cgattgcaag ccagctgaag cttcgtacgc 60 60 <210> 32 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GSF2 deletion <400> 32 aataaaaaaa aagtctggat ggtagtgttt tggttttaca agcataggcc actagtggat 60 60 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH1 deletion <400> 33 caccatatcc gcaatgac 18 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH1 deletion <400> 34 gtgttgtcct ctgaggac 18 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH2 deletion <400> 35 accgggcatc tccaactt 18 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH2 deletion <400> 36 ccatgtctac agtttagagg 20 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH3 deletion <400> 37 atgagcagca gccattttg 19 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH3 deletion <400> 38 tgatggtgat aatgtctctc a 21 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH4 deletion <400> 39 aagaactagt ttttagttcg cg 22 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH4 deletion <400> 40 agaacttccg ttcttctttt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH5 deletion <400> 41 ctgctatctg cttgtagaag 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH5 deletion <400> 42 gaaacgtttg tataggttgt 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD1 deletion <400> 43 cgccttgctt ctctcccctt 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD1 deletion <400> 44 ccgacagcct ctgaatgagt 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD2 deletion <400> 45 tacggaccta ttgccattgt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD2 deletion <400> 46 ttaagggcta tagataacag 20 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GSF2 deletion <400> 47 ggtcactcct tgttcttct 19 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GSF2 deletion <400> 48 agtagattcg tgaggaattg 20 <210> 49 <211> 1764 <212> DNA <213> Saccharomyces cerevisiae DLD1 <400> 49 atgttgtgga agcgtacttg cacaaggcta ataaagccta ttgcacaacc tagaggaagg 60 ctggtgagaa gatcatgcta cagatacgcc tcaacaggca caggcagcac cgacagcagc 120 agccagtggt taaaatactc tgtcatcgcc tcttcagcta ctctattcgg ttatttgttc 180 gctaagaacc tctattctag ggagactaag gaagatttga tagagaagct ggaaatggtc 240 aaaaagatcg acccagtaaa ttctacgtta aagctgtcct cattggactc accagactat 300 ttgcacgacc cggttaagat cgataaggtt gttgaggacc tgaagcaggt gctgggaaac 360 aagcctgaaa actactctga tgcgaaatcc gatttggacg cccattcaga tacctacttc 420 aacacgcatc acccctctcc cgagcaaaga cctaggatta tattattccc tcatactacc 480 gaagaagttt ccaaaatttt gaaaatatgt cacgataaca acatgccagt tgtacccttc 540 tcgggcggaa cgtccttgga ggggcacttc ctgcctacaa gaattggaga taccataacc 600 gtagacctgt ccaagtttat gaataacgtc gtaaaatttg acaagctgga cctggacatc 660 accgtgcagg ccggtctacc ctgggaggat ttgaatgact atttgagcga ccacggtttg 720 atgtttggct gtgaccctgg tccaggtgca cagattggtg gttgcattgc taattcttgt 780 tcaggaacca acgcctaccg ttacggtacc atgaaggaga atattataaa catgactata 840 gtgttgccgg acgggaccat tgtcaagacg aagaaaagac ccagaaagtc gagcgctggc 900 tataacttaa atggtttatt tgtgggaagt gaaggtacct taggtattgt tactgaagct 960 actgtcaagt gtcatgtcaa gcccaaagct gaaactgttg cggtggtatc ctttgatact 1020 atcaaggatg cggccgcatg tgcttctaat ctgactcaga gtggtattca tttgaacgcc 1080 atggagttac tggatgaaaa tatgatgaag ttgatcaacg catctgaatc cacggacaga 1140 tgtgattggg tagagaaacc aactatgttt ttcaagattg gtgggagatc tcccaacatt 1200 gtcaatgctc ttgtggatga agttaaggct gtcgcccagt taaatcactg caacagtttt 1260 cagtttgcta aagatgatga cgaaaaattg gaattatggg aagctagaaa ggtcgcgcta 1320 tggtctgtgc tagacgctga taagagcaaa gacaaatcag ctaaaatttg gacaactgat 1380 gtagctgttc ctgtgtcgca gttcgacaag gttattcacg aaactaaaaa ggacatgcaa 1440 gctagtaagc tgatcaacgc cattgttggt catgcaggtg atggtaactt ccatgcattc 1500 atcgtctaca gaacccctga agaacacgaa acctgtagcc aacttgttga cagaatggtc 1560 aagagagcac tgaacgcaga aggcacttgc acgggtgaac acggtgttgg tattggtaaa 1620 agagagtact tgctcgaaga attaggtgaa gcacccgtcg atttgatgag aaagattaag 1680 ctagctattg acccaaagag aatcatgaac ccggacaaaa tctttaaaac tgatccaaac 1740 gagcccgcta atgattacag gtga 1764 <210> 50 <211> 587 <212> PRT <213> Saccharomyces cerevisiae DLD1 <400> 50 Met Leu Trp Lys Arg Thr Cys Thr Arg Leu Ile Lys Pro Ile Ala Gln 1 5 10 15 Pro Arg Gly Arg Leu Val Arg Arg Ser Cys Tyr Arg Tyr Ala Ser Thr 20 25 30 Gly Thr Gly Ser Thr Asp Ser Ser Ser Gln Trp Leu Lys Tyr Ser Val 35 40 45 Ile Ala Ser Ser Ala Thr Leu Phe Gly Tyr Leu Phe Ala Lys Asn Leu 50 55 60 Tyr Ser Arg Glu Thr Lys Glu Asp Leu Ile Glu Lys Leu Glu Met Val 65 70 75 80 Lys Lys Ile Asp Pro Val Asn Ser Thr Leu Lys Leu Ser Ser Leu Asp 85 90 95 Ser Pro Asp Tyr Leu His Asp Pro Val Lys Ile Asp Lys Val Val Glu 100 105 110 Asp Leu Lys Gln Val Leu Gly Asn Lys Pro Glu Asn Tyr Ser Asp Ala 115 120 125 Lys Ser Asp Leu Asp Ala His Ser Asp Thr Tyr Phe Asn Thr His His 130 135 140 Pro Ser Pro Glu Gln Arg Pro Arg Ile Ile Leu Phe Pro His Thr Thr 145 150 155 160 Glu Glu Val Ser Lys Ile Leu Lys Ile Cys His Asp Asn Asn Met Pro 165 170 175 Val Val Pro Phe Ser Gly Gly Thr Ser Leu Glu Gly His Phe Leu Pro 180 185 190 Thr Arg Ile Gly Asp Thr Ile Thr Val Asp Leu Ser Lys Phe Met Asn 195 200 205 Asn Val Val Lys Phe Asp Lys Leu Asp Leu Asp Ile Thr Val Gln Ala 210 215 220 Gly Leu Pro Trp Glu Asp Leu Asn Asp Tyr Leu Ser Asp His Gly Leu 225 230 235 240 Met Phe Gly Cys Asp Pro Gly Pro Gly Ala Gln Ile Gly Gly Cys Ile 245 250 255 Ala Asn Ser Cys Ser Gly Thr Asn Ala Tyr Arg Tyr Gly Thr Met Lys 260 265 270 Glu Asn Ile Ile Asn Met Thr Ile Val Leu Pro Asp Gly Thr Ile Val 275 280 285 Lys Thr Lys Lys Arg Pro Arg Lys Ser Ser Ala Gly Tyr Asn Leu Asn 290 295 300 Gly Leu Phe Val Gly Ser Glu Gly Thr Leu Gly Ile Val Thr Glu Ala 305 310 315 320 Thr Val Lys Cys His Val Lys Pro Lys Ala Glu Thr Val Ala Val Val 325 330 335 Ser Phe Asp Thr Ile Lys Asp Ala Ala Ala Cys Ala Ser Asn Leu Thr 340 345 350 Gln Ser Gly Ile His Leu Asn Ala Met Glu Leu Leu Asp Glu Asn Met 355 360 365 Met Lys Leu Ile Asn Ala Ser Glu Ser Thr Asp Arg Cys Asp Trp Val 370 375 380 Glu Lys Pro Thr Met Phe Phe Lys Ile Gly Gly Arg Ser Pro Asn Ile 385 390 395 400 Val Asn Ala Leu Val Asp Glu Val Lys Ala Val Ala Gln Leu Asn His 405 410 415 Cys Asn Ser Phe Gln Phe Ala Lys Asp Asp Asp Glu Lys Leu Glu Leu 420 425 430 Trp Glu Ala Arg Lys Val Ala Leu Trp Ser Val Leu Asp Ala Asp Lys 435 440 445 Ser Lys Asp Lys Ser Ala Lys Ile Trp Thr Thr Asp Val Ala Val Pro 450 455 460 Val Ser Gln Phe Asp Lys Val Ile His Glu Thr Lys Lys Asp Met Gln 465 470 475 480 Ala Ser Lys Leu Ile Asn Ala Ile Val Gly His Ala Gly Asp Gly Asn 485 490 495 Phe His Ala Phe Ile Val Tyr Arg Thr Pro Glu Glu His Glu Thr Cys 500 505 510 Ser Gln Leu Val Asp Arg Met Val Lys Arg Ala Leu Asn Ala Glu Gly 515 520 525 Thr Cys Thr Gly Glu His Gly Val Gly Ile Gly Lys Arg Glu Tyr Leu 530 535 540 Leu Glu Glu Leu Gly Glu Ala Pro Val Asp Leu Met Arg Lys Ile Lys 545 550 555 560 Leu Ala Ile Asp Pro Lys Arg Ile Met Asn Pro Asp Lys Ile Phe Lys 565 570 575 Thr Asp Pro Asn Glu Pro Ala Asn Asp Tyr Arg 580 585 <210> 51 <211> 1851 <212> DNA <213> Saccharomyces cerevisiae JEN1 <400> 51 atgtcgtcgt caattacaga tgagaaaata tctggtgaac agcaacaacc tgctggcaga 60 aaactatact ataacacaag tacatttgca gagcctcctc tagtggacgg agaaggtaac 120 cctataaatt atgagccgga agtttacaac ccggatcacg aaaagctata ccataaccca 180 tcactgcctg cacaatcaat tcaggataca agagatgatg aattgctgga aagagtttat 240 agccaggatc aaggtgtaga gtatgaggaa gatgaagagg ataagccaaa cctaagcgct 300 gcgtccatta aaagttatgc tttaacgaga tttacgtcct tactgcacat ccacgagttt 360 tcttgggaga atgtcaatcc catacccgaa ctgcgcaaaa tgacatggca gaattggaac 420 tattttttta tgggttattt tgcgtggttg tctgcggctt gggccttctt ttgcgtttca 480 gtatcagtcg ctccattggc tgaactatat gacagaccaa ccaaggacat cacctggggg 540 ttgggattgg tgttatttgt tcgttcagca ggtgctgtca tatttggttt atggacagat 600 aagtcttcca gaaagtggcc gtacattaca tgtttgttct tatttgtcat tgcacaactc 660 tgtactccat ggtgtgacac atacgagaaa tttctgggcg taaggtggat aaccggtatt 720 gctatgggag gaatttacgg atgtgcttct gcaacagcga ttgaagatgc acctgtgaaa 780 gcacgttcgt tcctatcagg tctatttttt tctgcttacg ctatggggtt catatttgct 840 atcatttttt acagagcctt tggctacttt agggatgatg gctggaaaat attgttttgg 900 tttagtattt ttctaccaat tctactaatt ttctggagat tgttatggcc tgaaacgaaa 960 tacttcacca aggttttgaa agcccgtaaa ttaatattga gtgacgcagt gaaagctaat 1020 ggtggcgagc ctctaccaaa agccaacttt aaacaaaaga tggtatccat gaagagaaca 1080 gttcaaaagt actggttgtt gttcgcatat ttggttgttt tattggtggg tccaaattac 1140 ttgactcatg cttctcaaga cttgttgcca accatgctgc gtgcccaatt aggcctatcc 1200 aaggatgctg tcactgtcat tgtagtggtt accaacatcg gtgctatttg tgggggtatg 1260 atatttggac agttcatgga agttactgga agaagattag gcctattgat tgcatgcaca 1320 atgggtggtt gcttcaccta ccctgcattt atgttgagaa gcgaaaaggc tatattaggt 1380 gccggtttca tgttatattt ttgtgtcttt ggtgtctggg gtatcctgcc cattcacctt 1440 gcagagttgg cccctgctga tgcaagggct ttggttgccg gtttatctta ccagctaggt 1500 aatctagctt ctgcagcggc ttccacgatt gagacacagt tagctgatag atacccatta 1560 gaaagagatg cctctggtgc tgtgattaaa gaagattatg ccaaagttat ggctatcttg 1620 actggttctg ttttcatctt cacatttgct tgtgtttttg ttggccatga gaaattccat 1680 cgtgatttgt cctctcctgt tatgaagaaa tatataaacc aagtggaaga atacgaagcc 1740 gatggtcttt cgattagtga cattgttgaa caaaagacgg aatgtgcttc agtgaagatg 1800 attgattcga acgtctcaaa gacatatgag gagcatattg agaccgttta a 1851 <210> 52 <211> 616 <212> PRT <213> Saccharomyces cerevisiae JEN1 <400> 52 Met Ser Ser Ser Ile Thr Asp Glu Lys Ile Ser Gly Glu Gln Gln Gln 1 5 10 15 Pro Ala Gly Arg Lys Leu Tyr Tyr Asn Thr Ser Thr Phe Ala Glu Pro 20 25 30 Pro Leu Val Asp Gly Glu Gly Asn Pro Ile Asn Tyr Glu Pro Glu Val 35 40 45 Tyr Asn Pro Asp His Glu Lys Leu Tyr His Asn Pro Ser Leu Pro Ala 50 55 60 Gln Ser Ile Gln Asp Thr Arg Asp Asp Glu Leu Leu Glu Arg Val Tyr 65 70 75 80 Ser Gln Asp Gln Gly Val Glu Tyr Glu Glu Asp Glu Glu Asp Lys Pro 85 90 95 Asn Leu Ser Ala Ala Ser Ile Lys Ser Tyr Ala Leu Thr Arg Phe Thr 100 105 110 Ser Leu Leu His Ile His Glu Phe Ser Trp Glu Asn Val Asn Pro Ile 115 120 125 Pro Glu Leu Arg Lys Met Thr Trp Gln Asn Trp Asn Tyr Phe Phe Met 130 135 140 Gly Tyr Phe Ala Trp Leu Ser Ala Ala Trp Ala Phe Phe Cys Val Ser 145 150 155 160 Val Ser Val Ala Pro Leu Ala Glu Leu Tyr Asp Arg Pro Thr Lys Asp 165 170 175 Ile Thr Trp Gly Leu Gly Leu Val Leu Phe Val Arg Ser Ala Gly Ala 180 185 190 Val Ile Phe Gly Leu Trp Thr Asp Lys Ser Ser Arg Lys Trp Pro Tyr 195 200 205 Ile Thr Cys Leu Phe Leu Phe Val Ile Ala Gln Leu Cys Thr Pro Trp 210 215 220 Cys Asp Thr Tyr Glu Lys Phe Leu Gly Val Arg Trp Ile Thr Gly Ile 225 230 235 240 Ala Met Gly Gly Ile Tyr Gly Cys Ala Ser Ala Thr Ala Ile Glu Asp 245 250 255 Ala Pro Val Lys Ala Arg Ser Phe Leu Ser Gly Leu Phe Phe Ser Ala 260 265 270 Tyr Ala Met Gly Phe Ile Phe Ala Ile Ile Phe Tyr Arg Ala Phe Gly 275 280 285 Tyr Phe Arg Asp Asp Gly Trp Lys Ile Leu Phe Trp Phe Ser Ile Phe 290 295 300 Leu Pro Ile Leu Leu Ile Phe Trp Arg Leu Leu Trp Pro Glu Thr Lys 305 310 315 320 Tyr Phe Thr Lys Val Leu Lys Ala Arg Lys Leu Ile Leu Ser Asp Ala 325 330 335 Val Lys Ala Asn Gly Gly Glu Pro Leu Pro Lys Ala Asn Phe Lys Gln 340 345 350 Lys Met Val Ser Met Lys Arg Thr Val Gln Lys Tyr Trp Leu Leu Phe 355 360 365 Ala Tyr Leu Val Val Leu Leu Val Gly Pro Asn Tyr Leu Thr His Ala 370 375 380 Ser Gln Asp Leu Leu Pro Thr Met Leu Arg Ala Gln Leu Gly Leu Ser 385 390 395 400 Lys Asp Ala Val Thr Val Ile Val Val Val Thr Asn Ile Gly Ala Ile 405 410 415 Cys Gly Gly Met Ile Phe Gly Gln Phe Met Glu Val Thr Gly Arg Arg 420 425 430 Leu Gly Leu Leu Ile Ala Cys Thr Met Gly Gly Cys Phe Thr Tyr Pro 435 440 445 Ala Phe Met Leu Arg Ser Glu Lys Ala Ile Leu Gly Ala Gly Phe Met 450 455 460 Leu Tyr Phe Cys Val Phe Gly Val Trp Gly Ile Leu Pro Ile His Leu 465 470 475 480 Ala Glu Leu Ala Pro Ala Asp Ala Arg Ala Leu Val Ala Gly Leu Ser 485 490 495 Tyr Gln Leu Gly Asn Leu Ala Ser Ala Ala Ala Ser Thr Ile Glu Thr 500 505 510 Gln Leu Ala Asp Arg Tyr Pro Leu Glu Arg Asp Ala Ser Gly Ala Val 515 520 525 Ile Lys Glu Asp Tyr Ala Lys Val Met Ala Ile Leu Thr Gly Ser Val 530 535 540 Phe Ile Phe Thr Phe Ala Cys Val Phe Val Gly His Glu Lys Phe His 545 550 555 560 Arg Asp Leu Ser Ser Pro Val Met Lys Lys Tyr Ile Asn Gln Val Glu 565 570 575 Glu Tyr Glu Ala Asp Gly Leu Ser Ile Ser Asp Ile Val Glu Gln Lys 580 585 590 Thr Glu Cys Ala Ser Val Lys Met Ile Asp Ser Asn Val Ser Lys Thr 595 600 605 Tyr Glu Glu His Ile Glu Thr Val 610 615 <210> 53 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for DLD1 deletion <400> 53 tgtacatcat tccgatccag ctggaaacaa aagcaagaac acagctgaag cttcgtacgc 60 60 <210> 54 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for DLD1 deletion <400> 54 tttcaggttt acgtgaaggg tgaaaaagga aaatcagata cgcataggcc actagtggat 60 60 <210> 55 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for JEN1 deletion <400> 55 aaagtttttc ctcaaagaga ttaaatactg ctactgaaaa tcagctgaag cttcgtacgc 60 60 <210> 56 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for JEN1 deletion <400> 56 tttcaggttt acgtgaaggg tgaaaaagga aaatcagata cgcataggcc actagtggat 60 60 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of DLD1 deletion <400> 57 tcttgtcaac ccaggtccgt 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of DLD1 deletion <400> 58 aggaagtgat gtaagctgct 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of JEN1 deletion <400> 59 aacggtcttt tgccccccct 20 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of JEN1 deletion <400> 60 cttgctagtg ttaacggc 18 <210> 61 <211> 996 <212> DNA <213> Leuconostoc mesenteroides ldhA <400> 61 atgaagattt ttgcttacgg cattcgtgat gatgaaaagc catcacttga agaatggaaa 60 gcggctaacc cagagattga agtggactac acacaagaat tattgacacc tgaaacagct 120 aagttggctg agggatcaga ttcagctgtt gtttatcaac aattggacta tacacgtgaa 180 acattgacag ctttagctaa cgttggtgtt actaacttgt cattgcgtaa cgttggtaca 240 gataacattg attttgatgc agcacgtgaa tttaacttta acatttcaaa tgttcctgtt 300 tattcaccaa atgctattgc agaacactca atgattcaat tatctcgttt gctacgtcgc 360 acgaaagcat tggatgccaa aattgctaag cacgacttgc gttgggcacc aacaattgga 420 cgtgaaatgc gtatgcaaac agttggtgtt attggtacag gtcatattgg ccgtgttgct 480 attaacattt tgaaaggctt tggggccaag gttattgctt atgacaagta cccaaatgct 540 gaattacaag cagaaggttt gtacgttgac acattagacg aattatatgc acaagctgat 600 gcaatttcat tgtatgttcc tggtgtacct gaaaaccatc atctaatcaa tgcagatgct 660 attgctaaga tgaaggatgg tgtggttatc atgaacgctg cgcgtggtaa tttgatggac 720 attgacgcta ttattgatgg tttgaattct ggtaagattt cagacttcgg tatggacgtt 780 tatgaaaatg aagttggctt gttcaatgaa gattggtctg gtaaagaatt cccagatgct 840 aagattgctg acttgattgc acgcgaaaat gtattggtta cgccacacac ggctttctat 900 acaactaaag ctgttctaga aatggttcac caatcatttg atgcagcagt tgctttcgcc 960 aagggtgaga agccagctat tgctgttgaa tattaa 996 <210> 62 <211> 331 <212> PRT <213> Leuconostoc mesenteroides ldhA <400> 62 Met Lys Ile Phe Ala Tyr Gly Ile Arg Asp Asp Glu Lys Pro Ser Leu 1 5 10 15 Glu Glu Trp Lys Ala Ala Asn Pro Glu Ile Glu Val Asp Tyr Thr Gln 20 25 30 Glu Leu Leu Thr Pro Glu Thr Ala Lys Leu Ala Glu Gly Ser Asp Ser 35 40 45 Ala Val Val Tyr Gln Gln Leu Asp Tyr Thr Arg Glu Thr Leu Thr Ala 50 55 60 Leu Ala Asn Val Gly Val Thr Asn Leu Ser Leu Arg Asn Val Gly Thr 65 70 75 80 Asp Asn Ile Asp Phe Asp Ala Ala Arg Glu Phe Asn Phe Asn Ile Ser 85 90 95 Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ser Met Ile 100 105 110 Gln Leu Ser Arg Leu Leu Arg Arg Thr Lys Ala Leu Asp Ala Lys Ile 115 120 125 Ala Lys His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Met Arg 130 135 140 Met Gln Thr Val Gly Val Ile Gly Thr Gly His Ile Gly Arg Val Ala 145 150 155 160 Ile Asn Ile Leu Lys Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp Lys 165 170 175 Tyr Pro Asn Ala Glu Leu Gln Ala Glu Gly Leu Tyr Val Asp Thr Leu 180 185 190 Asp Glu Leu Tyr Ala Gln Ala Asp Ala Ile Ser Leu Tyr Val Pro Gly 195 200 205 Val Pro Glu Asn His His Leu Ile Asn Ala Asp Ala Ile Ala Lys Met 210 215 220 Lys Asp Gly Val Val Ile Met Asn Ala Ala Arg Gly Asn Leu Met Asp 225 230 235 240 Ile Asp Ala Ile Ile Asp Gly Leu Asn Ser Gly Lys Ile Ser Asp Phe 245 250 255 Gly Met Asp Val Tyr Glu Asn Glu Val Gly Leu Phe Asn Glu Asp Trp 260 265 270 Ser Gly Lys Glu Phe Pro Asp Ala Lys Ile Ala Asp Leu Ile Ala Arg 275 280 285 Glu Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr Lys Ala 290 295 300 Val Leu Glu Met Val His Gln Ser Phe Asp Ala Ala Val Ala Phe Ala 305 310 315 320 Lys Gly Glu Lys Pro Ala Ile Ala Val Glu Tyr 325 330 <210> 63 <211> 1692 <212> DNA <213> Saccharomyces cerevisiae PDC1 <400> 63 atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60 accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120 gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180 tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300 gtcccatcca tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420 gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480 agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540 ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600 attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720 ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780 ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840 ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960 ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgaccaatat tgctgacgcc 1020 gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080 gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140 ggtgatgttg tcattgctga aaccggtacc tccgctttcg gtatcaacca aaccactttc 1200 ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260 gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440 cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500 actttcggtg ctaaggacta cgaaacccac agagtcgcta ccaccggtga atgggacaag 1560 ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga ggttatgttg 1620 ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680 gctaagcaat aa 1692 <210> 64 <211> 563 <212> PRT <213> Saccharomyces cerevisiae PDC1 <400> 64 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F primer for ldhA <400> 65 ggcgggatcc atgaagattt ttgcttacgg 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R primer for ldhA <400> 66 ggcgctgcag ttaatattca acagcaatag 30 <210> 67 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> TEF1 promoter <400> 67 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg 300 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360 ttgctcatta gaaagaaagc atagcaatct aatctaagtt t 401 <210> 68 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> CYC1 terminator <400> 68 aaaaagaatc atgattgaat gaagatatta tttttttgaa ttatattttt taaattttat 60 ataaagacat ggtttttctt ttcaactcaa ataaagattt ataagttact taaataacat 120 acattttata aggtattcta taaaaagagt attatgttat tgttaacctt tttgtctcca 180 attgtcgtca taacgatgag gtgttgcatt tttggaaacg agattgacat agagtcaaaa 240 tttgctaaat ttgatccctc ccatcgcaag ataatcttcc ctcaaggtta tcatgattat 300 caggatggcg aaaggatacg ctaaaaattc aataaaaaat tcaatataat tttcgtttcc 360 caagaactaa cttggaaggt tatacatggg tac 393 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Lm.ldhA_INT F <400> 69 ggcggctagc gggaacaaaa gctggagctc 30 <210> 70 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Lm.ldhA_INT R <400> 70 ggcggggccc gggggatgtg ctgcaagg 28 <210> 71 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for PDC1 deletion/lhdA insertion <400> 71 ctcataacct cacgcaaaat aacacagtca aatcaatcaa agggaacaaa agctggagct 60 60 <210> 72 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for PDC1 deletion/lhdA insertion <400> 72 aatgcttata aaactttaac taataattag agattaaatc gcataggcca ctagtggat 59 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of PDC1 deletion/lhdA insertion <400> 73 gacttttcgt gtgatgaggc 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of PDC1 deletion/lhdA insertion <400> 74 tttacatgga ccgcaccaag 20 <110> SNU R & DB Foundation <120> Genetically engineered yeast cells having enhanced growth rate and          method of producing target materials using the same <130> PN112274 <160> 80 <170> Kopatentin 2.0 <210> 1 <211> 1212 <212> DNA <213> Saccharomyces cerevisiae GSF2 <400> 1 atggagattt acattagact taacgcagat gtcgagcacg actatgcgtt tcaggtgtca 60 aatgaagaca ccataaataa caaaattaag aagattttcc cttccaagac gggtttggcg 120 gacttaatgg tgctgagacc atcaattttc catgagaagg agcccgtaaa gttctataag 180 tctatccatc cagggtacct gtccgaaggt ggttgtttga tgttccatta tgaagctgat 240 aacgaagaaa atctcgagga gttgaatgac tccaagccgc ttattgacca gttgtggcct 300 ggtcagctag ttgttccgga gtggaagcta tccaagaaaa acatatgggt atacaccatt 360 attatgttgg cctggctata taccgatttg cccgacgcca tttctccaac cccaggtatt 420 tgtttgacta atcaattgtc tagactattg attcctgtag ccaagcacat ggatttacct 480 gaaattgccg ctaaactcga acaagaagtt caggcaaact attctagtct cgtagctcaa 540 tggcttttct ttgtcatgca cattttcaaa gtgggcataa ttactttatt cctcaaatta 600 ggcattgcca accccatcag cttcaatcct tacaagttat ggagtttgag agatttaact 660 tctccttctg ctaacggcgc taagaattct ggcggcaata acaacactac tgacttaaag 720 acacgtttgc gctctctggg ctggattggt gctaagagag ctacttacga cgattaccag 780 actaactatt acaactatgt catagataag atgggtggtg cagttgctgc ttatagagcg 840 ggcgcgatta ggaaagctgc agcaccaggt atccaacttg tggctggaga gggtttccaa 900 agtcctctag aagataggtt cactgcttcc actttcacgg ctattaaaac cgaacggaaa 960 ttcattttaa gtgaagaata ctttgtggag ctggaaaaca atctaaagaa gatcttggaa 1020 gagtatgacg gggatattgg taagatgaat gctgaaatta ggaggtttag aaggtttggt 1080 atttatgaac cagatgaaaa actggcctcg ctagttaagc tcagaagaga aattgctgat 1140 gagaaagaga aagcatcaaa caatgatgct acttttggta taaaaaagaa tgatttgaaa 1200 aaatctaatt aa 1212 <210> 2 <211> 403 <212> PRT <213> Saccharomyces cerevisiae GSF2 <400> 2 Met Glu Ile Tyr Ile Arg Leu Asn Ala Asp Val Glu His Asp Tyr Ala   1 5 10 15 Phe Gln Val Ser Asn Glu Asp Thr Ile Asn Asn Lys Ile Lys Lys Ile              20 25 30 Phe Pro Ser Lys Thr Gly Leu Ala Asp Leu Met Val Leu Arg Pro Ser          35 40 45 Ile Phe His Glu Lys Glu Pro Val Lys Phe Tyr Lys Ser Ile His Pro      50 55 60 Gly Tyr Leu Ser Glu Gly Gly Cys Leu Met Phe His Tyr Glu Ala Asp  65 70 75 80 Asn Glu Glu Asn Leu Glu Glu Leu Asn Asp Ser Lys Pro Leu Ile Asp                  85 90 95 Gln Leu Trp Pro Gly Gln Leu Val Val Pro Glu Trp Lys Leu Ser Lys             100 105 110 Lys Asn Ile Trp Val Tyr Thr Ile Ile Met Leu Ala Trp Leu Tyr Thr         115 120 125 Asp Leu Pro Asp Ala Ile Ser Pro Thr Pro Gly Ile Cys Leu Thr Asn     130 135 140 Gln Leu Ser Arg Leu Leu Ile Pro Val Ala Lys His Met Asp Leu Pro 145 150 155 160 Glu Ile Ala Ala Lys Leu Glu Gln Glu Val Gln Ala Asn Tyr Ser Ser                 165 170 175 Leu Val Ala Gln Trp Leu Phe Phe Val Met His Ile Phe Lys Val Gly             180 185 190 Ile Ile Thr Leu Phe Leu Lys Leu Gly Ile Ala Asn Pro Ile Ser Phe         195 200 205 Asn Pro Tyr Lys Leu Trp Ser Leu Arg Asp Leu Thr Ser Pro Ser Ala     210 215 220 Asn Gly Ala Lys Asn Ser Gly Gly Asn Asn Asn Thr Thr Asp Leu Lys 225 230 235 240 Thr Arg Leu Arg Ser Leu Gly Trp Ile Gly Ala Lys Arg Ala Thr Tyr                 245 250 255 Asp Asp Tyr Gln Thr Asn Tyr Tyr Asn Tyr Val Ile Asp Lys Met Gly             260 265 270 Gly Ala Val Ala Ala Tyr Arg Ala Gly Ala Ile Arg Lys Ala Ala Ala         275 280 285 Pro Gly Ile Gln Leu Val Ala Gly Glu Gly Phe Gln Ser Pro Leu Glu     290 295 300 Asp Arg Phe Thr Ala Ser Thr Phe Thr Ala Ile Lys Thr Glu Arg Lys 305 310 315 320 Phe Ile Leu Ser Glu Glu Tyr Phe Val Glu Leu Glu Asn Asn Leu Lys                 325 330 335 Lys Ile Leu Glu Glu Tyr Asp Gly Asp Ile Gly Lys Met Asn Ala Glu             340 345 350 Ile Arg Arg Phe Arg Arg Phe Gly Ile Tyr Glu Pro Asp Glu Lys Leu         355 360 365 Ala Ser Leu Val Lys Leu Arg Arg Glu Ile Ala Asp Glu Lys Glu Lys     370 375 380 Ala Ser Asn Asn Asp Ala Thr Phe Gly Ile Lys Lys Asn Asp Leu Lys 385 390 395 400 Lys Ser Asn             <210> 3 <211> 1047 <212> DNA <213> Saccharomyces cerevisiae ADH1 <400> 3 atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 4 <211> 348 <212> PRT <213> Saccharomyces cerevisiae ADH1 <400> 4 Met Ser Ile Pro Glu Thr Gln Lys Gly Val Ile Phe Tyr Glu Ser His   1 5 10 15 Gly Lys Leu Glu Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn              20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu          35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val      50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val  65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly                  85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys             100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln         115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr     130 135 140 Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile                 165 170 175 Ser Gly Ala Gly Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys             180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu         195 200 205 Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys     210 215 220 Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala Thr Asp Gly Gly Ala 225 230 235 240 His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser                 245 250 255 Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro             260 265 270 Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser         275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu     290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly                 325 330 335 Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys             340 345 <210> 5 <211> 1047 <212> DNA <213> Saccharomyces cerevisiae ADH2 <400> 5 atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60 cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120 tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180 ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480 aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540 gt; attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660 gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720 cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780 agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840 gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900 gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960 gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 6 <211> 348 <212> PRT <213> Saccharomyces cerevisiae ADH2 <400> 6 Met Ser Ile Pro Glu Thr Gln Lys Ala Ile Ile Phe Tyr Glu Ser Asn   1 5 10 15 Gly Lys Leu Glu His Lys Asp Ile Pro Val Pro Lys Pro Lys Pro Asn              20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu          35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Thr Lys Leu Pro Leu Val      50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val  65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly                  85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys             100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Glu         115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr     130 135 140 Asp Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Arg Ala Gly His Trp Ala Ala Ile                 165 170 175 Ser Gly Ala Gly Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys             180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Pro Gly Lys Glu         195 200 205 Glu Leu Phe Thr Ser Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys     210 215 220 Glu Lys Asp Ile Val Ser Ala Val Val Lys Ala Thr Asn Gly Gly Ala 225 230 235 240 His Gly Ile Ile Asn Val Ser Ser Ser Glu Ala Ala Ile Glu Ala Ser                 245 250 255 Thr Arg Tyr Cys Arg Ala Asn Gly Thr Val Val Leu Val Gly Leu Pro             260 265 270 Ala Gly Ala Lys Cys Ser Ser Asp Val Phe Asn His Val Val Lys Ser         275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu     290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Ser Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly                 325 330 335 Gln Ile Ala Gly Arg Tyr Val Val Asp Thr Ser Lys             340 345 <210> 7 <211> 1128 <212> DNA <213> Saccharomyces cerevisiae ADH3 <400> 7 atgttgagaa cgtcaacatt gttcaccagg cgtgtccaac caagcctatt ttctagaaac 60 attcttagat tgcaatccac agctgcaatc cctaagactc aaaaaggtgt catcttttat 120 gagaataagg ggaagctgca ttacaaagat atccctgtcc ccgagcctaa gccaaatgaa 180 attttaatca acgttaaata ttctggtgta tgtcacaccg atttacatgc ttggcacggc 240 gattggccat tacctgttaa actaccatta gtaggtggtc atgaaggtgc tggtgtagtt 300 gtcaaactag gttccaatgt caagggctgg aaagtcggtg atttagcagg tatcaaatgg 360 ctgaacggtt cttgtatgac atgcgaattc tgtgaatcag gtcatgaatc aaattgtcca 420 gatgctgatt tatctggtta cactcatgat ggttctttcc aacaatttgc gaccgctgat 480 gctattcaag ccgccaaaat tcaacagggt accgacttgg ccgaagtagc cccaatatta 540 tgtgctggtg ttactgtata taaagcacta aaagaggcag acttgaaagc tggtgactgg 600 gttgccatct ctggtgctgc aggtggcttg ggttccttgg ccgttcaata tgcaactgcg 660 atgggttaca gagttctagg tattgatgca ggtgaggaaa aggaaaaact tttcaagaaa 720 ttggggggtg aagtattcat cgactttact aaaacaaaga atatggtttc tgacattcaa 780 gaagctacca aaggtggccc tcatggtgtc attaacgttt ccgtttctga agccgctatt 840 tctctatcta cggaatatgt tagaccatgt ggtaccgtcg ttttggttgg tttgcccgct 900 aacgcctacg ttaaatcaga ggtattctct catgtggtga agtccatcaa tatcaagggt 960 tcttatgttg gtaacagagc tgatacgaga gaagccttag acttctttag cagaggtttg 1020 atcaaatcac caatcaaaat tgttggatta tctgaattac caaaggttta tgacttgatg 1080 gaaaagggca agattttggg tagatacgtc gtcgatacta gtaaataa 1128 <210> 8 <211> 375 <212> PRT <213> Saccharomyces cerevisiae ADH3 <400> 8 Met Leu Arg Thr Ser Thr Leu Phe Thr Arg Arg Val Gln Pro Ser Leu   1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Thr Ala Ala Ile Pro Lys              20 25 30 Thr Gln Lys Gly Val Ile Phe Tyr Glu Asn Lys Gly Lys Leu His Tyr          35 40 45 Lys Asp Ile Pro Val Pro Glu Pro Lys Pro Asn Glu Ile Leu Ile Asn      50 55 60 Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu His Ala Trp His Gly  65 70 75 80 Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val Gly Gly His Glu Gly                  85 90 95 Ala Gly Val Val Val Lys Leu Gly Ser Asn Val Lys Gly Trp Lys Val             100 105 110 Gly Asp Leu Ala Gly Ile Lys Trp Leu Asn Gly Ser Cys Met Thr Cys         115 120 125 Glu Phe Cys Glu Ser Gly His Glu Ser Asn Cys Pro Asp Ala Asp Leu     130 135 140 Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln Phe Ala Thr Ala Asp 145 150 155 160 Ala Ile Gln Ala Ala Lys Ile Gln Gln Gly Thr Asp Leu Ala Glu Val                 165 170 175 Ala Pro Ile Leu Cys Ala Gly Val Thr Val Tyr Lys Ala Leu Lys Glu             180 185 190 Ala Asp Leu Lys Ala Gly Asp Trp Val Ala Ile Ser Gly Ala Ala Gly         195 200 205 Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Thr Ala Met Gly Tyr Arg     210 215 220 Val Leu Gly Ile Asp Ala Gly Glu Glu Lys Glu Lys Leu Phe Lys Lys 225 230 235 240 Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys Thr Lys Asn Met Val                 245 250 255 Ser Asp Ile Gln Glu Ala Thr Lys Gly Gly Pro His Gly Val Ile Asn             260 265 270 Val Ser Val Ser Glu Ala Ala Ile Ser Leu Ser Thr Glu Tyr Val Arg         275 280 285 Pro Cys Gly Thr Val Val Leu Val Gly Leu Pro Ala Asn Ala Tyr Val     290 295 300 Lys Ser Glu Val Phe Ser His Val Val Lys Ser Ile Asn Ile Lys Gly 305 310 315 320 Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu Ala Leu Asp Phe Phe                 325 330 335 Ser Arg Gly Leu Ile Lys Ser Pro Ile Lys Ile Val Gly Leu Ser Glu             340 345 350 Leu Pro Lys Val Tyr Asp Leu Met Glu Lys Gly Lys Ile Leu Gly Arg         355 360 365 Tyr Val Val Asp Thr Ser Lys     370 375 <210> 9 <211> 1149 <212> DNA <213> Saccharomyces cerevisiae ADH4 <400> 9 atgtcttccg ttactgggtt ttacattcca ccaatctctt tctttggtga aggtgcttta 60 gaagaaaccg ctgattacat caaaaacaag gattacaaaa aggctttgat cgttactgat 120 cctggtattg cagctattgg tctctccggt agagtccaaa agatgttgga agaacgtggc 180 ttaaacgttg ctatctatga caaaactcaa ccaaacccaa atattgccaa tgtcacagct 240 ggtttgaagg ttttgaagga agaaaactct gaaattgtcg tttccattgg tggtggttct 300 gctcacgaca atgctaaggc cattgcttta ttggctacta acggtgggga aattggagat 360 tatgaaggtg tcaaccaatc taagaaggct gctttaccgc tatttgccat caacactact 420 gctggtactg cttccgagat gaccagattc actattatct ctaatgaaga aaagaaaatc 480 aagatggcca tcattgacaa caacgtcact ccagctgttg ctgtcaacga cccatctacc 540 atgtttggtt tgccacctgc tttgactgct gctactggtc tagatgcttt gactcactgt 600 atcgaagctt acgtttccac cgcctctaac ccaatcaccg atgcttgtgc tttgaagggt 660 attgatttga tcaatgaaag cttggtcgcc gcatacaaag acggtaaaga caagaaggcc 720 agaactgata tgtgttacgc agaatacttg gcaggtatgg ctttcaacaa tgcttctcta 780 ggttatgttc atgcccttgc tcatcaactt ggtggtttct accacttgcc tcatggtgtt 840 tgtaacgctg tcttgttgcc tcatgttcaa gaggccaaca tgcaatgtcc aaaggccaag 900 aagagattag gtgaaattgc cttgcattgc ggtgcttctc aagaagatcc agaagaaacc 960 atcaaggctt tgcacgtttt aaacagaacc atgaacattc caagaaactt gaaagactta 1020 ggtgttaaaa ccgaagattt tgacattttg gctgaacacg ccatgcatga tgcctgccat 1080 ttgactaacc cagttcaatt caccaaagaa caagtggttg ccattatcaa gaaagcctat 1140 gaatattaa 1149 <210> 10 <211> 382 <212> PRT <213> Saccharomyces cerevisiae ADH4 <400> 10 Met Ser Ser Val Thr Gly Phe Tyr Ile Pro Pro Ile Ser Phe Phe Gly   1 5 10 15 Glu Gly Ala Leu Glu Glu Thr Ala Asp Tyr Ile Lys Asn Lys Asp Tyr              20 25 30 Lys Lys Ala Leu Ile Val Thr Asp Pro Gly Ile Ala Ala Ile Gly Leu          35 40 45 Ser Gly Arg Val Gln Lys Met Leu Glu Glu Arg Gly Leu Asn Val Ala      50 55 60 Ile Tyr Asp Lys Thr Gln Pro Asn Pro Asn Ile Ala Asn Val Thr Ala  65 70 75 80 Gly Leu Lys Val Leu Lys Glu Glu Asn Ser Glu Ile Val Val Ser Ile                  85 90 95 Gly Gly Gly Ser Ala His Asp Asn Ala Lys Ala Ile Ala Leu Ala             100 105 110 Thr Asn Gly Gly Glu Ile Gly Asp Tyr Glu Gly Val Asn Gln Ser Lys         115 120 125 Lys Ala Ala Leu Pro Leu Phe Ala Ile Asn Thr Thr Ala Gly Thr Ala     130 135 140 Ser Glu Met Thr Arg Phe Thr Ile Ile Ser Asn Glu Glu Lys Lys Ile 145 150 155 160 Lys Met Ale Ile Ile Asp Asn Asn Ale Val Ale Val Ale Val Asn                 165 170 175 Asp Pro Ser Thr Met Phe Gly Leu Pro Pro Ala Leu Thr Ala Ala Thr             180 185 190 Gly Leu Asp Ala Leu Thr His Cys Ile Glu Ala Tyr Val Ser Thr Ala         195 200 205 Ser Asn Pro Ile Thr Asp Ala Cys Ala Leu Lys Gly Ile Asp Leu Ile     210 215 220 Asn Glu Ser Leu Val Ala Ala Tyr Lys Asp Gly Lys Asp Lys Lys Ala 225 230 235 240 Arg Thr Asp Met Cys Tyr Ala Glu Tyr Leu Ala Gly Met Ala Phe Asn                 245 250 255 Asn Ala Ser Leu Gly Tyr Val His Ala Leu Ala His Gln Leu Gly Gly             260 265 270 Phe Tyr His Leu Pro His Gly Val Cys Asn Ala Val Leu Leu Pro His         275 280 285 Val Gln Glu Ala Asn Met Gln Cys Pro Lys Ala Lys Lys Arg Leu Gly     290 295 300 Glu Ile Ala Leu His Cys Gly Ala Ser Gln Glu Asp Pro Glu Glu Thr 305 310 315 320 Ile Lys Ala Leu His Val Leu Asn Arg Thr Met Asn Ile Pro Arg Asn                 325 330 335 Leu Lys Asp Leu Gly Val Lys Thr Glu Asp Phe Asp Ile Leu Ala Glu             340 345 350 His Ala Met His Asp Ala Cys His Leu Thr Asn Pro Val Gln Phe Thr         355 360 365 Lys Glu Gln Val Val Ala Ile Ile Lys Lys Ala Tyr Glu Tyr     370 375 380 <210> 11 <211> 1056 <212> DNA <213> Saccharomyces cerevisiae ADH5 <400> 11 atgccttcgc aagtcattcc tgaaaaacaa aaggctattg tcttttatga gacagatgga 60 aaattggaat ataaagacgt cacagttccg gaacctaagc ctaacgaaat tttagtccac 120 gttaaatatt ctggtgtttg tcatagtgac ttgcacgcgt ggcacggtga ttggccattt 180 caattgaaat ttccattaat cggtggtcac gaaggtgctg gtgttgttgt taagttggga 240 tctaacgtta agggctggaa agtcggtgat tttgcaggta taaaatggtt gaatgggact 300 tgcatgtcct gtgaatattg tgaagtaggt aatgaatctc aatgtcctta tttggatggt 360 actggcttca cacatgatgg tacttttcaa gaatacgcaa ctgccgatgc cgttcaagct 420 gcccatattc caccaaacgt caatcttgct gaagttgccc caatcttgtg tgcaggtatc 480 actgtttata aggcgttgaa aagagccaat gtgataccag gccaatgggt cactatatcc 540 ggtgcatgcg gtggcttggg ttctctggca atccaatacg cccttgctat gggttacagg 600 gtcattggta tcgatggtgg taatgccaag cgaaagttat ttgaacaatt aggcggagaa 660 atattcatcg atttcacgga agaaaaagac attgttggtg ctataataaa ggccactaat 720 gt; aggtattgta ggcccaatgg tactgtcgtc ctggttggta tgccagctca tgcttactgc 840 aattccgatg ttttcaatca agttgtaaaa tcaatctcca tcgttggatc ttgtgttgga 900 aatagagctg atacaaggga ggctttagat ttcttcgcca gaggtttgat caaatctccg 960 atccacttag ctggcctatc ggatgttcct gaaatttttg caaagatgga gaagggtgaa 1020 attgttggta gatatgttgt tgagacttct aaatga 1056 <210> 12 <211> 351 <212> PRT <213> Saccharomyces cerevisiae ADH5 <400> 12 Met Pro Ser Gln Val Ile Pro Glu Lys Gln Lys Ala Ile Val Phe Tyr   1 5 10 15 Glu Thr Asp Gly Lys Leu Glu Tyr Lys Asp Val Thr Val Pro Glu Pro              20 25 30 Lys Pro Asn Glu Ile Leu Val His Val Lys Tyr Ser Gly Val Cys His          35 40 45 Ser Asp Leu His Ala Trp His Gly Asp Trp Pro Phe Gln Leu Lys Phe      50 55 60 Pro Leu Ile Gly Gly His Glu Gly Ala Gly Val Val Val Lys Leu Gly  65 70 75 80 Ser Asn Val Lys Gly Trp Lys Val Gly Asp Phe Ala Gly Ile Lys Trp                  85 90 95 Leu Asn Gly Thr Cys Met Ser Cys Glu Tyr Cys Glu Val Gly Asn Glu             100 105 110 Ser Gln Cys Pro Tyr Leu Asp Gly Thr Gly Phe Thr His Asp Gly Thr         115 120 125 Phe Gln Glu Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro     130 135 140 Pro Asn Val Asn Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile 145 150 155 160 Thr Val Tyr Lys Ala Leu Lys Arg Ala Asn Val Ile Pro Gly Gln Trp                 165 170 175 Val Thr Ile Ser Gly Ala Cys Gly Gly Leu Gly Ser Leu Ala Ile Gln             180 185 190 Tyr Ala Leu Ala Met Gly Tyr Arg Val Ile Gly Ile Asp Gly Gly Asn         195 200 205 Ala Lys Arg Lys Leu Phe Glu Gln Leu Gly Gly Glu Ile Phe Ile Asp     210 215 220 Phe Thr Glu Glu Lys Asp Ile Val Gly Ala Ile Ile Lys Ala Thr Asn 225 230 235 240 Gly Gly Ser His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile                 245 250 255 Glu Ala Ser Thr Arg Tyr Cys Arg Pro Asn Gly Thr Val Val Leu Val             260 265 270 Gly Met Pro Ala His Ala Tyr Cys Asn Ser Asp Val Phe Asn Gln Val         275 280 285 Val Lys Ser Ile Ser Ile Val Gly Ser Cys Val Gly Asn Arg Ala Asp     290 295 300 Thr Arg Glu Ala Leu Asp Phe Phe Ala Arg Gly Leu Ile Lys Ser Pro 305 310 315 320 Ile His Leu Ala Gly Leu Ser Asp Val Pro Glu Ile Phe Ala Lys Met                 325 330 335 Glu Lys Gly Glu Ile Val Gly Arg Tyr Val Val Glu Thr Ser Lys             340 345 350 <210> 13 <211> 1176 <212> DNA <213> Saccharomyces cerevisiae GPD1 <400> 13 atgtctgctg ctgctgatag attaaactta acttccggcc acttgaatgc tggtagaaag 60 agaagttcct cttctgtttc tttgaaggct gccgaaaagc ctttcaaggt tactgtgatt 120 ggatctggta actggggtac tactattgcc aaggtggttg ccgaaaattg taagggatac 180 ccagaagttt tcgctccaat agtacaaatg tgggtgttcg aagaagagat caatggtgaa 240 aaattgactg aaatcataaa tactagacat caaaacgtga aatacttgcc tggcatcact 300 ctacccgaca atttggttgc taatccagac ttgattgatt cagtcaagga tgtcgacatc 360 atcgttttca acattccaca tcaatttttg ccccgtatct gtagccaatt gaaaggtcat 420 gttgattcac acgtcagagc tatctcctgt ctaaagggtt ttgaagttgg tgctaaaggt 480 gtccaattgc tatcctctta catcactgag gaactaggta ttcaatgtgg tgctctatct 540 ggtgctaaca ttgccaccga agtcgctcaa gaacactggt ctgaaacaac agttgcttac 600 cacattccaa aggatttcag aggcgagggc aaggacgtcg accataaggt tctaaaggcc 660 ttgttccaca gaccttactt ccacgttagt gtcatcgaag atgttgctgg tatctccatc 720 tgtggtgctt tgaagaacgt tgttgcctta ggttgtggtt tcgtcgaagg tctaggctgg 780 ggtaacaacg cttctgctgc catccaaaga gtcggtttgg gtgagatcat cagattcggt 840 caaatgtttt tcccagaatc tagagaagaa acatactacc aagagtctgc tggtgttgct 900 gatttgatca ccacctgcgc tggtggtaga aacgtcaagg ttgctaggct aatggctact 960 tctggtaagg acgcctggga atgtgaaaag gagttgttga atggccaatc cgctcaaggt 1020 ttaattacct gcaaagaagt tcacgaatgg ttggaaacat gtggctctgt cgaagacttc 1080 ccattatttg aagccgtata ccaaatcgtt tacaacaact acccaatgaa gaacctgccg 1140 gacatgattg aagaattaga tctacatgaa gattag 1176 <210> 14 <211> 391 <212> PRT <213> Saccharomyces cerevisiae GPD1 <400> 14 Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn   1 5 10 15 Ala Gly Arg Lys Arg Ser Ser Ser Val Ser Leu Lys Ala Ala Glu              20 25 30 Lys Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr          35 40 45 Ile Ala Lys Val Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe      50 55 60 Ala Pro Ile Val Gln Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu  65 70 75 80 Lys Leu Thr Glu Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr Leu                  85 90 95 Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile             100 105 110 Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gln         115 120 125 Phe Leu Pro Arg Ile Cys Ser Gln Leu Lys Gly His Val Asp Ser His     130 135 140 Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly 145 150 155 160 Val Gln Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gln Cys                 165 170 175 Gly Ala Leu Ser Gly Ala Asn Ile Ala Thr Glu Val Ala Gln Glu His             180 185 190 Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys Asp Phe Arg Gly         195 200 205 Glu Gly Lys Asp Val Asp His Lys Val Leu Lys Ala Leu Phe His Arg     210 215 220 Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile 225 230 235 240 Cys Gly Ala Leu Lys Asn Val Val Ala Leu Gly Cys Gly Phe Val Glu                 245 250 255 Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Val Gly             260 265 270 Leu Gly Glu Ile Ile Arg Phe Gly Gln Met Phe Phe Pro Glu Ser Arg         275 280 285 Glu Glu Thr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile Thr     290 295 300 Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr 305 310 315 320 Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gln                 325 330 335 Ser Ala Gln Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu             340 345 350 Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val Tyr Gln         355 360 365 Ile Val Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Asp Met Ile Glu     370 375 380 Glu Leu Asp Leu His Glu Asp 385 390 <210> 15 <211> 1323 <212> DNA <213> Saccharomyces cerevisiae GPD2 <400> 15 atgcttgctg tcagaagatt aacaagatac acattcctta agcgaacgca tccggtgtta 60 tatactcgtc gtgcatataa aattttgcct tcaagatcta ctttcctaag aagatcatta 120 ttacaaacac aactgcactc aaagatgact gctcatacta atatcaaaca gcacaaacac 180 tgtcatgagg accatcctat cagaagatcg gactctgccg tgtcaattgt acatttgaaa 240 cgtgcgccct tcaaggttac agtgattggt tctggtaact gggggaccac catcgccaaa 300 gtcattgcgg aaaacacaga attgcattcc catatcttcg agccagaggt gagaatgtgg 360 gtttttgatg aaaagatcgg cgacgaaaat ctgacggata tcataaatac aagacaccag 420 aacgttaaat atctacccaa tattgacctg ccccataatc tagtggccga tcctgatctt 480 ttacactcca tcaagggtgc tgacatcctt gttttcaaca tccctcatca atttttacca 540 aacatagtca aacaattgca aggccacgtg gcccctcatg taagggccat ctcgtgtcta 600 aaagggttcg agttgggctc caagggtgtg caattgctat cctcctatgt tactgatgag 660 ttaggaatcc aatgtggcgc actatctggt gcaaacttgg caccggaagt ggccaaggag 720 cattggtccg aaaccaccgt ggcttaccaa ctaccaaagg attatcaagg tgatggcaag 780 gatgtagatc ataagatttt gaaattgctg ttccacagac cttacttcca cgtcaatgtc 840 atcgatgatg ttgctggtat atccattgcc ggtgccttga agaacgtcgt ggcacttgca 900 tgtggtttcg tagaaggtat gggatggggt aacaatgcct ccgcagccat tcaaaggctg 960 ggtttaggtg aaattatcaa gttcggtaga atgtttttcc cagaatccaa agtcgagacc 1020 tactatcaag aatccgctgg tgttgcagat ctgatcacca cctgctcagg cggtagaaac 1080 gtcaaggttg ccacatacat ggccaagacc ggtaagtcag ccttggaagc agaaaaggaa 1140 ttgcttaacg gtcaatccgc ccaagggata atcacatgca gagaagttca cgagtggcta 1200 caaacatgtg agttgaccca agaattccca ttattcgagg cagtctacca gatagtctac 1260 aacaacgtcc gcatggaaga cctaccggag atgattgaag agctagacat cgatgacgaa 1320 tag 1323 <210> 16 <211> 440 <212> PRT <213> Saccharomyces cerevisiae GPD2 <400> 16 Met Leu Ala Val Arg Arg Leu Thr Arg Tyr Thr Phe Leu Lys Arg Thr   1 5 10 15 His Pro Val Leu Tyr Thr Arg Arg Ala Tyr Lys Ile Leu Pro Ser Arg              20 25 30 Ser Thr Phe Leu Arg Arg Ser Leu Leu Gln Thr Gln Leu His Ser Lys          35 40 45 Met Thr Ala His Thr Asn Ile Lys Gln His Lys His Cys His Glu Asp      50 55 60 His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys  65 70 75 80 Arg Ala Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr                  85 90 95 Thr Ile Ala Lys Val Ile Ala Glu Asn Thr Glu Leu His Ser His Ile             100 105 110 Phe Glu Pro Glu Val Arg Met Trp Val Phe Asp Glu Lys Ile Gly Asp         115 120 125 Glu Asn Leu Thr Asp Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr     130 135 140 Leu Pro Asn Ile Asp Leu Pro His Asn Leu Val Ala Asp Pro Asp Leu 145 150 155 160 Leu His Ser Ile Lys Gly Ala Asp Ile Leu Val Phe Asn Ile Pro His                 165 170 175 Gln Phe Leu Pro Asn Ile Val Lys Gln Leu Gln Gly His Val Ala Pro             180 185 190 His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys         195 200 205 Gly Val Gln Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly Ile Gln     210 215 220 Cys Gly Ala Leu Ser Gly Ala Asn Leu Ala Pro Glu Val Ala Lys Glu 225 230 235 240 His Trp Ser Glu Thr Thr Val Ala Tyr Gln Leu Pro Lys Asp Tyr Gln                 245 250 255 Gly Asp Gly Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His             260 265 270 Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser         275 280 285 Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val     290 295 300 Glu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Leu 305 310 315 320 Gly Leu Gly Glu Ile Ile Lys Phe Gly Arg Met Phe Phe Pro Glu Ser                 325 330 335 Lys Val Glu Thr Tyr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile             340 345 350 Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala         355 360 365 Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu Leu Leu Asn Gly     370 375 380 Gln Ser Ala Gln Gly Ile Ile Thr Cys Arg Glu Val His Glu Trp Leu 385 390 395 400 Gln Thr Cys Glu Leu Thr Gln Glu Phe Pro Leu Phe Glu Ala Val Tyr                 405 410 415 Gln Ile Val Tyr Asn Asn Val Arg Met Glu Asp Leu Pro Glu Met Ile             420 425 430 Glu Glu Leu Asp Ile Asp Asp Glu         435 440 <210> 17 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH1 deletion <400> 17 ttcaagctat accaagcata caatcaacta tctcatatac acagctgaag cttcgtacgc 60                                                                           60 <210> 18 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH1 deletion <400> 18 cttatttaat aataaaaatc ataaatcata agaaattcgc gcataggcca ctagtggat 59 <210> 19 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH2 deletion <400> 19 tacaatcaac tatcaactat taactatatc gtaatacaca cagctgaagc ttcgtacgc 59 <210> 20 <211> 59 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for ADH2 deletion <400> 20 ataatgaaaa ctataaatcg taaagacata agagatccgc gcataggcca ctagtggat 59 <210> 21 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH3 deletion <400> 21 gttaaaacta ggaatagtat agtcataagt taacaccatc cagctgaagc ttcgtacgc 59 <210> 22 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH3 deletion <400> 22 acaaagactt tcataaaaag tttgggtgcg taacacgcta gcataggcca ctagtggat 59 <210> 23 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH4 deletion <400> 23 caagtttaca tttgcaacaa ctaatagtca aataagaaaa cagctgaagc ttcgtacgc 59 <210> 24 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH4 deletion <400> 24 gcacacgcat aattgacgtt tatgagttcg ttcgattttt gcataggcca ctagtggat 59 <210> 25 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH5 deletion <400> 25 agaaaattat ttaactacat atctacaaaa tcaaagcatc cagctgaagc ttcgtacgc 59 <210> 26 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH5 deletion <400> 26 taaaaagtaa aaatatattc atcaaattcg ttacaaaaga gcataggcca ctagtggat 59 <210> 27 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD1 deletion <400> 27 cacccccccc ctccacaaac acaaatattg ataatataaa gcagctgaag cttcgtacgc 60                                                                           60 <210> 28 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD1 deletion <400> 28 aagtggggga aagtatgata tgttatcttt ctccaataaa tgcataggcc actagtggat 60                                                                           60 <210> 29 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD2 deletion <400> 29 tctctttccc tttccttttc cttcgctccc cttccttatc acagctgaag cttcgtacgc 60                                                                           60 <210> 30 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD2 deletion <400> 30 ggcaacagga aagatcagag ggggaggggg ggggagagtg tgcataggcc actagtggat 60                                                                           60 <210> 31 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GSF2 deletion <400> 31 gagatcggtg gactttgttt tgatagaggg cgattgcaag ccagctgaag cttcgtacgc 60                                                                           60 <210> 32 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GSF2 deletion <400> 32 aataaaaaaa aagtctggat ggtagtgttt tggttttaca agcataggcc actagtggat 60                                                                           60 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH1 deletion <400> 33 caccatatcc gcaatgac 18 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH1 deletion <400> 34 gtgttgtcct ctgaggac 18 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH2 deletion <400> 35 accgggcatc tccaactt 18 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of ADH2 deletion <400> 36 ccatgtctac agtttagagg 20 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH3 deletion <400> 37 atgagcagca gccattttg 19 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of ADH3 deletion <400> 38 tgatggtgat aatgtctctc a 21 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH4 deletion <400> 39 aagaactagt ttttagttcg cg 22 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH4 deletion <400> 40 agaacttccg ttcttctttt 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH5 deletion <400> 41 ctgctatctg cttgtagaag 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of ADH5 deletion <400> 42 gaaacgtttg tataggttgt 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD1 deletion <400> 43 cgccttgctt ctctcccctt 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD1 deletion <400> 44 ccgacagcct ctgaatgagt 20 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD2 deletion <400> 45 tacggaccta ttgccattgt 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD2 deletion <400> 46 ttaagggcta tagataacag 20 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GSF2 deletion <400> 47 ggtcactcct tgttcttct 19 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GSF2 deletion <400> 48 agtagattcg tgaggaattg 20 <210> 49 <211> 1764 <212> DNA <213> Saccharomyces cerevisiae DLD1 <400> 49 atgttgtgga agcgtacttg cacaaggcta ataaagccta ttgcacaacc tagaggaagg 60 ctggtgagaa gatcatgcta cagatacgcc tcaacaggca caggcagcac cgacagcagc 120 agccagtggt taaaatactc tgtcatcgcc tcttcagcta ctctattcgg ttatttgttc 180 gctaagaacc tctattctag ggagactaag gaagatttga tagagaagct ggaaatggtc 240 aaaaagatcg acccagtaaa ttctacgtta aagctgtcct cattggactc accagactat 300 ttgcacgacc cggttaagat cgataaggtt gttgaggacc tgaagcaggt gctgggaaac 360 aagcctgaaa actactctga tgcgaaatcc gatttggacg cccattcaga tacctacttc 420 aacacgcatc acccctctcc cgagcaaaga cctaggatta tattattccc tcatactacc 480 gaagaagttt ccaaaatttt gaaaatatgt cacgataaca acatgccagt tgtacccttc 540 tcgggcggaa cgtccttgga ggggcacttc ctgcctacaa gaattggaga taccataacc 600 gtagacctgt ccaagtttat gaataacgtc gtaaaatttg acaagctgga cctggacatc 660 accgtgcagg ccggtctacc ctgggaggat ttgaatgact atttgagcga ccacggtttg 720 atgtttggct gtgaccctgg tccaggtgca cagattggtg gttgcattgc taattcttgt 780 tcaggaacca acgcctaccg ttacggtacc atgaaggaga atattataaa catgactata 840 gtgttgccgg acgggaccat tgtcaagacg aagaaaagac ccagaaagtc gagcgctggc 900 tataacttaa atggtttatt tgtgggaagt gaaggtacct taggtattgt tactgaagct 960 actgtcaagt gtcatgtcaa gcccaaagct gaaactgttg cggtggtatc ctttgatact 1020 atcaaggatg cggccgcatg tgcttctaat ctgactcaga gtggtattca tttgaacgcc 1080 atggagttac tggatgaaaa tatgatgaag ttgatcaacg catctgaatc cacggacaga 1140 tgtgattggg tagagaaacc aactatgttt ttcaagattg gtgggagatc tcccaacatt 1200 gtcaatgctc ttgtggatga agttaaggct gtcgcccagt taaatcactg caacagtttt 1260 cagtttgcta aagatgatga cgaaaaattg gaattatggg aagctagaaa ggtcgcgcta 1320 tggtctgtgc tagacgctga taagagcaaa gacaaatcag ctaaaatttg gacaactgat 1380 gtagctgttc ctgtgtcgca gttcgacaag gttattcacg aaactaaaaa ggacatgcaa 1440 gctagtaagc tgatcaacgc cattgttggt catgcaggtg atggtaactt ccatgcattc 1500 atcgtctaca gaacccctga agaacacgaa acctgtagcc aacttgttga cagaatggtc 1560 aagagagcac tgaacgcaga aggcacttgc acgggtgaac acggtgttgg tattggtaaa 1620 agagagtact tgctcgaaga attaggtgaa gcacccgtcg atttgatgag aaagattaag 1680 ctagctattg acccaaagag aatcatgaac ccggacaaaa tctttaaaac tgatccaaac 1740 gagcccgcta atgattacagta gtga 1764 <210> 50 <211> 587 <212> PRT <213> Saccharomyces cerevisiae DLD1 <400> 50 Met Leu Trp Lys Arg Thr Cys Thr Arg Leu Ile Lys Pro Ile Ala Gln   1 5 10 15 Pro Arg Gly Arg Leu Val Arg Arg Ser Cys Tyr Arg Tyr Ala Ser Thr              20 25 30 Gly Thr Gly Ser Thr Asp Ser Ser Ser Gln Trp Leu Lys Tyr Ser Val          35 40 45 Ile Ala Ser Ala Thr Leu Phe Gly Tyr Leu Phe Ala Lys Asn Leu      50 55 60 Tyr Ser Arg Glu Thr Lys Glu Asp Leu Ile Glu Lys Leu Glu Met Val  65 70 75 80 Lys Lys Ile Asp Pro Val Asn Ser Thr Leu Lys Leu Ser Ser Leu Asp                  85 90 95 Ser Pro Asp Tyr Leu His Asp Pro Val Lys Ile Asp Lys Val Val Glu             100 105 110 Asp Leu Lys Gln Val Leu Gly Asn Lys Pro Glu Asn Tyr Ser Asp Ala         115 120 125 Lys Ser Asp Leu Asp Ala His Ser Asp Thr Tyr Phe Asn Thr His His     130 135 140 Pro Ser Pro Glu Gln Arg Pro Ile Ile Leu Phe Pro His Thr Thr 145 150 155 160 Glu Glu Val Ser Lys Ile Leu Lys Ile Cys His Asp Asn Asn Met Pro                 165 170 175 Val Val Pro Phe Ser Gly Gly Thr Ser Leu Glu Gly His Phe Leu Pro             180 185 190 Thr Arg Ile Gly Asp Thr Ile Thr Val Asp Leu Ser Lys Phe Met Asn         195 200 205 Asn Val Val Lys Phe Asp Lys Leu Asp Leu Asp Ile Thr Val Gln Ala     210 215 220 Gly Leu Pro Trp Glu Asp Leu Asn Asp Tyr Leu Ser Asp His Gly Leu 225 230 235 240 Met Phe Gly Cys Asp Pro Gly Pro Gly Ala Gln Ile Gly Gly Cys Ile                 245 250 255 Ala Asn Ser Cys Ser Gly Thr Asn Ala Tyr Arg Tyr Gly Thr Met Lys             260 265 270 Glu Asn Ile Asn Met Thr Ile Val Leu Pro Asp Gly Thr Ile Val         275 280 285 Lys Thr Lys Lys Arg Pro Arg Lys Ser Ser Ala Gly Tyr Asn Leu Asn     290 295 300 Gly Leu Phe Val Gly Ser Glu Gly Thr Leu Gly Ile Val Thr Glu Ala 305 310 315 320 Thr Val Lys Cys His Val Lys Pro Lys Ala Glu Thr Val Ala Val Val                 325 330 335 Ser Phe Asp Thr Ile Lys Asp Ala Ala Cys Ala Ser Asn Leu Thr             340 345 350 Gln Ser Gly Ile His Leu Asn Ala Met Glu Leu Leu Asp Glu Asn Met         355 360 365 Met Lys Leu Ile Asn Ala Ser Glu Ser Thr Asp Arg Cys Asp Trp Val     370 375 380 Glu Lys Pro Thr Met Phe Phe Lys Ile Gly Gly Arg Ser Pro Asn Ile 385 390 395 400 Val Asn Ala Leu Val Asp Glu Val Lys Ala Val Ala Gln Leu Asn His                 405 410 415 Cys Asn Ser Phe Gln Phe Ala Lys Asp Asp Asp Glu Lys Leu Glu Leu             420 425 430 Trp Glu Ala Arg Lys Val Ala Leu Trp Ser Val Leu Asp Ala Asp Lys         435 440 445 Ser Lys Asp Lys Ser Ala Lys Ile Trp Thr Thr Asp Val Ala Val Pro     450 455 460 Val Ser Gln Phe Asp Lys Val Ile His Glu Thr Lys Lys Asp Met Gln 465 470 475 480 Ala Ser Lys Leu Ile Asn Ala Ile Val Gly His Ala Gly Asp Gly Asn                 485 490 495 Phe His Ala Phe Ile Val Tyr Arg Thr Pro Glu Glu His Glu Thr Cys             500 505 510 Ser Gln Leu Val Asp Arg Met Val Lys Arg Ala Leu Asn Ala Glu Gly         515 520 525 Thr Cys Thr Gly Glu His Gly Val Gly Ile Gly Lys Arg Glu Tyr Leu     530 535 540 Leu Glu Glu Leu Gly Glu Ala Pro Val Asp Leu Met Arg Lys Ile Lys 545 550 555 560 Leu Ala Ile Asp Pro Lys Arg Ile Met Asn Pro Asp Lys Ile Phe Lys                 565 570 575 Thr Asp Pro Asn Glu Pro Ala Asn Asp Tyr Arg             580 585 <210> 51 <211> 1851 <212> DNA <213> Saccharomyces cerevisiae JEN1 <400> 51 atgtcgtcgt caattacaga tgagaaaata tctggtgaac agcaacaacc tgctggcaga 60 aaactatact ataacacaag tacatttgca gagcctcctc tagtggacgg agaaggtaac 120 cctataaatt atgagccgga agtttacaac ccggatcacg aaaagctata ccataaccca 180 tcactgcctg cacaatcaat tcaggataca agagatgatg aattgctgga aagagtttat 240 agccaggatc aaggtgtaga gtatgaggaa gatgaagagg ataagccaaa cctaagcgct 300 gcgtccatta aaagttatgc tttaacgaga tttacgtcct tactgcacat ccacgagttt 360 tcttgggaga atgtcaatcc catacccgaa ctgcgcaaaa tgacatggca gaattggaac 420 tattttttta tgggttattt tgcgtggttg tctgcggctt gggccttctt ttgcgtttca 480 gtatcagtcg ctccattggc tgaactatat gacagaccaa ccaaggacat cacctggggg 540 ttgggattgg tgttatttgt tcgttcagca ggtgctgtca tatttggttt atggacagat 600 aagtcttcca gaaagtggcc gtacattaca tgtttgttct tatttgtcat tgcacaactc 660 tgtactccat ggtgtgacac atacgagaaa tttctgggcg taaggtggat aaccggtatt 720 gctatgggag gaatttacgg atgtgcttct gcaacagcga ttgaagatgc acctgtgaaa 780 gcacgttcgt tcctatcagg tctatttttt tctgcttacg ctatggggtt catatttgct 840 atcatttttt acagagcctt tggctacttt agggatgatg gctggaaaat attgttttgg 900 tttagtattt ttctaccaat tctactaatt ttctggagat tgttatggcc tgaaacgaaa 960 tacttcacca aggttttgaa agcccgtaaa ttaatattga gtgacgcagt gaaagctaat 1020 ggtggcgagc ctctaccaaa agccaacttt aaacaaaaga tggtatccat gaagagaaca 1080 gttcaaaagt actggttgtt gttcgcatat ttggttgttt tattggtggg tccaaattac 1140 ttgactcatg cttctcaaga cttgttgcca accatgctgc gtgcccaatt aggcctatcc 1200 aaggatgctg tcactgtcat tgtagtggtt accaacatcg gtgctatttg tgggggtatg 1260 atatttggac agttcatgga agttactgga agaagattag gcctattgat tgcatgcaca 1320 atgggtggtt gcttcaccta ccctgcattt atgttgagaa gcgaaaaggc tatattaggt 1380 gccggtttca tgttatattt ttgtgtcttt ggtgtctggg gtatcctgcc cattcacctt 1440 gcagagttgg cccctgctga tgcaagggct ttggttgccg gtttatctta ccagctaggt 1500 aatctagctt ctgcagcggc ttccacgatt gagacacagt tagctgatag atacccatta 1560 gaaagagatg cctctggtgc tgtgattaaa gaagattatg ccaaagttat ggctatcttg 1620 actggttctg ttttcatctt cacatttgct tgtgtttttg ttggccatga gaaattccat 1680 cgtgatttgt cctctcctgt tatgaagaaa tatataaacc aagtggaaga atacgaagcc 1740 gatggtcttt cgattagtga cattgttgaa caaaagacgg aatgtgcttc agtgaagatg 1800 attgattcga acgtctcaaa gacatatgag gagcatattg agaccgttta a 1851 <210> 52 <211> 616 <212> PRT <213> Saccharomyces cerevisiae JEN1 <400> 52 Met Ser Ser Ile Thr Asp Glu Lys Ile Ser Gly Glu Gln Gln Gln   1 5 10 15 Pro Ala Gly Arg Lys Leu Tyr Tyr Asn Thr Ser Thr Phe Ala Glu Pro              20 25 30 Pro Leu Val Asp Gly Glu Gly Asn Pro Ile Asn Tyr Glu Pro Glu Val          35 40 45 Tyr Asn Pro Asp His Glu Lys Leu Tyr His Asn Pro Ser Leu Pro Ala      50 55 60 Gln Ser Ile Gln Asp Thr Arg Asp Asp Glu Leu Leu Glu Arg Val Tyr  65 70 75 80 Ser Gln Asp Gln Gly Val Glu Tyr Glu Glu Asp Glu Glu Asp Lys Pro                  85 90 95 Asn Leu Ser Ala Ala Ser Ile Lys Ser Tyr Ala Leu Thr Arg Phe Thr             100 105 110 Ser Leu Leu His Ile His Glu Phe Ser Trp Glu Asn Val Asn Pro Ile         115 120 125 Pro Glu Leu Arg Lys Met Thr Trp Gln Asn Trp Asn Tyr Phe Phe Met     130 135 140 Gly Tyr Phe Ala Trp Leu Ser Ala Ala Trp Ala Phe Phe Cys Val Ser 145 150 155 160 Val Ser Val Ala Pro Leu Ala Glu Leu Tyr Asp Arg Pro Thr Lys Asp                 165 170 175 Ile Thr Trp Gly Leu Gly Leu Val Leu Phe Val Arg Ser Ala Gly Ala             180 185 190 Val Ile Phe Gly Leu Trp Thr Asp Lys Ser Ser Arg Lys Trp Pro Tyr         195 200 205 Ile Thr Cys Leu Phe Leu Phe Val Ile Ala Gln Leu Cys Thr Pro Trp     210 215 220 Cys Asp Thr Tyr Glu Lys Phe Leu Gly Val Arg Trp Ile Thr Gly Ile 225 230 235 240 Ala Met Gly Gly Ile Tyr Gly Cys Ala Ser Ala Thr Ala Ile Glu Asp                 245 250 255 Ala Pro Val Lys Ala Arg Ser Phe Leu Ser Gly Leu Phe Phe Ser Ala             260 265 270 Tyr Ala Met Gly Phe Ile Phe Ala Ile Ile Phe Tyr Arg Ala Phe Gly         275 280 285 Tyr Phe Arg Asp Asp Gly Trp Lys Ile Leu Phe Trp Phe Ser Ile Phe     290 295 300 Leu Pro Ile Leu Leu Ile Phe Trp Arg Leu Leu Trp Pro Glu Thr Lys 305 310 315 320 Tyr Phe Thr Lys Val Leu Lys Ala Arg Lys Leu Ile Leu Ser Asp Ala                 325 330 335 Val Lys Ala Asn Gly Gly Glu Pro Leu Pro Lys Ala Asn Phe Lys Gln             340 345 350 Lys Met Val Ser Met Lys Arg Thr Val Gln Lys Tyr Trp Leu Leu Phe         355 360 365 Ala Tyr Leu Val Val Leu Leu Val Gly Pro Asn Tyr Leu Thr His Ala     370 375 380 Ser Gln Asp Leu Leu Pro Thr Met Leu Arg Ala Gln Leu Gly Leu Ser 385 390 395 400 Lys Asp Ala Val Thr Val Ile Val Val Val Thr Asn Ile Gly Ala Ile                 405 410 415 Cys Gly Gly Met Ile Phe Gly Gln Phe Met Glu Val Thr Gly Arg Arg             420 425 430 Leu Gly Leu Leu Ile Ala Cys Thr Met Gly Gly Cys Phe Thr Tyr Pro         435 440 445 Ala Phe Met Leu Arg Ser Glu Lys Ala Ile Leu Gly Ala Gly Phe Met     450 455 460 Leu Tyr Phe Cys Val Phe Gly Val Trp Gly Ile Leu Pro Ile His Leu 465 470 475 480 Ala Glu Leu Ala Pro Ala Asp Ala Arg Ala Leu Val Ala Gly Leu Ser                 485 490 495 Tyr Gln Leu Gly Asn Leu Ala Ser Ala Ala Ser Thr Ile Glu Thr             500 505 510 Gln Leu Ala Asp Arg Tyr Pro Leu Glu Arg Asp Ala Ser Gly Ala Val         515 520 525 Ile Lys Glu Asp Tyr Ala Lys Val Met Ala Ile Leu Thr Gly Ser Val     530 535 540 Phe Ile Phe Thr Phe Ala Cys Val Phe Val Gly His Glu Lys Phe His 545 550 555 560 Arg Asp Leu Ser Ser Pro Val Met Lys Lys Tyr Ile Asn Gln Val Glu                 565 570 575 Glu Tyr Glu Ala Asp Gly Leu Ser Ile Ser Asp Ile Val Glu Gln Lys             580 585 590 Thr Glu Cys Ala Ser Val Lys Met Ile Asp Ser Asn Val Ser Lys Thr         595 600 605 Tyr Glu Glu His Ile Glu Thr Val     610 615 <210> 53 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for DLD1 deletion <400> 53 tgtacatcat tccgatccag ctggaaacaa aagcaagaac acagctgaag cttcgtacgc 60                                                                           60 <210> 54 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for DLD1 deletion <400> 54 tttcaggttt acgtgaaggg tgaaaaagga aaatcagata cgcataggcc actagtggat 60                                                                           60 <210> 55 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for JEN1 deletion <400> 55 aaagtttttc ctcaaagaga ttaaatactg ctactgaaaa tcagctgaag cttcgtacgc 60                                                                           60 <210> 56 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for JEN1 deletion <400> 56 tttcaggttt acgtgaaggg tgaaaaagga aaatcagata cgcataggcc actagtggat 60                                                                           60 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of DLD1 deletion <400> 57 tcttgtcaac ccaggtccgt 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of DLD1 deletion <400> 58 aggaagtgat gtaagctgct 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of JEN1 deletion <400> 59 aacggtcttt tgccccccct 20 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of JEN1 deletion <400> 60 cttgctagtg ttaacggc 18 <210> 61 <211> 996 <212> DNA <213> Leuconostoc mesenteroides ldhA <400> 61 atgaagattt ttgcttacgg cattcgtgat gatgaaaagc catcacttga agaatggaaa 60 gcggctaacc cagagattga agtggactac acacaagaat tattgacacc tgaaacagct 120 aagttggctg agggatcaga ttcagctgtt gtttatcaac aattggacta tacacgtgaa 180 acattgacag ctttagctaa cgttggtgtt actaacttgt cattgcgtaa cgttggtaca 240 gataacattg attttgatgc agcacgtgaa tttaacttta acatttcaaa tgttcctgtt 300 tattcaccaa atgctattgc agaacactca atgattcaat tatctcgttt gctacgtcgc 360 acgaaagcat tggatgccaa aattgctaag cacgacttgc gttgggcacc aacaattgga 420 cgtgaaatgc gtatgcaaac agttggtgtt attggtacag gtcatattgg ccgtgttgct 480 attaacattt tgaaaggctt tggggccaag gttattgctt atgacaagta cccaaatgct 540 gaattacaag cagaaggttt gtacgttgac acattagacg aattatatgc acaagctgat 600 gcaatttcat tgtatgttcc tggtgtacct gaaaaccatc atctaatcaa tgcagatgct 660 attgctaaga tgaaggatgg tgtggttatc atgaacgctg cgcgtggtaa tttgatggac 720 attgacgcta ttattgatgg tttgaattct ggtaagattt cagacttcgg tatggacgtt 780 tatgaaaatg aagttggctt gttcaatgaa gattggtctg gtaaagaatt cccagatgct 840 aagattgctg acttgattgc acgcgaaaat gtattggtta cgccacacac ggctttctat 900 acaactaaag ctgttctaga aatggttcac caatcatttg atgcagcagt tgctttcgcc 960 aagggtgaga agccagctat tgctgttgaa tattaa 996 <210> 62 <211> 331 <212> PRT <213> Leuconostoc mesenteroides ldhA <400> 62 Met Lys Ile Phe Ala Tyr Gly Ile Arg Asp Asp Glu Lys Pro Ser Leu   1 5 10 15 Glu Glu Trp Lys Ala Ala Asn Pro Glu Ile Glu Val Asp Tyr Thr Gln              20 25 30 Glu Leu Leu Thr Pro Glu Thr Ala Lys Leu Ala Glu Gly Ser Asp Ser          35 40 45 Ala Val Val Tyr Gln Gln Leu Asp Tyr Thr Arg Glu Thr Leu Thr Ala      50 55 60 Leu Ala Asn Val Gly Val Thr Asn Leu Ser Leu Arg Asn Val Gly Thr  65 70 75 80 Asp Asn Ile Asp Phe Asp Ala Ala Arg Glu Phe Asn Phe Asn Ile Ser                  85 90 95 Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ser Met Ile             100 105 110 Gln Leu Ser Arg Leu Leu Arg Arg Thr Lys Ala Leu Asp Ala Lys Ile         115 120 125 Ala Lys His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Met Arg     130 135 140 Met Gln Thr Val Gly Val Ile Gly Thr Gly His Ile Gly Arg Val Ala 145 150 155 160 Ile Asn Ile Leu Lys Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp Lys                 165 170 175 Tyr Pro Asn Ala Glu Leu Gln Ala Glu Gly Leu Tyr Val Asp Thr Leu             180 185 190 Asp Glu Leu Tyr Ala Gln Ala Asp Ala Ile Ser Leu Tyr Val Pro Gly         195 200 205 Val Pro Glu Asn His His Leu Ile Asn Ala Asp Ala Ile Ala Lys Met     210 215 220 Lys Asp Gly Val Val Ile Met Asn Ala Ala Arg Gly Asn Leu Met Asp 225 230 235 240 Ile Asp Ale Ile Ile Asp Gly Leu Asn Ser Gly Lys Ile Ser Asp Phe                 245 250 255 Gly Met Asp Val Tyr Glu Asn Glu Val Gly Leu Phe Asn Glu Asp Trp             260 265 270 Ser Gly Lys Glu Phe Pro Asp Ala Lys Ile Ala Asp Leu Ile Ala Arg         275 280 285 Glu Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr Lys Ala     290 295 300 Val Leu Glu Met Val His Gln Ser Phe Asp Ala Ala Val Ala Phe Ala 305 310 315 320 Lys Gly Glu Lys Pro Ala Ile Ala Val Glu Tyr                 325 330 <210> 63 <211> 1692 <212> DNA <213> Saccharomyces cerevisiae PDC1 <400> 63 atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60 accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120 gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180 tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300 gtcccatcca tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420 gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480 agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540 ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600 attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720 ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780 ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840 ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960 ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgaccaatat tgctgacgcc 1020 gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080 gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140 gt; ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260 gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440 cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500 actttcggtg ctaaggacta cgaaacccac agagtcgcta ccaccggtga atgggacaag 1560 ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga ggttatgttg 1620 ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680 gctaagcaat aa 1692 <210> 64 <211> 563 <212> PRT <213> Saccharomyces cerevisiae PDC1 <400> 64 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln   1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser              20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn          35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile      50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser  65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                  85 90 95 His Val Val Gly Val Ser Ser Ser Ser Ala Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val                 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn             180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val         195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg     210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His                 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val             260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr                 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Ala Arg             340 345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu         355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val     370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu                 485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val             500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn         515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp     530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F primer for ldhA <400> 65 ggcgggatcc atgaagattt ttgcttacgg 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for ldhA <400> 66 ggcgctgcag ttaatattca acagcaatag 30 <210> 67 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> TEF1 promoter <400> 67 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg 300 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360 ttgctcatta gaaagaaagc atagcaatct aatctaagtt t 401 <210> 68 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> CYC1 terminator <400> 68 aaaaagaatc atgattgaat gaagatatta tttttttgaa ttatattttt taaattttat 60 ataaagacat ggtttttctt ttcaactcaa ataaagattt ataagttact taaataacat 120 acattttata aggtattcta taaaaagagt attatgttat tgttaacctt tttgtctcca 180 attgtcgtca taacgatgag gtgttgcatt tttggaaacg agattgacat agagtcaaaa 240 tttgctaaat ttgatccctc ccatcgcaag ataatcttcc ctcaaggtta tcatgattat 300 caggatggcg aaaggatacg ctaaaaattc aataaaaaat tcaatataat tttcgtttcc 360 caagaactaa cttggaaggt tatacatggg tac 393 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Lm.ldhA_INT F <400> 69 ggcggctagc gggaacaaaa gctggagctc 30 <210> 70 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Lm.ldhA_INT R <400> 70 ggcggggccc gggggatgtg ctgcaagg 28 <210> 71 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for PDC1 deletion / lhdA insertion <400> 71 ctcataacct cacgcaaaat aacacagtca aatcaatcaa agggaacaaa agctggagct 60                                                                           60 <210> 72 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for PDC1 deletion / lhdA insertion <400> 72 aatgcttata aaactttaac taataattag agattaaatc gcataggcca ctagtggat 59 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of PDC1 deletion / lhdA insertion <400> 73 gacttttcgt gtgatgaggc 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> R primer for identification of PDC1 deletion / lhdA insertion <400> 74 tttacatgga ccgcaccaag 20

Claims (14)

모세포에 비하여 글루코오스 신호 인자(glucose signaling factor: GSF)의 활성이 감소되어 있는, 상기 모세포에 비하여 생장 속도가 증대된 유전적으로 조작된 효모 세포로서, 상기 모세포는 유전적 조작 전의 세포에 비하여 알코올 탈수소효소(alcohol dehydrogenase: ADH), 글리세롤-3-인산 탈수소효소(glycerol-3-phosphate dehydrogenase: GPD), 젖산 생산능을 갖지 않는 균주의 D형 젖산 탈수소효소(D-lactate dehydrogenase: DLD), 모노카복실레이트/수소 동시수송단백질(monocarboxylate/proton symporter: JEN), 또는 피루브산 탈탄산효소(pyruvate decarboxylase: PDC)의 활성이 감소되어 있는 것인, 효모 세포.
The present invention relates to genetically engineered yeast cells whose growth rate is increased as compared to the parent cells in which the activity of glucose signaling factor (GSF) is decreased as compared to the parent cells, (D-lactate dehydrogenase (DLD)), a monocarboxylic acid dehydrogenase (DLD), an alcohol dehydrogenase (ADH), a glycerol-3-phosphate dehydrogenase (GPD) / Hydrogen co-transport protein (monocarboxylate / proton symporter: JEN), or pyruvate decarboxylase (PDC).
청구항 1에 있어서, 상기 모세포는 유전적 조작 전의 세포에 비하여 젖산, 숙신산, 에탄올, 이소부탄올, 2,3-부탄다이올, 아세토인, 또는 그들의 조합의 생산이 증대되도록 유전적으로 조작된 효모 세포인 것인 효모 세포. The method according to claim 1, wherein the parent cell is a genetically engineered yeast cell that is increased in production of lactic acid, succinic acid, ethanol, isobutanol, 2,3-butanediol, acetone, Yeast cells. 삭제delete 청구항 1에 있어서, 상기 모세포는 유전적 조작 전의 세포에 비하여 아세토락테이트 신타아제(acetolactate synthase), 아세토락테이트 디카복실레이즈(acetolactate decarboxylase), 2,3-부탄다이올 탈수소효소, 젖산 생산능을 갖는 균주의 D형 젖산 탈수소효소, 또는 NADH 산화효소(NADH oxidase)의 활성이 증가되어 있는, 유전적으로 조작된 효모 세포인 것인 효모 세포. [2] The method of claim 1, wherein the parent cells are selected from the group consisting of acetolactate synthase, acetolactate decarboxylase, 2,3-butanediol dehydrogenase, Wherein the yeast cell is a genetically engineered yeast cell having increased activity of the D-type lactate dehydrogenase, or NADH oxidase, of the strains of interest. 청구항 1에 있어서, 상기 글루코오스 신호 인자는 글루코오스 신호 인자 1(glucose signaling factor 1: GSF1) 또는 글루코오스 신호 인자 2(glucose signaling factor 2: GSF2)인 것인 효모 세포. The yeast cell according to claim 1, wherein the glucose signaling factor is glucose signaling factor 1 (GSF1) or glucose signaling factor 2 (GSF2). 청구항 1에 있어서, 상기 효모 세포는 글루코오스 신호 인자를 코딩하는 유전자가 제거 또는 파괴된 것인 효모 세포. The yeast cell according to claim 1, wherein the yeast cell is one in which a gene coding for a glucose signaling factor is deleted or destroyed. 청구항 6에 있어서, 상기 글루코오스 신호 인자를 코딩하는 유전자는 서열번호 1의 뉴클레오티드 서열로 이루어지는 것인 효모 세포. 7. The yeast cell according to claim 6, wherein the gene coding for the glucose signaling factor is a nucleotide sequence of SEQ ID NO: 1. 청구항 1에 있어서, 상기 글루코오스 신호 인자는 서열번호 2의 아미노산 서열로 이루어지는 것인 효모 세포. 2. The yeast cell according to claim 1, wherein the glucose signaling factor comprises the amino acid sequence of SEQ ID NO: 2. 청구항 1에 있어서, 상기 효모 세포는 모세포에 비하여 포도당 소비량이 증대된 것인 효모 세포. The yeast cell according to claim 1, wherein the yeast cell has increased glucose consumption as compared to a parent cell. 청구항 1에 있어서, 상기 효모 세포는 모세포에 비하여 목적 물질의 생산이 증대된 것인 효모 세포. The yeast cell according to claim 1, wherein the yeast cell has increased production of a target substance as compared to a parent cell. 청구항 1에 있어서, 상기 효모 세포는 사카로마이세스(Saccharomyces) 속에 속하는 것인 효모 세포.The yeast cell according to claim 1, wherein the yeast cell belongs to the genus Saccharomyces . 청구항 11에 있어서, 상기 사카로마이세스속은 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 사카로마이세스 바야누스(S. bayanus), 사카로마이세스 파라독서스 (S. paradoxus), 사카로마이세스 미카테(S. mikatae), 및 사카로마이세스 쿠드리아브제비(S. kudriavzevii)로 이루어진 군으로부터 선택된 어느 하나인 효모 세포. The method according to claim 11, wherein the saccharide with three Levy jiae My process as MY access genus Saccharomyces (Saccharomyces S. cerevisiae , S. bayanus , S. paradoxus , S. mikatae , and Saccharomyces kudryavuzi ( S. cerevisiae ), S. bayanus , S. paradoxus , S. kudriavzevii ). 청구항 1, 2, 4 내지 12 중 어느 한 항의 효모 세포를 배지에서 배양하는 단계;및
배양된 효모 세포, 상기 효모 세포를 배양한 배지 또는 그의 조합으로부터 목적 물질을 분리하는 단계를 포함하는 목적 물질을 생산하는 방법.
Culturing the yeast cells of any one of claims 1, 2 and 4 to 12 in a medium;
Separating the target substance from the cultured yeast cells, the culture medium in which the yeast cells are cultured, or a combination thereof.
청구항 13에 있어서, 상기 목적 물질은 젖산, 숙신산, 에탄올, 이소부탄올, 2,3-부탄다이올, 또는 아세토인인 것인 방법. 14. The method of claim 13, wherein the target material is lactic acid, succinic acid, ethanol, isobutanol, 2,3-butanediol, or acetone.
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