KR20070096348A - Mutants having a producing ability of 1,4-butanediol and method for preparing 1,4-bdo using the same - Google Patents

Mutants having a producing ability of 1,4-butanediol and method for preparing 1,4-bdo using the same Download PDF

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KR20070096348A
KR20070096348A KR1020060026599A KR20060026599A KR20070096348A KR 20070096348 A KR20070096348 A KR 20070096348A KR 1020060026599 A KR1020060026599 A KR 1020060026599A KR 20060026599 A KR20060026599 A KR 20060026599A KR 20070096348 A KR20070096348 A KR 20070096348A
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gene encoding
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succinic acid
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박시재
이상엽
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주식회사 엘지화학
한국과학기술원
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Abstract

Mutants having highly producing ability of 1,4-butanediol and a method for producing 1,4-butanediol by using the same mutants are provided to mass produce 1,4-butanediol from microorganisms by inserting enzyme genes producing 1,4-butanediol from succinate and deleting a gene producing succinate from succinate semialdehyde, so that the 1,4-butandiol is importantly used in chemical industry. The mutants capable of producing a large quantity of 1,4-butanediol contain an inserted gene encoding an enzyme converting succinate into 4-hydroxybutyrate(4HB), an inserted gene encoding an enzyme converting the 4-hydroxybutyrate into 1,4-butanediol(1,4-BDO) and a deleted gene associated with conversion of succinate semialdehyde into succinate and include bacterium, yeast and fungus, wherein the gene encoding the enzyme converting succinate into 4-hydroxybutyrate is selected from Cat1(succinyl-CoA transferase) gene of SEQ ID NO:1, SucD(succinate semialdehyde dehydrogenase) gene SEQ ID NO:2, 4hbD(4-hydroxybutyrate dehydrogenase) gene SEQ ID NO:3 and GHB(4-hydroxybutyrate dehydrogenase) gene SEQ ID NO:4; the gene encoding the enzyme converting succinate into 4-hydroxybutyrate is Cat2(4-hydroxybutyrate-CoA transferase) gene of SEQ ID NO:5 or the gene encoding alcohol dehydrogenase reducing 4HB-CoA of SEQ ID NO:6 or SEQ ID NO:7; and the gene associated with conversion of succinate semialdehyde into succinate is GabD(succinic semialdehyde dehydrogenase) gene of SEQ ID NO:8.

Description

1,4―ｂｕｔａｎｅｄｉｏｌ〔１，４―ＢＤＯ〕생성능을 가지는 변이체 및 이를 이용한 １，４―ＢＤＯ의 제조방법 {Mutants Having a Producing Ability of 1,4―butanediol and Method for Preparing 1,4―BDO Using the Same}Mutants Having a Producing Ability of 1,4-butanediol and Method for Preparing 1,4-BDO Using the 1,4―BDO Using the Same}

도 1은 숙신산으로부터 4HB를 생산하는 경로를 나타낸 모식도이다.1 is a schematic diagram showing a route for producing 4HB from succinic acid.

도 2는 숙신산으로부터 4HB를 통하여 1,4-BDO를 생산하는 경로를 나타낸 모식도이다. Figure 2 is a schematic diagram showing a pathway for producing 1,4-BDO from succinic acid through 4HB.

본 발명은 1,4-BDO(1,4-butanediol) 생성능을 가지는 변이체 및 이를 이용한 1,４-BDO의 제조방법에 관한 것으로, 보다 구체적으로는, 숙신산 고생성능을 가지는 미생물에서, 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자와 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자가 도입 또는 증폭되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자가 결실되어 있는 것을 특징으로 하는 1,4-BDO 고생성능을 가지는 변이체 및 상기 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 1,4-BDO(1,4-butanediol)를 수득하는 것을 특징으로 하는 1,4-BDO의 제조방법에 관한 것이다.The present invention relates to a variant having 1,4-BDO (1,4-butanediol) generating ability and a method for preparing 1,4-BDO using the same, and more specifically, in a microorganism having high succinic acid, succinate (succinate) ) Genes of enzymes that convert 4HB to 4-hydroxybutyrate and enzymes that convert 4HB to 1,4-BDO (1,4-butanediol) are introduced or amplified, and succinate semialdehyde The 1,4-BDO high-performance variant characterized in that the gene involved in the conversion to succinic acid is deleted, and the variant was cultured in a medium containing carbohydrate, and then 1,4-BDO (1, It relates to a method for producing 1,4-BDO characterized in that 4-butanediol) is obtained.

생분해성 고분자 물질은 심각한 공해문제의 한 축을 이루는 합성고분자 소재를 대체할 수 있는 대안으로 제시되고 있으며, 이에 따라 다양한 생분해성 고분자 물질이 개발되고 있다. 그 중 하나인, 폴리-베타-하이드록시부탄산(poly-β-hydroxybutyrate)은 영양불균형 상태에서 다양한 미생물들이 축적하는 생분해성 고분자 물질로서 생분해성, 내습성, 압전성 그리고 생체 적합성 등 우수한 특성을 갖고 있다. 그 중, 4HB(4-hydroxybutyrate)는 폴리하이드록시알카노에이트(PHA)의 대표적인 예로, 폴리에스테르와 유사한 특성을 지니고 있어, 결정질의 플라스틱으로부터 고탄성의 고무에 이르기까지 폭넓은 물성을 나타내므로, 미생물 분해성 플라스틱으로서 많은 연구가 진행중이다. Biodegradable polymer materials have been suggested as an alternative to synthetic polymer materials, which constitute a major axis of the pollution problem. Accordingly, various biodegradable polymer materials have been developed. One of them, poly-β-hydroxybutyrate, is a biodegradable polymer that accumulates in various microorganisms under nutritional imbalance, and has excellent properties such as biodegradability, moisture resistance, piezoelectricity, and biocompatibility. have. Among them, 4HB (4-hydroxybutyrate) is a representative example of polyhydroxyalkanoate (PHA), and has similar characteristics to polyester, and exhibits a wide range of physical properties ranging from crystalline plastics to high elastic rubber. Much research is underway as degradable plastics.

또한, 4HB는 1,4-BDO(1,4-butanediol), gammabutyrolactone(GBL) 및 THF 등의 다양한 탄소수 4개의 화학물질로 쉽게 전환될 수 있다. 특히, 상기 1,4-BDO는 다양한 화학물질은 고분자, 솔벤트, 정밀화학 중간물질 등으로 화학산업 전반에 걸쳐 중요하게 쓰이고 있다. 현재 대부분의 탄소수 4개 화학물질은 1,4-butanediol, maleic anhydride 등으로부터 유래되어 합성되고 있지만, 유가가 올라감에 따라 생산비용이 증대되고 있어 화학생산공정을 보완 및 대체하는 공정의 개발이 요구되고 있는 실정이다. 이에, 상기 화학생산공정의 대안으로 생물학적 공정이 제시되고 있다. In addition, 4HB can be easily converted to various 4 carbon atoms such as 1,4-BDO (1,4-butanediol), gammabutyrolactone (GBL) and THF. In particular, the 1,4-BDO is a variety of chemicals are used throughout the chemical industry as a polymer, solvent, fine chemical intermediates. Currently, most of the four carbon-carbon chemicals are synthesized from 1,4-butanediol, maleic anhydride, etc., but as the oil price rises, the production cost is increasing, and development of a process to supplement and replace the chemical production process is required. There is a situation. Thus, a biological process has been proposed as an alternative to the chemical production process.

한편, 숙신산은 탄소수 4개의 dicarboxylic acid로서 미생물이 혐기조건에서 배양될 때 생산되는 유기산의 일종이다. 현재 다양한 미생물이 숙신산 생산균주로서 이용되어지고 있으며, 효율적인 발효공정 및 분리정제공정의 개발에 따라 생산가격이 낮아지고 있는 추세이다. 또한, 숙신산으로부터 4HB가 생성될 수 있으며, 일단 생성된 4HB는 다양한 탄소수 4개의 유기산이 유도될 수 있다. On the other hand, succinic acid is a dicarboxylic acid having 4 carbon atoms and is a kind of organic acid produced when microorganisms are cultured under anaerobic conditions. Currently, various microorganisms are used as succinic acid producing strains, and production prices are decreasing due to the development of efficient fermentation and separation and purification processes. In addition, 4HB can be generated from succinic acid, and once produced, 4HB can be derived from various organic acids having 4 carbon atoms.

숙신산의 생산 효율을 높이기 위한 방법과 관련된 대표적인 특허 출원으로는 본 출원인의 출원인 국제 특허 출원 공개번호 WO 2005/052135이 있고, 상기 특허 출원에서는 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생산하는 루멘 박테리아 변이균주 및 이를 이용한 숙신산의 제조방법에 대하여 개시하고 있다. 또한, 대한민국 특허출원 제 10-2004-60149호에서는, 숙신산을 고농도로 생산할 수 있는 대장균 변이체의 제조방법이 개시되어 있고, 대한민국 특허출원 10-2005-0076301호, 10-2005-0076317호 및 10-2005-0076348호에서는 신규 유전자를 이용한 숙신산의 제조방법에 대하여 개시되어 있다. Representative patent applications related to the method for increasing the production efficiency of succinic acid include the applicant's international patent application publication number WO 2005/052135, in which the lumen producing high concentration of succinic acid with little other organic acid produced. Disclosed is a bacterial strain and a method for producing succinic acid using the same. In addition, Korean Patent Application Nos. 10-2004-60149 disclose a method for producing E. coli variants capable of producing succinic acid at high concentrations, and Korean Patent Applications 10-2005-0076301, 10-2005-0076317, and 10- In 2005-0076348, a method for producing succinic acid using a novel gene is disclosed.

이에, 당업계에서는 상기 특허에 개시된 숙신산을 고농도로 생산할 수 있는 미생물을 이용하여, 화학산업 전반에 걸쳐 중요하게 사용되고 있는 탄소수 4개의 화학물질인 1,4-BDO(1,4-butanediol) 생성능을 가지는 변이체 및 이를 이용한 1,4-BDO의 생물학적 제조방법이 절실하게 요구되고 있는 실정이다. Therefore, in the art, by using a microorganism capable of producing a high concentration of succinic acid disclosed in the patent, it is possible to produce 1,4-BDO (1,4-butanediol), which is an important four-carbon chemical used throughout the chemical industry. Eggplant variants and a method for biological production of 1,4-BDO using the same situation is urgently needed.

이에 본 발명자들은 숙신산을 고농도로 생산할 수 있는 미생물을 이용하여 1,4-butanediol(1,4-BDO)를 제조하고자 예의 노력한 결과, 클로스트리디움(Clostridium) 균주로부터 유래된 4HB 생합성 관련 유전자 및 1,4-BDO 생합성 관련 유전자를 숙신산을 대량으로 생산할 수 있는 미생물에 도입시켜, 1,4-BDO 생성 미생물 변이체를 수득하고, 상기 미생물 변이체가 1,4-BDO를 효율적으로 생산하는 것을 확인하고 본 발명을 완성하게 되었다.Accordingly, the present inventors have made efforts to prepare 1,4-butanediol (1,4-BDO) using microorganisms capable of producing high concentrations of succinic acid. As a result, 4HB biosynthesis-related genes derived from Clostridium strain and 1 , 4-BDO biosynthesis-related genes were introduced into microorganisms capable of producing large amounts of succinic acid to obtain 1,4-BDO-producing microbial variants, which confirmed that the microbial variants efficiently produced 1,4-BDO. The invention was completed.

결국, 본 발명의 주된 목적은 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자 및 4HB를 1,4-BDO로 전환하는 효소의 유전자가 도입 또는 증폭되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자가 결실되어 있는 것을 특징으로 하는 1,4-BDO 고생성능을 가지는 변이체를 제공하는데 있다.As a result, the main object of the present invention is the introduction of a gene of an enzyme for converting succinate to 4HB (4-hydroxybutyrate) and a gene for an enzyme for converting 4HB to 1,4-BDO. To provide a variant having a high 1,4-BDO performance, characterized in that the gene involved in the conversion of succinate semialdehyde) to succinic acid is deleted.

본 발명의 다른 목적은 상기 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 1,4-BDO(4-butanediol)를 수득하는 것을 특징으로 하는 1,4-BDO의 제조방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing 1,4-BDO characterized in that after culturing the variant in a medium containing a carbohydrate, 1,4-BDO (4-butanediol) from the culture solution. have.

상기 목적을 달성하기 위하여, 본 발명은 숙신산 고생성능을 가지는 미생물에서, 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자와 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자가 도입 또는 증폭되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관 여하는 유전자가 결실되어 있는 것을 특징으로 하는 1,4-BDO 고생성능을 가지는 변이체를 제공한다. In order to achieve the above object, the present invention is a microorganism having high succinic acid performance, the gene of the enzyme to convert succinate (succinate) to 4HB (4-hydroxybutyrate) and 4HB 1,4-BDO (1,4-butanediol) It provides a variant having a high performance 1,4-BDO characterized in that the gene of the enzyme converting to or is introduced or amplified, and the gene involved in converting succinate semialdehyde into succinic acid is deleted. .

본 발명에 있어서, 상기 숙신산 고생성능을 가지는 미생물은 박테리아, 효모 및 곰팡이로 구성된 군에서 선택되는 것을 특징으로 할 수 있고, 상기 박테리아는 루멘박테리아, 코리네박테리움(Corynebacterium) 속, 브레비박테리움(Brevibacterium) 속 및 대장균으로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. In the present invention, the microorganism having high succinic acid performance may be selected from the group consisting of bacteria, yeast and fungi, and the bacteria are lumen bacteria, Corynebacterium genus, Brevibacterium ( Brevibacterium ) genus and E. coli may be selected from the group consisting of.

본 발명에 있어서, 상기 루멘박테리아는, 젖산 탈수소화효소를 코딩하는 유전자(ldhA)와 피루브산-개미산 분해효소를 코딩하는 유전자(pfl)가 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 할 수 있다. In the present invention, the lumen bacteria, a gene encoding lactic acid dehydrogenase ( ldhA ) and a gene encoding pyruvate-formic acid degrading enzyme ( pfl ) is deleted, and other organic acids are hardly produced under anaerobic conditions. It may be characterized by having the characteristic of producing succinic acid at high concentration.

본 발명에 있어서, 상기 루멘박테리아는, 젖산 탈수소화효소를 코딩하는 유전자(ldhA), 피루브산-개미산 분해효소를 코딩하는 유전자(pfl), 포스포트랜스아세틸화효소를 코딩하는 유전자(pta) 및 아세트산 키나제를 코딩하는 유전자(ackA)가 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 할 수 있다. In the present invention, the lumen bacterium, a gene encoding lactic acid dehydrogenase ( ldhA ), a gene encoding pyruvate-formic acid degrading enzyme ( pfl ), a gene encoding phosphotransacetylase ( pta ) and acetic acid The gene encoding the kinase ( ackA ) is deleted, and in anaerobic conditions, it can be characterized in that it has a characteristic of generating succinic acid at high concentration with little generation of other organic acids.

본 발명에 있어서, 상기 루멘박테리아는, 젖산 탈수소화효소를 코딩하는 유전자(ldhA), 피루브산-개미산 분해효소를 코딩하는 유전자(pfl) 및 포스포피루브산 카르복실라제를 코딩하는 유전자(ppc)가 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 할 수 있다. In the present invention, the lumen bacteria, the gene encoding lactic acid dehydrogenase ( ldhA ), the gene encoding pyruvate-formic acid degrading enzyme ( pfl ) and the gene encoding phosphopyruvic acid carboxylase ( ppc ) is deleted In the anaerobic condition, the organic acid may be characterized by having a high concentration of succinic acid with little generation of other organic acids.

본 발명에 있어서, 상기 루멘 박테리아는 맨하이미아 속(Mannheimia sp.), 액티노바실러스 속(Actinobacillus sp.) 및 언에어로바이오스피리륨 속(Anaerobiospirillum sp.)으로 구성된 군에서 선택되는 것을 특징으로 할 수 있으나, 이에 국한되는 것은 아니다. 상기 루멘 박테리아의 경우, 맨하이미아 속(Mannheimia sp.)인 것을 특징으로 할 수 있고, 바람직하게는, 맨하이미아 숙시니시프로듀슨스 MBEL55E(KCTC 0769BP), 맨하이미아 속 LPK(KCTC 10558BP), LPK4 및 LPK7(KCTC 10626BP)로 구성된 군에서 선택되는 것을 특징으로 할 수 있다. In the present invention, the lumen bacteria may be selected from the group consisting of the genus Mannheimia sp. , Actinobacillus sp. And genus Anaerobiospirillum sp. May be, but is not limited to. In the case of the lumen bacteria, it may be characterized by the genus Mannheimia sp. , And preferably, the succinsis producers MBEL55E (KCTC 0769BP) and the genus LPK (KCTC 10558BP) , LPK4 and LPK7 (KCTC 10626BP) may be selected from the group consisting of.

본 발명에 있어서, 상기 대장균은 포도당 인산전이효소를 코딩하는 유전자(ptsG) 및 피루베이트 키나아제를 코딩하는 유전자(pykA 및 pykF)가 모두 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 할 수 있고, 바람직하게는 상기 대장균 변이체는 W3110GFA인 것을 특징으로 할 수 있다. In the present invention, the E. coli is deleted both the gene encoding the glucose phosphatase ( ptsG ) and the gene encoding pyruvate kinase ( pykA and pykF ), while generating little other organic acid under anaerobic conditions It may be characterized by having a characteristic of producing a high concentration of succinic acid, preferably, the E. coli variant may be characterized as W3110GFA.

본 발명에 있어서, 상기 숙신산을 4HB로 전환하는 효소의 유전자와 상기 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자는 클로스트리디움 클루이베리(Clostridium kluyveri) 유래인 것을 특징으로 할 수 있고, 상기 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자는 클로스트리디움 아세토부틸리쿰(Clostridium acetobutylicum) 유래인 것을 특징으로 할 수 있다.In the present invention, the gene of the enzyme for converting the succinic acid to 4HB and the gene involved in converting the succinate semialdehyde to succinic acid may be characterized in that it is derived from Clostridium kluyveri . The gene of the enzyme for converting 4HB into 1,4-BDO (1,4-butanediol) may be characterized in that it is derived from Clostridium acetobutylicum .

본 발명에 있어서, 상기 숙신산을 4HB로 전환하는 효소의 유전자는 Cat1(succinyl-CoA transferase)를 코딩하는 유전자, SucD(succinate semialdehyde dehydrogenase)를 코딩하는 유전자, 4hbD(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자 및 GHB(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자로 구성된 군에서 선택되는 것을 특징으로 할 수 있고, 더욱 바람직하게는, 상기 Cat1를 코딩하는 유전자는 서열번호 1, SucD를 코딩하는 유전자는 서열번호 2, 4hbD를 코딩하는 유전자는 서열번호 3 및 GHB를 코딩하는 유전자는 서열번호 4의 염기서열을 가지는 것을 특징으로 할 수 있다.In the present invention, the gene for converting the succinic acid into 4HB is a gene encoding Cat1 (succinyl-CoA transferase), a gene encoding SucD (succinate semialdehyde dehydrogenase), a gene encoding 4hbD (4-hydroxybutyrate dehydrogenase) And GHB (4-hydroxybutyrate dehydrogenase) can be characterized in that it is selected from the group consisting of, more preferably, the gene encoding Cat1 is SEQ ID NO: 1, the gene encoding SucD is SEQ ID NO: 2 The gene encoding 4hbD may be characterized by having a nucleotide sequence of SEQ ID NO: 4 and a gene encoding GHB.

본 발명에 있어서, 상기 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자는 4-hydroxybutyrate-CoA transferase를 코딩하는 유전자 또는 4HB-CoA를 환원하는 alcohol dehydrogenase를 코딩하는 유전자인 것을 특징으로 할 수 있고, 더욱 바람직하게는, 상기 alcohol dehydrogenase는 클로스트리디움 아세토부틸리쿰(Clostridium acetobutylicum) 유래 butyl-CoA dehydrogenase인 것을 특징으로 할 수 있다. In the present invention, the gene of the enzyme for converting 4HB to 1,4-BDO (1,4-butanediol) is a gene encoding 4-hydroxybutyrate-CoA transferase or a gene encoding alcohol dehydrogenase reducing 4HB-CoA It may be characterized in that, more preferably, the alcohol dehydrogenase may be characterized in that the butyl-CoA dehydrogenase derived from Clostridium acetobutylicum ( Clostridium acetobutylicum ).

본 발명에 있어서, 상기 4-hydroxybutyrate-CoA transferase를 코딩하는 유전자는 서열번호 5의 염기서열을 가지는 것을 특징으로 할 수 있고, 상기 butyl-CoA dehydrogenase를 코딩하는 유전자는 서열번호 6의 염기서열(CAP0035) 또는 서열번호 7의 염기서열(CAP0162)을 가지는 것을 특징으로 할 수 있다. In the present invention, the gene encoding the 4-hydroxybutyrate-CoA transferase may be characterized by having a nucleotide sequence of SEQ ID NO: 5, the gene encoding the butyl-CoA dehydrogenase is a nucleotide sequence of SEQ ID NO: 6 (CAP0035 ) Or the nucleotide sequence of SEQ ID NO: 7 (CAP0162).

본 발명에 있어서, 상기 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자는 GabD(succinic semialdehyde dehydrogenase)를 코딩하는 유전자인 것을 특징으로 할 수 있고, 더욱 바람직하게 는, 상기 GabD를 코딩하는 유전자는 서열번호 8의 염기서열을 가지는 것을 특징으로 할 수 있다. In the present invention, the gene involved in converting succinate semialdehyde to succinic acid may be a gene encoding Succinic semialdehyde dehydrogenase (GabD), and more preferably, The gene may be characterized by having the nucleotide sequence of SEQ ID NO: 8.

본 발명에 있어서, 숙신산의 운송에 관여하는 DctA(C4-dicarboxylate transport protein)를 코딩하는 유전자가 추가로 도입 또는 증폭되어 있는 것을 특징으로 할 수 있고, 더욱 바람직하게는 상기 DctA를 코딩하는 유전자는 서열번호 9의 염기서열을 가지는 것을 특징으로 할 수 있다. In the present invention, the gene encoding the DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid may be further introduced or amplified. More preferably, the gene encoding the DctA is a sequence. It may be characterized by having the nucleotide sequence of the number 9.

본 발명은 또한, 숙신산 고생성능을 가지는 미생물에서 GabD를 코딩하는 유전자가 결실되어 있고, Cat1을 코딩하는 유전자, SucD를 코딩하는 유전자, 4hbD를 코딩하는 유전자, GHB를 코딩하는 유전자, 4-hydroxybutyrate-CoA transferase를 코딩하는 유전자 및 butyl-CoA dehydrogenase를 코딩하는 유전자가 모두 도입 또는 증폭되어 있는 것을 특징으로 하는 1,4-BDO 고생성능을 가지는 미생물 변이체를 제공한다. The present invention also provides a gene that encodes GabD in a microorganism having high succinic acid performance, is a gene encoding Cat1, a gene encoding SucD, a gene encoding 4hbD, a gene encoding GHB, 4-hydroxybutyrate- Provided is a microbial variant having high 1,4-BDO performance, characterized in that both a gene encoding CoA transferase and a gene encoding butyl-CoA dehydrogenase are introduced or amplified.

본 발명에 있어서, 숙신산의 운송에 관여하는 DctA(C4-dicarboxylate transport protein)를 코딩하는 유전자가 추가로 도입 또는 증폭되어 있는 것을 특징으로 할 수 있다.In the present invention, the gene encoding the DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid may be further introduced or amplified.

본 발명은 또한, 상기 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 1,4-BDO(1,4-butanediol)를 수득하는 것을 특징으로 하는 1,4-BDO의 제조방법을 제공한다.The present invention also provides a method for producing 1,4-BDO, comprising culturing the variant in a medium containing a carbohydrate, and then obtaining 1,4-BDO (1,4-butanediol) from the culture solution. do.

본 발명은 또한, 서열번호 6의 염기서열을 가지는 CAP0035(butyl-CoA dehydrogenase) 유전자를 제공하고, 서열번호 7의 염기서열을 가지는 CAP0162(butyl-CoA dehydrogenase)를 제공한다. The present invention also provides a CAP0035 (butyl-CoA dehydrogenase) gene having a nucleotide sequence of SEQ ID NO: 6, and provides a CAP0162 (butyl-CoA dehydrogenase) having a nucleotide sequence of SEQ ID NO: 7.

본 발명은 또한, 상기 유전자를 함유하는 재조합벡터를 제공한다. The present invention also provides a recombinant vector containing the gene.

이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.

본 발명의 숙신산 고생성 미생물 중 루멘박테리아는 본 출원인의 특허 출원인 WO 2005/052135에 개시된 방법대로 제조하였다. 즉, 루멘 박테리아의 일종인 Mannheimia succiniciproducens 55E에서 젖산 탈수소화효소 유전자(ldhA)와 피루브산-개미산 분해효소 유전자(pfl)를 결실시켜 변이균주인 Mannheimia sp. LPK(KCTC 10558BP)를 제작하였고, 상기 LPK 균주에서 포스포트랜스아세틸화효소 유전자(pta)와 아세트산 키나제 유전자(ackA) 및 포스포피루브산 카르복실라제 유전자(ppc)를 각각 결실시켜 변이균주들(Mannheimia sp. LPK7 및 LPK4)을 제작한 다음, 이를 혐기적 조건에서 배양한 결과, 숙신산이 고수율로 생산되는 것을 확인하였다. Lumen bacteria among the succinic acid producing microorganisms of the present invention were prepared according to the method disclosed in the applicant's patent application WO 2005/052135. In other words, in the lumen bacterium Mannheimia succiniciproducens 55E, the lactic acid dehydrogenase gene ( ldhA ) and pyruvate- formatease gene ( pfl ) were deleted, resulting in mutant strain Mannheimia sp. LPK (KCTC 10558BP) was produced, and mutant strains ( Mannheimia ) were deleted from the phosphotransacetylacetylase gene ( pta ), acetic acid kinase gene ( ackA ) and phosphopyruvic acid carboxylase gene ( ppc ), respectively, in the LPK strain. sp. LPK7 and LPK4) were produced, and then cultured under anaerobic conditions, it was confirmed that succinic acid was produced in high yield.

또한, 본 발명의 숙신산 고생성 미생물 중, 대장균은 본 출원인의 출원인 국내 특허 공개번호 9-2006-0011345에 개시된 방법대로 제조하였다. 즉, 박테리오파아지 레드오페론(red operon, exo-beta-gam)을 발현하는 재조합 발현벡터 pTrcEBG로 형질전환된 W3110 균주에서 포도당 인산전이효소(glucose phophotransferase)를 코딩하는　유전자(ptsG)와 파이루베이트 키나제(pyruvate kinase)를 코딩하는 두 개의 유전자(pykA, pykF)가 결실시켜, 대장균 변이균주 W3110GFA를 수득하여, 이를 혐기적 조건에서 배양한 결과, 모균주인 W3110 균주를 사용하는 경우보다, W3110GFA를 사용하는 경우 생산성이 크게 향상되는 것을 확인하였다. In addition, among the succinic acid-producing microorganisms of the present invention, E. coli was prepared according to the method disclosed in the applicant's domestic patent publication No. 9-2006-0011345. In other words, in the W3110 strain transformed with the recombinant expression vector pTrcEBG expressing the bacteriophage red operon (exo-beta-gam), the gene (ptsG) and pyruvate kinase encoding glucose phophotransferase Two genes (pykA, pykF) encoding (pyruvate kinase) were deleted to obtain Escherichia coli mutant strain W3110GFA, which was cultured under anaerobic conditions, and W3110GFA was used rather than using the parent strain W3110 strain. When it was confirmed that the productivity is greatly improved.

본 발명에 있어서, 숙신산을 4HB로 전환하는 효소는 클로스트리디움 클루이베리(Clostridium kluyveri)에서 유래된 4HB(4-hydroxybutyate) 생합성에 이용되는 효소들이고, 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소는 클로스트리디움 아세토부틸리쿰(Clostridium acetobutylicum)에서 유래된 1,4-BDO 생합성에 이용되는 효소들이다. 물론 클로스트리디움 클루이베리 및 클로스트리디움 아세토부틸리쿰이 4HB 및 1,4-BDO를 생산하는 것은 아니지만 상기 균주에서 클로닝된 효소들이 4HB 및 1,4-BDO 생산에 중요한 역할을 하는 것을 알 수 있었다. In the present invention, enzymes for converting succinic acid to 4HB are enzymes used for 4HB (4-hydroxybutyate) biosynthesis derived from Clostridium kluyveri , and 4HB is 1,4-BDO (1,4- butanediol) are enzymes used for 1,4-BDO biosynthesis derived from Clostridium acetobutylicum . Of course, although Clostridium cloverberry and Clostridium acetobutylicum do not produce 4HB and 1,4-BDO, it can be seen that the enzymes cloned in the strain play an important role in 4HB and 1,4-BDO production. there was.

본 발명에 있어서, 숙신산을 4HB로 전환하는 효소는 도 1에 나타난 바와 같이, succinate를 succinyl-CoA로 전환하는 효소인 Cat1(succinyl-CoA transferase)와 succinyl-CoA를 succinate semialdehyde로 전환하는 SucD(succinate semialdehyde dehydrogenase) 및 succinate semialdehyde를 4-hydroxybutyrate로 전환하는 4hbD(4-hydroxybutyrate dehydrogenase) 및 GHB(4-hydroxybutyrate dehydrogenase)가 있다. In the present invention, the enzyme for converting succinic acid to 4HB is SucD (succinate) for converting succinate to succinyl-CoA and Cat1 (succinyl-CoA transferase) and succinyl-CoA to succinate semialdehyde 4-hydroxybutyrate dehydrogenase (GHB) and 4-hydroxybutyrate dehydrogenase (GHB), which convert semialdehyde dehydrogenase) and succinate semialdehyde into 4-hydroxybutyrate.

또한, 본 발명에 있어서, 4HB를 1,4-BDO로 전환하는 효소는 도 2에 나타난 바와 같이, 4HB를 4-hydroxybutyryl-CoA로 전환하는 4-HB-CoA transferase 및 4-hydroxybutyryl-CoA를 1,4-butanediol로 전환하는 alcohol dehydrogenase가 있다. In addition, in the present invention, the enzyme for converting 4HB to 1,4-BDO is 4-HB-CoA transferase and 4-hydroxybutyryl-CoA for converting 4HB to 4-hydroxybutyryl-CoA, as shown in Figure 2 And alcohol dehydrogenase, which converts into 4-butanediol.

또한, 본 발명의 목적을 목적을 달성하기 위하여, 숙신산을 효율적으로 이용하는 것이 매우 중요한 데 이것을 충족하기 위해, 숙신산 고생성 미생물은 Succinic semialdehyde를 Succininate로 전환하는데 관여하는 gabD(succinic semialdehyde dehydrogenase)가 소실시키는 것을 특징으로 한다. 또한, 미생물 체내로 빠져나온 숙신산의 효율적인 체내전달을 위해 숙신산의 전달(transport)에 관여하는 DctA 효소를 증폭시킬 수 있다.In addition, in order to achieve the object of the present invention, it is very important to efficiently use succinic acid. To meet this, succinic acid-producing microorganisms are depleted by succinic semialdehyde dehydrogenase (gabD) involved in converting Succinic semialdehyde into Succininate. It is characterized by. In addition, it is possible to amplify the DctA enzyme involved in the transport of succinic acid for efficient in vivo delivery of succinic acid released into the body of the microorganism.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

특히, 하기 실시예에서는 루멘박테리아 중, Mannheimia 속 균주로부터 유전자를 결실시킨 숙신산 고농도 생성 변이균주 Mannheimia sp. LPK(KCTC 10558BP), LPK7 및 LPK4과 대장균 변이균주 W3110GFA를 이용하여, 1,4-BOD를 제조하는 방법만을 예시하였으나, 다른 루멘 박테리아 균주를 사용하여 변이균주를 수득하고, 이를 이용하여 숙신산을 제조하는 것 역시 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. In particular, the following examples in which the genes deleted from the lumen of the bacteria, Mannheimia sp acid a high concentration generated mutant Mannheimia sp. Using only LPK (KCTC 10558BP), LPK7 and LPK4 and Escherichia coli mutant strain W3110GFA, only 1,4-BOD was prepared. It will also be apparent to one of ordinary skill in the art.

또한, 하기 실시예에서는 특정 배지와 배양방법만을 예시하였으나, 문헌에 보고된 바와 같이(Lee et al., Bioprocess Biosyst. Eng., 26:63, 2003; Lee et al., Appl. Microbiol. Biotechnol., 58:663, 2002; Lee et al., Biotechnol. Lett., 25:111, 2003; Lee et al., Appl. Microbiol. Biotechnol., 54:23, 2000; Lee et al., Biotechnol. Bioeng., 72:41, 2001), 유청(whey), CSL(corn steep liguor) 등의 당화액과 다른 배지를 사용한 경우나, 유가배양(fed-batch culture), 연속배양 등 다양한 방법을 사용하는 것도 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. In addition, the following examples exemplify specific media and culture methods, but as reported in the literature (Lee et al. , Bioprocess Biosyst. Eng. , 26:63, 2003; Lee et al. , Appl. Microbiol. Biotechnol. , 58: 663, 2002; Lee et al, Biotechnol Lett, 25:......... 111, 2003; Lee et al, Appl Microbiol Biotechnol, 54:23, 2000; Lee et al, Biotechnol Bioeng. 72:41, 2001), or other mediums such as whey and corn steep liguor, or other methods such as fed-batch culture and continuous culture. It will be obvious to those of ordinary skill in the industry.

실시예 1: 숙신산 고생성능을 가지는 미생물의 제조방법Example 1 Preparation of Microorganisms Having High Succinic Acid Performance

1-1 숙신산 고생성능을 가지는 루멘박테리아의 제조1-1 Preparation of Lumen Bacteria with High Succinic Acid Performance

본 발명의 숙신산 고생성 미생물인 루멘박테리아는 본 출원인의 특허 출원인 WO 2005/052135에 개시된 방법대로 제조하였다. 즉, 루멘 박테리아의 일종인 Mannheimia succiniciproducens 55E에서 젖산 탈수소화효소 유전자(ldhA)와 피루브산-개미산 분해효소 유전자(pfl)를 결실시켜 변이균주인 Mannheimia sp. LPK(KCTC 10558BP)를 제작하였고, 상기 LPK 균주에서 포스포트랜스아세틸화효소 유전자(pta)와 아세트산 키나제 유전자(ackA) 및 포스포피루브산 카르복실라제 유전자(ppc)를 각각 결실시켜 변이균주들(Mannheimia sp. LPK7 및 LPK4)을 제작하였다.Lumen bacteria, the succinic acid producing microorganisms of the present invention, were prepared according to the method disclosed in the applicant's patent application WO 2005/052135. In other words, in the lumen bacterium Mannheimia succiniciproducens 55E, the lactic acid dehydrogenase gene ( ldhA ) and pyruvate- formatease gene ( pfl ) were deleted, resulting in mutant strain Mannheimia sp. LPK (KCTC 10558BP) was produced, and mutant strains ( Mannheimia ) were deleted by phosphotransacetylacetylase gene ( pta ), acetic acid kinase gene ( ackA ) and phosphopyruvic acid carboxylase gene ( ppc ), respectively, in the LPK strain. sp. LPK7 and LPK4).

1-2 숙신산 고생성능을 가지는 대장균의 제조 Preparation of Escherichia Coli with High Performance of 1-2 Succinic Acid

본 발명의 숙신산 고생성 미생물인 대장균은 본 출원인의 출원인 국내 특허 공개번호 9-2006-0011345에 개시된 방법대로 제조하였다. 즉, 박테리오파아지 레드오페론(red operon, exo-beta-gam)을 발현하는 재조합 발현벡터 pTrcEBG로 형질전환된 W3110 균주에서 포도당 인산전이효소(glucose phophotransferase)를 코딩하는　유전자(ptsG)와 파이루베이트 키나제(pyruvate kinase)를 코딩하는 두 개의 유전자(pykA, pykF)가 결실시켜, 대장균 변이균주 W3110GFA를 수득하였다. E. coli, a succinic acid-producing microorganism of the present invention, was prepared according to the method disclosed in the applicant's applicant's Korean Patent Publication No. 9-2006-0011345. In other words, in the W3110 strain transformed with the recombinant expression vector pTrcEBG expressing the bacteriophage red operon (exo-beta-gam), the gene (ptsG) and pyruvate kinase encoding glucose phophotransferase Two genes (pykA, pykF) encoding (pyruvate kinase) were deleted to obtain E. coli mutant strain W3110GFA.

실시예 2: 1,4-BDO 전환 효소 클로닝Example 2: Cloning of 1,4-BDO Converting Enzyme

2-1 4HB 전환 효소(Cat1, SucD, 4hbD, GHB)를 코딩하는 유전자의 클로닝 2-1 Cloning of Genes Encoding 4HB Converting Enzymes (Cat1, SucD, 4hbD, GHB)

본 발명자들은 클로스트리디움 클루이베리 DSM 555로부터 Cat1을 코딩하는 유전자 cat1을 클로닝하고자 알려진 유전자 시퀀스 (L21902) 에 기초하여 합성된 올리고뉴클레오티드 프라이머를 사용하여, DNA 중합 효소반응 (polymerase chain reaction, PCR)으로 cat1 유전자를 증폭시켰다.The present inventors have performed DNA polymerase chain reaction (PCR) using oligonucleotide primers synthesized based on a known gene sequence (L21902) for cloning the gene cat1 encoding Cat1 from Clostridium cloverberry DSM 555. cat1 gene was amplified.

PCR 증폭실험에 사용한 프라이머는 다음과 같다. Primers used in the PCR amplification experiment are as follows.

[서열번호 10] cat1f-BspHI aaaaatcaat gagtaaaggg ataaagaatt cac [SEQ ID NO: 10] cat1f-BspHI aaaaatcaat gagtaaaggg ataaagaatt cac

[서열번호 11] cat1b-XbaI gctctagatt atttcatact accagttttt ataaa[SEQ ID NO 11] cat1b-XbaI gctctagatt atttcatact accagttttt ataaa

발현 벡터를 만들기 위해 상기 증폭된 cat1 유전자를 NcoI/XbaI 로 절단한pTrc99A (Amersham Pharmacia Biotech) 발현 벡터에 삽입함으로써 pTrc99Cat1를 제조하였다. PTrc99Cat1 was prepared by inserting the amplified cat1 gene into pTrc99A (Amersham Pharmacia Biotech) expression vector digested with Nco I / Xba I to make an expression vector.

또한 SucD, 4hbD operon 유전자를 클로닝하고자 알려진 유전자 시퀀스 (L21902) 에 기초하여 합성된 올리고뉴클레오티드 프라이머를 사용하여, DNA 중합 효소반응 (polymerase chain reaction, PCR)으로 sucD4hbD 유전자를 증폭시켰다. In addition, the DNA polymerase chain reaction (PCR) was performed using oligonucleotide primers synthesized based on a known gene sequence (L21902) to clone the SucD and 4hbD operon genes. sucD4hbD gene was amplified.

PCR 증폭실험에 사용한 프라이머는 다음과 같다. Primers used in the PCR amplification experiment are as follows.

[서열번호 12] SucDf-BspHI aaaaatcaat gagtaatgaa gtatctataa aag [SEQ ID NO: 12] SucDf-BspHI aaaaatcaat gagtaatgaa gtatctataa aag

[서열번호 13] 4hbDb-XabI gctctagatt agataaaaaa gaggacattt cacaatatgg[SEQ ID NO: 13] 4hbDb-XabI gctctagatt agataaaaaa gaggacattt cacaatatgg

발현 벡터를 만들기 위해 상기 증폭된 sucD4hbD 유전자를 NcoI/XbaI 로 절단한 pTrc99A (Amersham Pharmacia Biotech) 발현 벡터에 삽입함으로써 pTrc99SucD4hbD를 제조하였다.PTrc99SucD4hbD was prepared by inserting the amplified sucD4hbD gene into pTrc99A (Amersham Pharmacia Biotech) expression vector digested with Nco I / Xba I to make an expression vector.

또한, 서열번호 4의 GHB 유전자를 클로닝하고자 알려진 유전자 시퀀스 (L36817)에 기초하여 합성된 올리고뉴클레오티드 프라이머를 사용하여, DNA 중합효소반응 (polymerase chain reaction, PCR)으로 ghb 유전자를 증폭시켰다. In addition, using an oligonucleotide primer synthesized based on a known gene sequence (L36817) to clone the GHB gene of SEQ ID NO: 4, a DNA polymerase chain reaction (PCR) was performed. The ghb gene was amplified.

PCR 증폭실험에 사용한 프라이머는 다음과 같다. Primers used in the PCR amplification experiment are as follows.

[서열번호 14] H16 GHBf-BspHI aaaaatcaat ggcgtttatc tactatctg[SEQ ID NO: 14] H16 GHBf-BspHI aaaaatcaat ggcgtttatc tactatctg

[서열번호 15] H16 GHBb-XbaI gctctagatt acatggactg ctcaagcata c[SEQ ID NO: 15] H16 GHBb-XbaI gctctagatt acatggactg ctcaagcata c

2-2 숙신산의 운송에 관여하는 DctA를 코딩하는 유전자의 클로닝 2-2 Cloning of the Gene Encoding DctA Involved in the Transport of Succinic Acid

대장균에서 숙신산의 운송에 관여하는 서열번호 9의 DctA를 코딩하는 유전자의 클로닝을 위하여 대장균 W3110로부터 DctA을 코딩하는 유전자 dctA을 클로닝하고자 알려진 유전자 시퀀스 (NC_000913) 에 기초하여 합성된 올리고뉴클레오티드 프라이머를 사용하여, DNA 중합 효소반응 (polymerase chain reaction, PCR)으로 dctA 유전자를 증폭시켰다. For the cloning of the gene encoding the DctA of SEQ ID NO: 9 involved in the transport of succinic acid in Escherichia coli, using an oligonucleotide primer synthesized based on a known gene sequence (NC_000913) to clone the gene dctA encoding DctA from Escherichia coli W3110 DNA polymerase chain reaction (PCR) dctA gene was amplified.

PCR 증폭실험에 사용한 프라이머는 다음과 같다. Primers used in the PCR amplification experiment are as follows.

[서열번호 16] DctAf-EcoRI ggaattcatg aaaacctctc tgtttaaaag c[SEQ ID NO: 16] DctAf-EcoRI ggaattcatg aaaacctctc tgtttaaaag c

[서열번호 17] DctAb-XbaI gctctagatt aagaggataa ttcgtgcgtt ttgcc[SEQ ID NO: 17] DctAb-XbaI gctctagatt aagaggataa ttcgtgcgtt ttgcc

발현 벡터를 만들기 위해 상기 증폭된 dctA 유전자를 p10499A (Park et al. (2002) FEMS Microbiol. Lett 214: 217-222) 발현 벡터에 EcoRI/XbaI 로 절단해 삽입함으로서 p10499DctA를 제조하였다. P10499DctA was prepared by cutting and inserting the amplified dctA gene into p10499A (Park et al. (2002) FEMS Microbiol. Lett 214: 217-222) expression vector with Eco RI / Xba I to make an expression vector.

2-3 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소를 코딩하는 유전자의 클로닝 2-3 Cloning of a Gene Encoding an Enzyme That Converts 4HB to 1,4-BDO (1,4-butanediol)

클로스트리디움 아세토부틸리쿰(Clostridium acetobutylicum)에서 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소인, 서열번호 5의 4-hydroxybutyrate-CoA transferase를 코딩하는 유전자, 서열번호 6 및 서열번호 7의 butyryl-CoA dehydrogenase를 코딩하는 유전자를 각각 클로닝 하기 위해 알려진 유전자 시퀀스 (NC_003030) 에 기초하여 합성된 올리고뉴클레오티드 프라이머를 사용하여, DNA 중합 효소반응 (polymerase chain reaction, PCR)으로 cat 2, cap0035 및 cap0162 유전자를 증폭시켰다. Gene encoding the 4-hydroxybutyrate-CoA transferase of SEQ ID NO: 5, an enzyme that converts 4HB to 1,4-BDO (1,4-butanediol) in Clostridium acetobutylicum , SEQ ID NO: 6 And DNA 2 polymerase chain reaction (PCR) using oligonucleotide primers synthesized based on a known gene sequence (NC_003030) to clone genes encoding butyryl-CoA dehydrogenase of SEQ ID NO: 7, respectively. , The cap0035 and cap0162 genes were amplified.

PCR 증폭실험에 사용한 프라이머는 다음과 같다. Primers used in the PCR amplification experiment are as follows.

[서열번호 18] cat2f-NcoI aaacatgcca tggagtggga agagatatat aaagag [SEQ ID NO: 18] cat2f-NcoI aaacatgcca tggagtggga agagatatat aaagag

[서열번호 19] cat2b-BamHI cgggatcctt aaaatctctt tttaaattca ttcattaatg [SEQ ID NO 19] cat2b-BamHI cgggatcctt aaaatctctt tttaaattca ttcattaatg

[서열번호 20] CAP0035f-BspHI aaaaatcaat gaaagttaca aatcaaaaag aac[SEQ ID NO: 20] CAP0035f-BspHI aaaaatcaat gaaagttaca aatcaaaaag aac

[서열번호 21] CAP0035b1-XbaI gctctagatt aaaatgcttt tatatagata tc [SEQ ID NO 21] CAP0035b1-XbaI gctctagatt aaaatgcttt tatatagata tc

[서열번호 22] CAP0162f- BspHI aaaaatcaat gaaagtcaca acagtaaagg[SEQ ID NO 22] CAP0162f- BspHI aaaaatcaat gaaagtcaca acagtaaagg

[서열번호 23] CAP0162b1-XbaI gctctagatt aaggttgttt tttaaaacaa tt [SEQ ID NO 23] CAP0162b1-XbaI gctctagatt aaggttgttt tttaaaacaa tt

발현 벡터를 만들기 위해 상기 증폭된 cat2, cap0035 및 cap0162 유전자를 NcoI/XbaI 로 절단한 pTrc99A (Amersham Pharmacia Biotech) 발현 벡터에 각각 삽입함으로써 pTrc99Cat2, pTrc99CAP35 및 pTrc99CAP162 를 제조하였다.PTrc99Cat2, pTrc99CAP35 and pTrc99CAP162 were prepared by inserting the amplified cat2, cap0035 and cap0162 genes into pTrc99A (Amersham Pharmacia Biotech) expression vectors, respectively, digested with Nco I / Xba I to make expression vectors.

실시예 3: 1,4-BDO 고생성능을 가지는 변이체 제작Example 3 Manufacture of Variants Having High 1,4-BDO Performance

3-1 숙신산 고생능 미생물에서 GabD 코딩하는 유전자 결실3-1 Deletion of GabD-coding Genes in High-Performance Microorganisms

본 발명의 숙신산 고생성 미생물인 대장균은 본 출원인의 출원인 국내 특허 공개번호 제10-2006-0011345에 개시된 방법대로 제조하였다. 즉, 박테리오파아지 레드오페론(red operon, exo-beta-gam)을 발현하는 재조합 발현벡터 pTrcEBG로 형 질전환된 W3110GFA 균주에서 서열번호 8의 GabD 유전자를 소실시킨 대장균 변이균주 W3110GFA-1를 수득하였다. E. coli, a succinic acid-producing microorganism of the present invention, was prepared according to the method disclosed in Korean Patent Publication No. 10-2006-0011345 of the applicant. That is, E. coli mutant strain W3110GFA-1, which lost the GabD gene of SEQ ID NO: 8 in a W3110GFA strain transformed with a recombinant expression vector pTrcEBG expressing bacteriophage red operon (exo-beta-gam), was obtained.

3-2 실시예 2에서 제조된 유전자들의 도입3-2 Introduction of Genes Prepared in Example 2

실시예 2에서 클로닝한 4HB 전환 효소 클로닝를 3-1에서 제조된 GabD가 제거된 W3110GFA-1에 도입시켰다.The 4HB converting enzyme clone cloned in Example 2 was introduced into W3110GFA-1 from which GabD was prepared in 3-1 was removed.

실시예 4: 1,4-BDO 수득Example 4: Obtaining 1,4-BDO

실시예 2-1, 2-2 에서 제조한 4HB를 생산하는 재조합 미생물을 실시예 2-3에서 제조한 butytyl-CoA dehydrogenase를 발현하는 재조합 플라스미드로 형질전환한 다음 특허 10-2006-0011345에 제시한 대로 배양을 실시하였다 The recombinant microorganism producing 4HB prepared in Examples 2-1 and 2-2 was transformed with a recombinant plasmid expressing butytyl-CoA dehydrogenase prepared in Example 2-3, and then disclosed in Patent 10-2006-0011345. Culture was carried out as

즉, 상기 실시예 3에서 제작된 대장균 변이균주(W3110GFA)를 CaCO3 과량 함유된 배지조건에서 호기적으로 배양하고, 이로부터 생산되는 산물을 분석하였다.That is, the Escherichia coli mutant strain (W3110GFA) prepared in Example 3 was cultured aerobic under medium containing CaCO 3 excess, and the product produced therefrom was analyzed.

먼저, LB 배지 10㎖를 제조하고, 대장균을 접종한 후, 37℃에서 12시간동안 전배양을 수행하였다. 이어, 증류수 1리터당 30g glucose, 2g KH2PO4, 10g (NH4 )2SO4·7H2O, 10mg MnCl2·4H2O, 10mg FeSO4·4H2O, 20g CaCO3, 50mg FeSO4·7H2O, 10mg CaCl2, 11mg ZnSO4·7H2O, 2.5mg MnSO4·5H2O, 5mg CuSO4·5H2O, 0.5mg(NH4)Mo7O24·4H20, 0.1mg NaB4O7·10H2O 의 성분이 있고 KOH로 pH를 7.0으로 맞 춘 배지 100㎖을 함유한 250㎖ 플라스크에 대장균 W3110GFA 돌연변이주 5㎖을 접종하여, 30℃에서 120rpm으로 72시간 동안 배양한 후, 생성된 4HB는 황산으로 배양액의 pH를 2로 낮춘 후 클로로포름 또는 에틸아세테이트로 추출하였다. First, 10 ml of LB medium was prepared, and inoculated with E. coli, followed by preculture at 37 ° C. for 12 hours. Then, 30 g glucose, 2 g KH 2 PO 4 , 10 g (NH 4 ) 2 SO 4 · 7H 2 O, 10 mg MnCl 2 · 4H 2 O, 10 mg FeSO 4 · 4H 2 O, 20 g CaCO 3 , 50 mg FeSO 4 per liter of distilled water 7H 2 O, 10mg CaCl 2 , 11mg ZnSO 4 7H 2 O, 2.5mg MnSO 4 5H 2 O, 5mg CuSO 4 5H 2 O, 0.5mg (NH 4 ) Mo 7 O 24 4H 2 0, 0.1 5 ml of E. coli W3110GFA mutant strain was inoculated into a 250 ml flask containing 100 ml of medium prepared with NaH 4 O 7 · 10H 2 O and adjusted to pH 7.0 with KOH. After that, the produced 4HB was extracted with chloroform or ethyl acetate after lowering the pH of the culture solution to 2 with sulfuric acid.

이상 상세히 기술한 바와 같이, 본 발명에 따르면, 숙신산을 고농도로 생산할 수 있는 미생물을 이용하여, 화학산업 전반에 걸쳐 중요하게 사용되고 있는 탄소수 4개의 화학물질인 1,4-BDO(1,4-butanediol) 생성능을 가지는 변이체 및 이를 이용한 1,4-BDO의 생물학적 제조방법을 제공하는 효과가 있다. As described in detail above, according to the present invention, by using a microorganism capable of producing a high concentration of succinic acid, 1,4-BDO (1,4-butanediol), which is an important four-carbon chemical used throughout the chemical industry ) It has the effect of providing a variant having the ability to produce and a biological method for producing 1,4-BDO using the same.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> LG Chem,Ltd. <120> Mutants Having a Producing Ability of 1,4-Butanediol and Method for Preparing 1,4-BDO Using the Same <130> P06-B034 <160> 23 <170> KopatentIn 1.71 <210> 1 <211> 1617 <212> DNA <213> Clostridium kluyveri <400> 1 atgagtaaag ggataaagaa ttcacaattg aaaaaaaaga atgtaaaggc tagtaatgtg 60 gcagaaaaga ttgaagagaa agttgaaaaa acagataagg ttgttgaaaa ggcagctgag 120 gttactgaaa aacgaattag aaacttgaag cttcaggaaa aagttgtaac agcagatgtg 180 gcagctgata tgatagaaaa cggtatgatt gttgcaatta gcggatttac tccttccggg 240 tatcctaaag aagtacctaa agcattgact aaaaaagtta atgccttaga ggaagaattc 300 aaggtaacac tttatacagg ttcatctaca ggagccgata tagacggaga atgggcaaaa 360 gcaggaataa tagaaagaag aattccatat cagacaaatt ctgatatgag gaaaaaaata 420 aatgatggtt ctattaagta tgctgatatg catttaagcc atatggctca atatattaat 480 tattctgtaa ttcctaaagt agatatagct ataatagagg cagtagctat tacagaagaa 540 ggggatatta ttccttcaac aggaattgga aatacagcta cttttgtgga aaatgcagat 600 aaggtaatag tggaaattaa tgaggctcaa ccgcttgaat tggaaggtat ggcagatata 660 tatacattaa aaaaccctcc aagaagagag cccataccta tagttaatgc aggcaatagg 720 atagggacca catatgtgac ctgtggttct gaaaaaatat gcgctatagt gatgacaaat 780 acccaggata aaacaagacc tcttacagaa gtgtctcctg tatctcaggc tatatccgat 840 aatcttatag gatttttaaa taaagaggtt gaagagggaa aattacctaa gaacctgctt 900 cctatacagt caggagttgg aagtgtagca aatgcagttt tggccggact ttgtgaatca 960 aattttaaaa atttgagttg ttatacagaa gttatacagg attctatgct gaagcttata 1020 aaatgtggta aagcagatgt ggtgtcaggc acttccataa gtccttcacc ggagatgttg 1080 cctgagttca taaaggacat aaatttcttt agagaaaaga tagtattaag accacaggaa 1140 ataagtaata atccagagat agcaagaaga ataggagtta tatccataaa cactgctttg 1200 gaagtagata tatatggtaa tgtaaactcc actcatgtta tgggaagcaa aatgatgaat 1260 ggtataggcg gttctggaga ctttgccaga aatgcatatt tgactatatt cactacagag 1320 tctatcgcca aaaaaggaga tatatcatct atagttccta tggtatccca tgtggatcat 1380 acagaacatg atgtaatggt aattgttaca gaacagggag tagcagattt aagaggtctt 1440 tctcctaggg aaaaggccgt ggctataata gaaaattgtg ttcatcctga ttacaaggat 1500 atgcttatgg aatattttga agaggcttgt aagtcatcag gtggaaatac accacataat 1560 cttgaaaaag ctctttcctg gcatacaaaa tttataaaaa ctggtagtat gaaataa 1617 <210> 2 <211> 1419 <212> DNA <213> Clostridium kluyveri <400> 2 atgagtaatg aagtatctat aaaagaatta attgaaaagg caaaggcggc acaaaaaaaa 60 ttggaagcct atagtcaaga acaagttgat gtactagtaa aagcactagg aaaagtggtt 120 tatgataatg cagaaatgtt tgcaaaagaa gcagttgaag aaacagaaat gggtgtttat 180 gaagataaag tagctaaatg tcatttgaaa tcaggagcta tttggaatca tataaaagac 240 aagaaaactg taggcataat aaaagaagaa cctgaaaggg cacttgttta tgttgctaag 300 ccaaagggag ttgtggcagc tactacgcct ataactaatc cagtggtaac tcctatgtgt 360 aatgcaatgg ctgctataaa gggcagaaat acaataatag tagcaccaca tcctaaagca 420 aagaaagttt cagctcatac tgtagaactt atgaatgctg agcttaaaaa attgggagca 480 ccagaaaata tcatacagat agtagaagca ccatcaagag aagctgctaa ggaacttatg 540 gaaagtgctg atgtagttat tgctacaggc ggtgctggaa gagttaaagc tgcttactcc 600 agtggaagac cagcttatgg cgttggacct ggaaattcac aggtaatagt tgataaggga 660 tacgattata acaaagctgc acaggatata ataacaggaa gaaaatatga caatggaatt 720 atatgttctt cagagcaatc agttatagct cctgctgaag attatgataa ggtaatagca 780 gcttttgtag aaaatggggc attctatgta gaagatgagg aaacagtaga aaagtttaga 840 tcaactttat ttaaagatgg aaaaataaac agcaagatta taggtaaatc cgtccaaatt 900 attgcggatc ttgcaggagt aaaagtacca gaaggtacta aggttatagt acttaagggt 960 aaaggtgcag gagaaaaaga tgtactttgt aaagaaaaaa tgtgtccagt tttagtagca 1020 ttgaaatatg atacttttga agaagcagtt gaaatagcta tggctaatta tatgtatgaa 1080 ggagctggtc atacagcagg catacattct gacaatgacg agaacataag atatgcaaga 1140 actgtattac ctataagcag attagttgta aatcagcctg caactactgc tggaggaact 1200 gtattaccta taagcagatt agttgtaaat cagcctgcaa ctactgctgg aggaagtttc 1260 aataatggat ttaaccctac tactacacta ggctgcggat catggggcag aaacagtatt 1320 tcagaaaatc ttacttacga gcatcttata aatgtttcaa gaatagggta tttcaataaa 1380 gaagcaaaag ttcctagcta tgaggaaata tggggataa 1419 <210> 3 <211> 1116 <212> DNA <213> Clostridium kluyveri <400> 3 atgaagttat taaaattggc acctgatgtt tataaatttg atactgcaga ggagtttatg 60 aaatacttta aggttggaaa aggtgacttt atacttacta atgaattttt atataaacct 120 ttccttgaga aattcaatga tggtgcagat gctgtatttc aggagaaata tggactcggt 180 gaaccttctg atgaaatgat aaacaatata attaaggata ttggagataa acaatataat 240 agaattattg ctgtaggggg aggatctgta atagatatag ccaaaatcct cagtcttaag 300 tatactgatg attcattgga tttgtttgag ggaaaagtac ctcttgtaaa aaacaaagaa 360 ttaattatag ttccaactac atgtggaaca ggttcagaag ttacaaatgt atcagttgca 420 gaattaaaga gaagacatac taaaaaagga attgcttcag acgaattata tgcaacttat 480 gcagtacttg taccagaatt tataaaagga cttccatata agttttttgt aaccagctcc 540 gtagatgcct taatacatgc aacagaagct tatgtatctc caaatgcaaa tccttatact 600 gatatgttta gtgtaaaagc tatggagtta attttaaatg gatacatgca aatggtagag 660 aaaggaaatg attacagagt tgaaataatt gaggattttg ttataggcag caattatgca 720 ggtatagctt ttggaaatgc aggagtggga gcggttcacg cactctcata tccaataggc 780 ggaaattatc atgtgcctca tggagaagca aattatctgt tttttacaga aatatttaaa 840 acttattatg agaaaaatcc aaatggcaag attaaagatg taaataaact attagcaggc 900 atactaaaat gtgatgaaag tgaagcttat gacagtttat cacaactttt agataaatta 960 ttgtcaagaa aaccattaag agaatatgga atgaaagagg aagaaattga aacttttgct 1020 gattcagtaa tagaaggaca gcagagactg ttggtaaaca attatgaacc tttttcaaga 1080 gaagacatag taaacacata taaaaagtta tattaa 1116 <210> 4 <211> 1149 <212> DNA <213> Clostridium kluyveri <400> 4 atggcgttta tctactatct gacccacatc cacctggatt tcggcgcggt aagcctgctc 60 aagtccgaat gcgagcgcat cggcatccgc cgcccgttgc tggtgaccga caagggcgtg 120 gtcgccgcgg gagtggcgca gcgtgccatc gatgcaatgc agggcctgca ggttgcggta 180 ttcgatgaaa ccccgtcgaa cccgaccgag gccatggtgc gcaaggccgc cgcacaatac 240 cgcgaggccg gctgcgacgg gctggtggca gtgggcggcg gctcgtcgat cgacctcgcc 300 aagggcatcg ccatcctggc cacgcatgag ggcgagctga ccacctatgc caccatcgaa 360 ggcggcagcg ccaggatcac cgacaaggcg gcgccgctga tcgcggtgcc caccacctcg 420 ggcaccggca gcgaggtggc gcgcggcgcc atcatcatcc tggacgacgg ccgcaagctg 480 ggcttccatt cctggcattt gctgcccaag tccgccgtct gcgacccgga actgacgctg 540 gggctgccgg ccgggctgac cgcggccacc ggcatggatg cgatcgcgca ctgcatcgag 600 accttcctgg cccccgcctt caacccgccc gcggacggca ttgcgctgga cgggctggag 660 cgcggctggg gccatatcga acgcgccacc cgcgacggtc aggaccgcga cgcacgcctg 720 aacatgatga gcgcgtcgat gcagggcgca atggcgttcc agaaggggct gggctgcgtg 780 cattcgctgt cgcacccgct gggcgggctg aagatcgacg gccgcaccgg cctgcaccac 840 ggcacgctca acgcggtggt gatgccggcg gtgctgcgct tcaacgccga tgcgcccacg 900 gtggtgcgcg acgaccgcta cgcacgcctg cgccgcgcca tgcacctgcc cgacggcgcc 960 gatatcgcgc aggccgtgca cgacatgacc gtgcgcctgg gcctgcccac cgggctgcgt 1020 cagatgggtg tcaccgagga catgttcgac aaggtgattg ccggtgcgct ggtcgaccat 1080 tgccacaaga ccaacccgaa agaagccagc gccgcggatt atcggcgtat gcttgagcag 1140 tccatgtag 1149 <210> 5 <211> 1290 <212> DNA <213> Clostridium acetobutylicum <400> 5 atggagtggg aagagatata taaagagaaa ctggtaactg cagaaaaagc tgtttcaaaa 60 atagaaaacc atagcagggt agtttttgca catgcagtag gagaacccgt agatttagta 120 aatgcactag ttaaaaataa ggataattat ataggactag aaatagttca catggtagct 180 atgggcaaag gtgtatatac aaaagagggt atgcaaagac attttagaca taatgctttg 240 tttgtaggcg gatctactag agatgcagta aattcaggaa gagcagttta tacaccttgt 300 tttttctatg aagtgccaag tttgtttaaa gaaaaacgtt tgcctgtaga tgtagcactt 360 attcaggtaa gtgagccaga taaatatggc tactgcagtt ttggagtttc caatgactat 420 accaagccag cagcagaaag tgctaagctt gtaattgcag aagtgaataa aaacatgcca 480 agaactcttg gagattcttt tatacatgta tcagatattg attatatagt ggaagcttca 540 cacccattgt tagaattgca gcctcctaaa ttgggagatg tagaaaaagc cataggagaa 600 aactgtgcat ctttaattga agatggagct actcttcagc ttggaatagg tgctatacca 660 gatgcggtac ttttattctt aaagaacaaa aagaatttag gaatacattc tgagatgata 720 tcagatggtg tgatggaact ggtgaaggca ggggttatca ataacaagaa aaagaccctc 780 catccaggca aaatagttgt aacattttta atgggaacaa aaaaattata tgattttgta 840 aacaataatc caatggtaga aacttattct gtagattatg taaataatcc actggtaatt 900 atgaaaaatg acaatatggt ttcaataaat tcttgtgttc aagtagactt aatgggacaa 960 gtatgttctg aaagtatagg attgaaacag ataagtggag tgggaggcca ggtagatttt 1020 attagaggag ctaatctatc aaagggtgga aaggctatta tagctatacc ttccacagct 1080 ggaaaaggaa aagtttcaag aataactcca cttctagata ctggtgctgc agttacaact 1140 tctagaaatg aagtagatta tgtagttact gaatatggtg ttgctcatct taagggcaaa 1200 actttaagaa atagggcaag agctctaata aatatcgctc atccaaaatt cagagaatca 1260 ttaatgaatg aatttaaaaa gagattttag 1290 <210> 6 <211> 2577 <212> DNA <213> Clostridium acetobutylicum <400> 6 atgaaagtta caaatcaaaa agaactaaaa caaaagctaa atgaattgag agaagcgcaa 60 aagaagtttg caacctatac tcaagagcaa gttgataaaa tttttaaaca atgtgccata 120 gccgcagcta aagaaagaat aaacttagct aaattagcag tagaagaaac aggaataggt 180 cttgtagaag ataaaattat aaaaaatcat tttgcagcag aatatatata caataaatat 240 aaaaatgaaa aaacttgtgg cataatagac catgacgatt ctttaggcat aacaaaggtt 300 gctgaaccaa ttggaattgt tgcagccata gttcctacta ctaatccaac ttccacagca 360 attttcaaat cattaatttc tttaaaaaca agaaacgcaa tattcttttc accacatcca 420 cgtgcaaaaa aatctacaat tgctgcagca aaattaattt tagatgcagc tgttaaagca 480 ggagcaccta aaaatataat aggctggata gatgagccat caatagaact ttctcaagat 540 ttgatgagtg aagctgatat aatattagca acaggaggtc cttcaatggt taaagcggcc 600 tattcatctg gaaaacctgc aattggtgtt ggagcaggaa atacaccagc aataatagat 660 gagagtgcag atatagatat ggcagtaagc tccataattt tatcaaagac ttatgacaat 720 ggagtaatat gcgcttctga acaatcaata ttagttatga attcaatata cgaaaaagtt 780 aaagaggaat ttgtaaaacg aggatcatat atactcaatc aaaatgaaat agctaaaata 840 aaagaaacta tgtttaaaaa tggagctatt aatgctgaca tagttggaaa atctgcttat 900 ataattgcta aaatggcagg aattgaagtt cctcaaacta caaagatact tataggcgaa 960 gtacaatctg ttgaaaaaag cgagctgttc tcacatgaaa aactatcacc agtacttgca 1020 atgtataaag ttaaggattt tgatgaagct ctaaaaaagg cacaaaggct aatagaatta 1080 ggtggaagtg gacacacgtc atctttatat atagattcac aaaacaataa ggataaagtt 1140 aaagaatttg gattagcaat gaaaacttca aggacattta ttaacatgcc ttcttcacag 1200 ggagcaagcg gagatttata caattttgcg atagcaccat catttactct tggatgcggc 1260 acttggggag gaaactctgt atcgcaaaat gtagagccta aacatttatt aaatattaaa 1320 agtgttgctg aaagaaggga aaatatgctt tggtttaaag tgccacaaaa aatatatttt 1380 aaatatggat gtcttagatt tgcattaaaa gaattaaaag atatgaataa gaaaagagcc 1440 tttatagtaa cagataaaga tctttttaaa cttggatatg ttaataaaat aacaaaggta 1500 ctagatgaga tagatattaa atacagtata tttacagata ttaaatctga tccaactatt 1560 gattcagtaa aaaaaggtgc taaagaaatg cttaactttg aacctgatac tataatctct 1620 attggtggtg gatcgccaat ggatgcagca aaggttatgc acttgttata tgaatatcca 1680 gaagcagaaa ttgaaaatct agctataaac tttatggata taagaaagag aatatgcaat 1740 ttccctaaat taggtacaaa ggcgatttca gtagctattc ctacaactgc tggtaccggt 1800 tcagaggcaa caccttttgc agttataact aatgatgaaa caggaatgaa atacccttta 1860 acttcttatg aattgacccc aaacatggca ataatagata ctgaattaat gttaaatatg 1920 cctagaaaat taacagcagc aactggaata gatgcattag ttcatgctat agaagcatat 1980 gtttcggtta tggctacgga ttatactgat gaattagcct taagagcaat aaaaatgata 2040 tttaaatatt tgcctagagc ctataaaaat gggactaacg acattgaagc aagagaaaaa 2100 atggcacatg cctctaatat tgcggggatg gcatttgcaa atgctttctt aggtgtatgc 2160 cattcaatgg ctcataaact tggggcaatg catcacgttc cacatggaat tgcttgtgct 2220 gtattaatag aagaagttat taaatataac gctacagact gtccaacaaa gcaaacagca 2280 ttccctcaat ataaatctcc taatgctaag agaaaatatg ctgaaattgc agagtatttg 2340 aatttaaagg gtactagcga taccgaaaag gtaacagcct taatagaagc tatttcaaag 2400 ttaaagatag atttgagtat tccacaaaat ataagtgccg ctggaataaa taaaaaagat 2460 ttttataata cgctagataa aatgtcagag cttgcttttg atgaccaatg tacaacagct 2520 aatcctaggt atccacttat aagtgaactt aaggatatct atataaaatc attttaa 2577 <210> 7 <211> 2589 <212> DNA <213> Clostridium acetobutylicum <400> 7 atgaaagtca caacagtaaa ggaattagat gaaaaactca aggtaattaa agaagctcaa 60 aaaaaattct cttgttactc gcaagaaatg gttgatgaaa tctttagaaa tgcagcaatg 120 gcagcaatcg acgcaaggat agagctagca aaagcagctg ttttggaaac cggtatgggc 180 ttagttgaag acaaggttat aaaaaatcat tttgcaggcg aatacatcta taacaaatat 240 aaggatgaaa aaacctgcgg tataattgaa cgaaatgaac cctacggaat tacaaaaata 300 gcagaaccta taggagttgt agctgctata atccctgtaa caaaccccac atcaacaaca 360 atatttaaat ccttaatatc ccttaaaact agaaatggaa ttttcttttc gcctcaccca 420 agggcaaaaa aatccacaat actagcagct aaaacaatac ttgatgcagc cgttaagagt 480 ggtgccccgg aaaatataat aggttggata gatgaacctt caattgaact aactcaatat 540 ttaatgcaaa aagcagatat aacccttgca actggtggtc cctcactagt taaatctgct 600 tattcttccg gaaaaccagc aataggtgtt ggtccgggta acaccccagt aataattgat 660 gaatctgctc atataaaaat ggcagtaagt tcaattatat tatccaaaac ctatgataat 720 ggtgttatat gtgcttctga acaatctgta atagtcttaa aatccatata taacaaggta 780 aaagatgagt tccaagaaag aggagcttat ataataaaga aaaacgaatt ggataaagtc 840 cgtgaagtga tttttaaaga tggatccgta aaccctaaaa tagtcggaca gtcagcttat 900 actatagcag ctatggctgg cataaaagta cctaaaacca caagaatatt aataggagaa 960 gttacctcct taggtgaaga agaacctttt gcccacgaaa aactatctcc tgttttggct 1020 atgtatgagg ctgacaattt tgatgatgct ttaaaaaaag cagtaactct aataaactta 1080 ggaggcctcg gccatacctc aggaatatat gcagatgaaa taaaagcacg agataaaata 1140 gatagattta gtagtgccat gaaaaccgta agaacctttg taaatatccc aacctcacaa 1200 ggtgcaagtg gagatctata taattttaga ataccacctt ctttcacgct tggctgcgga 1260 ttttggggag gaaattctgt ttccgagaat gttggtccaa aacatctttt gaatattaaa 1320 accgtagctg aaaggagaga aaacatgctt tggtttagag ttccacataa agtatatttt 1380 aagttcggtt gtcttcaatt tgctttaaaa gatttaaaag atctaaagaa aaaaagagcc 1440 tttatagtta ctgatagtga cccctataat ttaaactatg ttgattcaat aataaaaata 1500 cttgagcacc tagatattga ttttaaagta tttaataagg ttggaagaga agctgatctt 1560 aaaaccataa aaaaagcaac tgaagaaatg tcctccttta tgccagacac tataatagct 1620 ttaggtggta cccctgaaat gagctctgca aagctaatgt gggtactata tgaacatcca 1680 gaagtaaaat ttgaagatct tgcaataaaa tttatggaca taagaaagag aatatatact 1740 ttcccaaaac tcggtaaaaa ggctatgtta gttgcaatta caacttctgc tggttccggt 1800 tctgaggtta ctccttttgc tttagtaact gacaataaca ctggaaataa gtacatgtta 1860 gcagattatg aaatgacacc aaatatggca attgtagatg cagaacttat gatgaaaatg 1920 ccaaagggat taaccgctta ttcaggtata gatgcactag taaatagtat agaagcatac 1980 acatccgtat atgcttcaga atacacaaac ggactagcac tagaggcaat acgattaata 2040 tttaaatatt tgcctgaggc ttacaaaaac ggaagaacca atgaaaaagc aagagagaaa 2100 atggctcacg cttcaactat ggcaggtatg gcatccgcta atgcatttct aggtctatgt 2160 cattccatgg caataaaatt aagttcagaa cacaatattc ctagtggcat tgccaatgca 2220 ttactaatag aagaagtaat aaaatttaac gcagttgata atcctgtaaa acaagcccct 2280 tgcccacaat ataagtatcc aaacaccata tttagatatg ctcgaattgc agattatata 2340 aagcttggag gaaatactga tgaggaaaag gtagatctct taattaacaa aatacatgaa 2400 ctaaaaaaag ctttaaatat accaacttca ataaaggatg caggtgtttt ggaggaaaac 2460 ttctattcct cccttgatag aatatctgaa cttgcactag atgatcaatg cacaggcgct 2520 aatcctagat ttcctcttac aagtgagata aaagaaatgt atataaattg ttttaaaaaa 2580 caaccttaa 2589 <210> 8 <211> 1449 <212> DNA <213> Escherichia coli <400> 8 atgaaactta acgacagtaa cttattccgc cagcaggcgt tgattaacgg ggaatggctg 60 gacgccaaca atggtgaagc catcgacgtc accaatccgg cgaacggcga caagctgggt 120 agcgtgccga aaatgggcgc ggatgaaacc cgcgccgcta tcgacgccgc caaccgcgcc 180 ctgcccgcct ggcgcgcgct caccgccaaa gaacgcgcca ccattctgcg caactggttc 240 aatttgatga tggagcatca ggacgattta gcgcgcctga tgaccctcga acagggtaaa 300 ccactggccg aagcgaaagg cgaaatcagc tacgccgcct cctttattga gtggtttgcc 360 gaagaaggca aacgcattta tggcgacacc attcctggtc atcaggccga taaacgcctg 420 attgttatca agcagccgat tggcgtcacc gcggctatca cgccgtggaa cttcccggcg 480 gcgatgatta cccgcaaagc cggtccggcg ctggcagcag gctgcaccat ggtgctgaag 540 cccgccagtc agacgccgtt ctctgcgctg gcgctggcgg agctggcgat ccgcgcgggc 600 gttccggctg gggtatttaa cgtggtcacc ggttcggcgg gcgcggtcgg taacgaactg 660 accagtaacc cgctggtgcg caaactgtcg tttaccggtt cgaccgaaat tggccgccag 720 ttaatggaac agtgcgcgaa agacatcaag aaagtgtcgc tggagctggg cggtaacgcg 780 ccgtttatcg tctttgacga tgccgacctc gacaaagccg tggaaggcgc gctggcctcg 840 aaattccgca acgccgggca aacctgcgtc tgcgccaacc gcctgtatgt gcaggacggc 900 gtgtatgacc gttttgccga aaaattgcag caggcagtga gcaaactgca catcggcgac 960 gggctggata acggcgtcac catcgggccg ctgatcgatg aaaaagcggt agcaaaagtg 1020 gaagagcata ttgccgatgc gctggagaaa ggcgcgcgcg tggtttgcgg cggtaaagcg 1080 cacgaacgcg gcggcaactt cttccagccg accattctgg tggacgttcc ggccaacgcc 1140 aaagtgtcga aagaagagac gttcggcccc ctcgccccgc tgttccgctt taaagatgaa 1200 gctgatgtga ttgcgcaagc caatgacacc gagtttggcc ttgccgccta tttctacgcc 1260 cgtgatttaa gccgcgtctt ccgcgtgggc gaagcgctgg agtacggcat cgtcggcatc 1320 aataccggca ttatttccaa tgaagtggcc ccgttcggcg gcatcaaagc ctcgggtctg 1380 ggtcgtgaag gttcgaagta tggcatcgaa gattacttag aaatcaaata tatgtgcatc 1440 ggtctttaa 1449 <210> 9 <211> 1287 <212> DNA <213> Escherichia coli <400> 9 atgaaaacct ctctgtttaa aagcctttac tttcaggtcc tgacagcgat agccattggt 60 attctccttg gccatttcta tcctgaaata ggcgagcaaa tgaaaccgct tggcgacggc 120 ttcgttaagc tcattaagat gatcatcgct cctgtcatct tttgtaccgt cgtaacgggc 180 attgcgggca tggaaagcat gaaggcggtc ggtcgtaccg gcgcagtcgc actgctttac 240 tttgaaattg tcagtaccat cgcgctgatt attggtctta tcatcgttaa cgtcgtgcag 300 cctggtgccg gaatgaacgt cgatccggca acgcttgatg cgaaagcggt agcggtttac 360 gccgatcagg cgaaagacca gggcattgtc gccttcatta tggatgtcat cccggcgagc 420 gtcattggcg catttgccag cggtaacatt ctgcaggtgc tgctgtttgc cgtactgttt 480 ggttttgcgc tccaccgtct gggcagcaaa ggccaactga tttttaacgt catcgaaagt 540 ttctcgcagg tcatcttcgg catcatcaat atgatcatgc gtctggcacc tattggtgcg 600 ttcggggcaa tggcgtttac catcggtaaa tacggcgtcg gcacactggt gcaactgggg 660 cagctgatta tctgtttcta cattacctgt atcctgtttg tggtgctggt attgggttca 720 atcgctaaag cgactggttt cagtatcttc aaatttatcc gctacatccg tgaagaactg 780 ctgattgtac tggggacttc atcttccgag tcggcgctgc cgcgtatgct cgacaagatg 840 gagaaactcg gctgccgtaa atcggtggtg gggctggtca tcccgacagg ctactcgttt 900 aaccttgatg gcacatcgat atacctgaca atggcggcgg tgtttatcgc ccaggccact 960 aacagtcaga tggatatcgt ccaccaaatc acgctgttaa tcgtgttgct gctttcttct 1020 aaaggggcgg caggggtaac gggtagtggc tttatcgtgc tggcggcgac gctctctgcg 1080 gtgggccatt tgccggtagc gggtctggcg ctgatcctcg gtatcgaccg ctttatgtca 1140 gaagctcgtg cgctgactaa cctggtcggt aacggcgtag cgaccattgt cgttgctaag 1200 tgggtgaaag aactggacca caaaaaactg gacgatgtgc tgaataatcg tgcgccggat 1260 ggcaaaacgc acgaattatc ctcttaa 1287 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aaaaatcaat gagtaaaggg ataaagaatt cac 33 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gctctagatt atttcatact accagttttt ataaa 35 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aaaaatcaat gagtaatgaa gtatctataa aag 33 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gctctagatt agataaaaaa gaggacattt cacaatatgg 40 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aaaaatcaat ggcgtttatc tactatctg 29 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gctctagatt acatggactg ctcaagcata c 31 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggaattcatg aaaacctctc tgtttaaaag c 31 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gctctagatt aagaggataa ttcgtgcgtt ttgcc 35 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 aaacatgcca tggagtggga agagatatat aaagag 36 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cgggatcctt aaaatctctt tttaaattca ttcattaatg 40 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 aaaaatcaat gaaagttaca aatcaaaaag aac 33 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gctctagatt aaaatgcttt tatatagata tc 32 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 aaaaatcaat gaaagtcaca acagtaaagg 30 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gctctagatt aaggttgttt tttaaaacaa tt 32 <110> LG Chem, Ltd. <120> Mutants Having a Producing Ability of 1,4-Butanediol and Method          for Preparing 1,4-BDO Using the Same <130> P06-B034 <160> 23 <170> KopatentIn 1.71 <210> 1 <211> 1617 <212> DNA <213> Clostridium kluyveri <400> 1 atgagtaaag ggataaagaa ttcacaattg aaaaaaaaga atgtaaaggc tagtaatgtg 60 gcagaaaaga ttgaagagaa agttgaaaaa acagataagg ttgttgaaaa ggcagctgag 120 gttactgaaa aacgaattag aaacttgaag cttcaggaaa aagttgtaac agcagatgtg 180 gcagctgata tgatagaaaa cggtatgatt gttgcaatta gcggatttac tccttccggg 240 tatcctaaag aagtacctaa agcattgact aaaaaagtta atgccttaga ggaagaattc 300 aaggtaacac tttatacagg ttcatctaca ggagccgata tagacggaga atgggcaaaa 360 gcaggaataa tagaaagaag aattccatat cagacaaatt ctgatatgag gaaaaaaata 420 aatgatggtt ctattaagta tgctgatatg catttaagcc atatggctca atatattaat 480 tattctgtaa ttcctaaagt agatatagct ataatagagg cagtagctat tacagaagaa 540 ggggatatta ttccttcaac aggaattgga aatacagcta cttttgtgga aaatgcagat 600 aaggtaatag tggaaattaa tgaggctcaa ccgcttgaat tggaaggtat ggcagatata 660 tatacattaa aaaaccctcc aagaagagag cccataccta tagttaatgc aggcaatagg 720 atagggacca catatgtgac ctgtggttct gaaaaaatat gcgctatagt gatgacaaat 780 acccaggata aaacaagacc tcttacagaa gtgtctcctg tatctcaggc tatatccgat 840 aatcttatag gatttttaaa taaagaggtt gaagagggaa aattacctaa gaacctgctt 900 cctatacagt caggagttgg aagtgtagca aatgcagttt tggccggact ttgtgaatca 960 aattttaaaa atttgagttg ttatacagaa gttatacagg attctatgct gaagcttata 1020 aaatgtggta aagcagatgt ggtgtcaggc acttccataa gtccttcacc ggagatgttg 1080 cctgagttca taaaggacat aaatttcttt agagaaaaga tagtattaag accacaggaa 1140 ataagtaata atccagagat agcaagaaga ataggagtta tatccataaa cactgctttg 1200 gaagtagata tatatggtaa tgtaaactcc actcatgtta tgggaagcaa aatgatgaat 1260 ggtataggcg gttctggaga ctttgccaga aatgcatatt tgactatatt cactacagag 1320 tctatcgcca aaaaaggaga tatatcatct atagttccta tggtatccca tgtggatcat 1380 acagaacatg atgtaatggt aattgttaca gaacagggag tagcagattt aagaggtctt 1440 tctcctaggg aaaaggccgt ggctataata gaaaattgtg ttcatcctga ttacaaggat 1500 atgcttatgg aatattttga agaggcttgt aagtcatcag gtggaaatac accacataat 1560 cttgaaaaag ctctttcctg gcatacaaaa tttataaaaa ctggtagtat gaaataa 1617 <210> 2 <211> 1419 <212> DNA <213> Clostridium kluyveri <400> 2 atgagtaatg aagtatctat aaaagaatta attgaaaagg caaaggcggc acaaaaaaaa 60 ttggaagcct atagtcaaga acaagttgat gtactagtaa aagcactagg aaaagtggtt 120 tatgataatg cagaaatgtt tgcaaaagaa gcagttgaag aaacagaaat gggtgtttat 180 gaagataaag tagctaaatg tcatttgaaa tcaggagcta tttggaatca tataaaagac 240 aagaaaactg taggcataat aaaagaagaa cctgaaaggg cacttgttta tgttgctaag 300 ccaaagggag ttgtggcagc tactacgcct ataactaatc cagtggtaac tcctatgtgt 360 aatgcaatgg ctgctataaa gggcagaaat acaataatag tagcaccaca tcctaaagca 420 aagaaagttt cagctcatac tgtagaactt atgaatgctg agcttaaaaa attgggagca 480 ccagaaaata tcatacagat agtagaagca ccatcaagag aagctgctaa ggaacttatg 540 gaaagtgctg atgtagttat tgctacaggc ggtgctggaa gagttaaagc tgcttactcc 600 agtggaagac cagcttatgg cgttggacct ggaaattcac aggtaatagt tgataaggga 660 tacgattata acaaagctgc acaggatata ataacaggaa gaaaatatga caatggaatt 720 atatgttctt cagagcaatc agttatagct cctgctgaag attatgataa ggtaatagca 780 gcttttgtag aaaatggggc attctatgta gaagatgagg aaacagtaga aaagtttaga 840 tcaactttat ttaaagatgg aaaaataaac agcaagatta taggtaaatc cgtccaaatt 900 attgcggatc ttgcaggagt aaaagtacca gaaggtacta aggttatagt acttaagggt 960 aaaggtgcag gagaaaaaga tgtactttgt aaagaaaaaa tgtgtccagt tttagtagca 1020 ttgaaatatg atacttttga agaagcagtt gaaatagcta tggctaatta tatgtatgaa 1080 ggagctggtc atacagcagg catacattct gacaatgacg agaacataag atatgcaaga 1140 actgtattac ctataagcag attagttgta aatcagcctg caactactgc tggaggaact 1200 gtattaccta taagcagatt agttgtaaat cagcctgcaa ctactgctgg aggaagtttc 1260 aataatggat ttaaccctac tactacacta ggctgcggat catggggcag aaacagtatt 1320 tcagaaaatc ttacttacga gcatcttata aatgtttcaa gaatagggta tttcaataaa 1380 gaagcaaaag ttcctagcta tgaggaaata tggggataa 1419 <210> 3 <211> 1116 <212> DNA <213> Clostridium kluyveri <400> 3 atgaagttat taaaattggc acctgatgtt tataaatttg atactgcaga ggagtttatg 60 aaatacttta aggttggaaa aggtgacttt atacttacta atgaattttt atataaacct 120 ttccttgaga aattcaatga tggtgcagat gctgtatttc aggagaaata tggactcggt 180 gaaccttctg atgaaatgat aaacaatata attaaggata ttggagataa acaatataat 240 agaattattg ctgtaggggg aggatctgta atagatatag ccaaaatcct cagtcttaag 300 tatactgatg attcattgga tttgtttgag ggaaaagtac ctcttgtaaa aaacaaagaa 360 ttaattatag ttccaactac atgtggaaca ggttcagaag ttacaaatgt atcagttgca 420 gaattaaaga gaagacatac taaaaaagga attgcttcag acgaattata tgcaacttat 480 gcagtacttg taccagaatt tataaaagga cttccatata agttttttgt aaccagctcc 540 gtagatgcct taatacatgc aacagaagct tatgtatctc caaatgcaaa tccttatact 600 gatatgttta gtgtaaaagc tatggagtta attttaaatg gatacatgca aatggtagag 660 aaaggaaatg attacagagt tgaaataatt gaggattttg ttataggcag caattatgca 720 ggtatagctt ttggaaatgc aggagtggga gcggttcacg cactctcata tccaataggc 780 ggaaattatc atgtgcctca tggagaagca aattatctgt tttttacaga aatatttaaa 840 acttattatg agaaaaatcc aaatggcaag attaaagatg taaataaact attagcaggc 900 atactaaaat gtgatgaaag tgaagcttat gacagtttat cacaactttt agataaatta 960 ttgtcaagaa aaccattaag agaatatgga atgaaagagg aagaaattga aacttttgct 1020 gattcagtaa tagaaggaca gcagagactg ttggtaaaca attatgaacc tttttcaaga 1080 gaagacatag taaacacata taaaaagtta tattaa 1116 <210> 4 <211> 1149 <212> DNA <213> Clostridium kluyveri <400> 4 atggcgttta tctactatct gacccacatc cacctggatt tcggcgcggt aagcctgctc 60 aagtccgaat gcgagcgcat cggcatccgc cgcccgttgc tggtgaccga caagggcgtg 120 gtcgccgcgg gagtggcgca gcgtgccatc gatgcaatgc agggcctgca ggttgcggta 180 ttcgatgaaa ccccgtcgaa cccgaccgag gccatggtgc gcaaggccgc cgcacaatac 240 cgcgaggccg gctgcgacgg gctggtggca gtgggcggcg gctcgtcgat cgacctcgcc 300 aagggcatcg ccatcctggc cacgcatgag ggcgagctga ccacctatgc caccatcgaa 360 ggcggcagcg ccaggatcac cgacaaggcg gcgccgctga tcgcggtgcc caccacctcg 420 ggcaccggca gcgaggtggc gcgcggcgcc atcatcatcc tggacgacgg ccgcaagctg 480 ggcttccatt cctggcattt gctgcccaag tccgccgtct gcgacccgga actgacgctg 540 gggctgccgg ccgggctgac cgcggccacc ggcatggatg cgatcgcgca ctgcatcgag 600 accttcctgg cccccgcctt caacccgccc gcggacggca ttgcgctgga cgggctggag 660 cgcggctggg gccatatcga acgcgccacc cgcgacggtc aggaccgcga cgcacgcctg 720 aacatgatga gcgcgtcgat gcagggcgca atggcgttcc agaaggggct gggctgcgtg 780 cattcgctgt cgcacccgct gggcgggctg aagatcgacg gccgcaccgg cctgcaccac 840 ggcacgctca acgcggtggt gatgccggcg gtgctgcgct tcaacgccga tgcgcccacg 900 gtggtgcgcg acgaccgcta cgcacgcctg cgccgcgcca tgcacctgcc cgacggcgcc 960 gatatcgcgc aggccgtgca cgacatgacc gtgcgcctgg gcctgcccac cgggctgcgt 1020 cagatgggtg tcaccgagga catgttcgac aaggtgattg ccggtgcgct ggtcgaccat 1080 tgccacaaga ccaacccgaa agaagccagc gccgcggatt atcggcgtat gcttgagcag 1140 tccatgtag 1149 <210> 5 <211> 1290 <212> DNA <213> Clostridium acetobutylicum <400> 5 atggagtggg aagagatata taaagagaaa ctggtaactg cagaaaaagc tgtttcaaaa 60 atagaaaacc atagcagggt agtttttgca catgcagtag gagaacccgt agatttagta 120 aatgcactag ttaaaaataa ggataattat ataggactag aaatagttca catggtagct 180 atgggcaaag gtgtatatac aaaagagggt atgcaaagac attttagaca taatgctttg 240 tttgtaggcg gatctactag agatgcagta aattcaggaa gagcagttta tacaccttgt 300 tttttctatg aagtgccaag tttgtttaaa gaaaaacgtt tgcctgtaga tgtagcactt 360 attcaggtaa gtgagccaga taaatatggc tactgcagtt ttggagtttc caatgactat 420 accaagccag cagcagaaag tgctaagctt gtaattgcag aagtgaataa aaacatgcca 480 agaactcttg gagattcttt tatacatgta tcagatattg attatatagt ggaagcttca 540 cacccattgt tagaattgca gcctcctaaa ttgggagatg tagaaaaagc cataggagaa 600 aactgtgcat ctttaattga agatggagct actcttcagc ttggaatagg tgctatacca 660 gatgcggtac ttttattctt aaagaacaaa aagaatttag gaatacattc tgagatgata 720 tcagatggtg tgatggaact ggtgaaggca ggggttatca ataacaagaa aaagaccctc 780 catccaggca aaatagttgt aacattttta atgggaacaa aaaaattata tgattttgta 840 aacaataatc caatggtaga aacttattct gtagattatg taaataatcc actggtaatt 900 atgaaaaatg acaatatggt ttcaataaat tcttgtgttc aagtagactt aatgggacaa 960 gtatgttctg aaagtatagg attgaaacag ataagtggag tgggaggcca ggtagatttt 1020 attagaggag ctaatctatc aaagggtgga aaggctatta tagctatacc ttccacagct 1080 ggaaaaggaa aagtttcaag aataactcca cttctagata ctggtgctgc agttacaact 1140 tctagaaatg aagtagatta tgtagttact gaatatggtg ttgctcatct taagggcaaa 1200 actttaagaa atagggcaag agctctaata aatatcgctc atccaaaatt cagagaatca 1260 ttaatgaatg aatttaaaaa gagattttag 1290 <210> 6 <211> 2577 <212> DNA <213> Clostridium acetobutylicum <400> 6 atgaaagtta caaatcaaaa agaactaaaa caaaagctaa atgaattgag agaagcgcaa 60 aagaagtttg caacctatac tcaagagcaa gttgataaaa tttttaaaca atgtgccata 120 gccgcagcta aagaaagaat aaacttagct aaattagcag tagaagaaac aggaataggt 180 cttgtagaag ataaaattat aaaaaatcat tttgcagcag aatatatata caataaatat 240 aaaaaatgaaa aaacttgtgg cataatagac catgacgatt ctttaggcat aacaaaggtt 300 gctgaaccaa ttggaattgt tgcagccata gttcctacta ctaatccaac ttccacagca 360 attttcaaat cattaatttc tttaaaaaca agaaacgcaa tattcttttc accacatcca 420 cgtgcaaaaa aatctacaat tgctgcagca aaattaattt tagatgcagc tgttaaagca 480 ggagcaccta aaaatataat aggctggata gatgagccat caatagaact ttctcaagat 540 ttgatgagtg aagctgatat aatattagca acaggaggtc cttcaatggt taaagcggcc 600 tattcatctg gaaaacctgc aattggtgtt ggagcaggaa atacaccagc aataatagat 660 gagagtgcag atatagatat ggcagtaagc tccataattt tatcaaagac ttatgacaat 720 ggagtaatat gcgcttctga acaatcaata ttagttatga attcaatata cgaaaaagtt 780 aaagaggaat ttgtaaaacg aggatcatat atactcaatc aaaatgaaat agctaaaata 840 aaagaaacta tgtttaaaaa tggagctatt aatgctgaca tagttggaaa atctgcttat 900 ataattgcta aaatggcagg aattgaagtt cctcaaacta caaagatact tataggcgaa 960 gtacaatctg ttgaaaaaag cgagctgttc tcacatgaaa aactatcacc agtacttgca 1020 atgtataaag ttaaggattt tgatgaagct ctaaaaaagg cacaaaggct aatagaatta 1080 ggtggaagtg gacacacgtc atctttatat atagattcac aaaacaataa ggataaagtt 1140 aaagaatttg gattagcaat gaaaacttca aggacattta ttaacatgcc ttcttcacag 1200 ggagcaagcg gagatttata caattttgcg atagcaccat catttactct tggatgcggc 1260 acttggggag gaaactctgt atcgcaaaat gtagagccta aacatttatt aaatattaaa 1320 agtgttgctg aaagaaggga aaatatgctt tggtttaaag tgccacaaaa aatatatttt 1380 aaatatggat gtcttagatt tgcattaaaa gaattaaaag atatgaataa gaaaagagcc 1440 tttatagtaa cagataaaga tctttttaaa cttggatatg ttaataaaat aacaaaggta 1500 ctagatgaga tagatattaa atacagtata tttacagata ttaaatctga tccaactatt 1560 gattcagtaa aaaaaggtgc taaagaaatg cttaactttg aacctgatac tataatctct 1620 attggtggtg gatcgccaat ggatgcagca aaggttatgc acttgttata tgaatatcca 1680 gaagcagaaa ttgaaaatct agctataaac tttatggata taagaaagag aatatgcaat 1740 ttccctaaat taggtacaaa ggcgatttca gtagctattc ctacaactgc tggtaccggt 1800 tcagaggcaa caccttttgc agttataact aatgatgaaa caggaatgaa atacccttta 1860 acttcttatg aattgacccc aaacatggca ataatagata ctgaattaat gttaaatatg 1920 cctagaaaat taacagcagc aactggaata gatgcattag ttcatgctat agaagcatat 1980 gtttcggtta tggctacgga ttatactgat gaattagcct taagagcaat aaaaatgata 2040 tttaaatatt tgcctagagc ctataaaaat gggactaacg acattgaagc aagagaaaaa 2100 atggcacatg cctctaatat tgcggggatg gcatttgcaa atgctttctt aggtgtatgc 2160 cattcaatgg ctcataaact tggggcaatg catcacgttc cacatggaat tgcttgtgct 2220 gtattaatag aagaagttat taaatataac gctacagact gtccaacaaa gcaaacagca 2280 ttccctcaat ataaatctcc taatgctaag agaaaatatg ctgaaattgc agagtatttg 2340 aatttaaagg gtactagcga taccgaaaag gtaacagcct taatagaagc tatttcaaag 2400 ttaaagatag atttgagtat tccacaaaat ataagtgccg ctggaataaa taaaaaagat 2460 ttttataata cgctagataa aatgtcagag cttgcttttg atgaccaatg tacaacagct 2520 aatcctaggt atccacttat aagtgaactt aaggatatct atataaaatc attttaa 2577 <210> 7 <211> 2589 <212> DNA <213> Clostridium acetobutylicum <400> 7 atgaaagtca caacagtaaa ggaattagat gaaaaactca aggtaattaa agaagctcaa 60 aaaaaattct cttgttactc gcaagaaatg gttgatgaaa tctttagaaa tgcagcaatg 120 gcagcaatcg acgcaaggat agagctagca aaagcagctg ttttggaaac cggtatgggc 180 ttagttgaag acaaggttat aaaaaatcat tttgcaggcg aatacatcta taacaaatat 240 aaggatgaaa aaacctgcgg tataattgaa cgaaatgaac cctacggaat tacaaaaata 300 gcagaaccta taggagttgt agctgctata atccctgtaa caaaccccac atcaacaaca 360 atatttaaat ccttaatatc ccttaaaact agaaatggaa ttttcttttc gcctcaccca 420 agggcaaaaa aatccacaat actagcagct aaaacaatac ttgatgcagc cgttaagagt 480 ggtgccccgg aaaatataat aggttggata gatgaacctt caattgaact aactcaatat 540 ttaatgcaaa aagcagatat aacccttgca actggtggtc cctcactagt taaatctgct 600 tattcttccg gaaaaccagc aataggtgtt ggtccgggta acaccccagt aataattgat 660 gaatctgctc atataaaaat ggcagtaagt tcaattatat tatccaaaac ctatgataat 720 ggtgttatat gtgcttctga acaatctgta atagtcttaa aatccatata taacaaggta 780 aaagatgagt tccaagaaag aggagcttat ataataaaga aaaacgaatt ggataaagtc 840 cgtgaagtga tttttaaaga tggatccgta aaccctaaaa tagtcggaca gtcagcttat 900 actatagcag ctatggctgg cataaaagta cctaaaacca caagaatatt aataggagaa 960 gttacctcct taggtgaaga agaacctttt gcccacgaaa aactatctcc tgttttggct 1020 atgtatgagg ctgacaattt tgatgatgct ttaaaaaaag cagtaactct aataaactta 1080 ggaggcctcg gccatacctc aggaatatat gcagatgaaa taaaagcacg agataaaata 1140 gatagattta gtagtgccat gaaaaccgta agaacctttg taaatatccc aacctcacaa 1200 ggtgcaagtg gagatctata taattttaga ataccacctt ctttcacgct tggctgcgga 1260 ttttggggag gaaattctgt ttccgagaat gttggtccaa aacatctttt gaatattaaa 1320 accgtagctg aaaggagaga aaacatgctt tggtttagag ttccacataa agtatatttt 1380 aagttcggtt gtcttcaatt tgctttaaaa gatttaaaag atctaaagaa aaaaagagcc 1440 tttatagtta ctgatagtga cccctataat ttaaactatg ttgattcaat aataaaaata 1500 cttgagcacc tagatattga ttttaaagta tttaataagg ttggaagaga agctgatctt 1560 aaaaccataa aaaaagcaac tgaagaaatg tcctccttta tgccagacac tataatagct 1620 ttaggtggta cccctgaaat gagctctgca aagctaatgt gggtactata tgaacatcca 1680 gaagtaaaat ttgaagatct tgcaataaaa tttatggaca taagaaagag aatatatact 1740 ttcccaaaac tcggtaaaaa ggctatgtta gttgcaatta caacttctgc tggttccggt 1800 tctgaggtta ctccttttgc tttagtaact gacaataaca ctggaaataa gtacatgtta 1860 gcagattatg aaatgacacc aaatatggca attgtagatg cagaacttat gatgaaaatg 1920 ccaaagggat taaccgctta ttcaggtata gatgcactag taaatagtat agaagcatac 1980 acatccgtat atgcttcaga atacacaaac ggactagcac tagaggcaat acgattaata 2040 tttaaatatt tgcctgaggc ttacaaaaac ggaagaacca atgaaaaagc aagagagaaa 2100 atggctcacg cttcaactat ggcaggtatg gcatccgcta atgcatttct aggtctatgt 2160 cattccatgg caataaaatt aagttcagaa cacaatattc ctagtggcat tgccaatgca 2220 ttactaatag aagaagtaat aaaatttaac gcagttgata atcctgtaaa acaagcccct 2280 tgcccacaat ataagtatcc aaacaccata tttagatatg ctcgaattgc agattatata 2340 aagcttggag gaaatactga tgaggaaaag gtagatctct taattaacaa aatacatgaa 2400 ctaaaaaaag ctttaaatat accaacttca ataaaggatg caggtgtttt ggaggaaaac 2460 ttctattcct cccttgatag aatatctgaa cttgcactag atgatcaatg cacaggcgct 2520 aatcctagat ttcctcttac aagtgagata aaagaaatgt atataaattg ttttaaaaaa 2580 caaccttaa 2589 <210> 8 <211> 1449 <212> DNA <213> Escherichia coli <400> 8 atgaaactta acgacagtaa cttattccgc cagcaggcgt tgattaacgg ggaatggctg 60 gacgccaaca atggtgaagc catcgacgtc accaatccgg cgaacggcga caagctgggt 120 agcgtgccga aaatgggcgc ggatgaaacc cgcgccgcta tcgacgccgc caaccgcgcc 180 ctgcccgcct ggcgcgcgct caccgccaaa gaacgcgcca ccattctgcg caactggttc 240 aatttgatga tggagcatca ggacgattta gcgcgcctga tgaccctcga acagggtaaa 300 ccactggccg aagcgaaagg cgaaatcagc tacgccgcct cctttattga gtggtttgcc 360 gaagaaggca aacgcattta tggcgacacc attcctggtc atcaggccga taaacgcctg 420 attgttatca agcagccgat tggcgtcacc gcggctatca cgccgtggaa cttcccggcg 480 gcgatgatta cccgcaaagc cggtccggcg ctggcagcag gctgcaccat ggtgctgaag 540 cccgccagtc agacgccgtt ctctgcgctg gcgctggcgg agctggcgat ccgcgcgggc 600 gttccggctg gggtatttaa cgtggtcacc ggttcggcgg gcgcggtcgg taacgaactg 660 accagtaacc cgctggtgcg caaactgtcg tttaccggtt cgaccgaaat tggccgccag 720 ttaatggaac agtgcgcgaa agacatcaag aaagtgtcgc tggagctggg cggtaacgcg 780 ccgtttatcg tctttgacga tgccgacctc gacaaagccg tggaaggcgc gctggcctcg 840 aaattccgca acgccgggca aacctgcgtc tgcgccaacc gcctgtatgt gcaggacggc 900 gtgtatgacc gttttgccga aaaattgcag caggcagtga gcaaactgca catcggcgac 960 gggctggata acggcgtcac catcgggccg ctgatcgatg aaaaagcggt agcaaaagtg 1020 gaagagcata ttgccgatgc gctggagaaa ggcgcgcgcg tggtttgcgg cggtaaagcg 1080 cacgaacgcg gcggcaactt cttccagccg accattctgg tggacgttcc ggccaacgcc 1140 aaagtgtcga aagaagagac gttcggcccc ctcgccccgc tgttccgctt taaagatgaa 1200 gctgatgtga ttgcgcaagc caatgacacc gagtttggcc ttgccgccta tttctacgcc 1260 cgtgatttaa gccgcgtctt ccgcgtgggc gaagcgctgg agtacggcat cgtcggcatc 1320 aataccggca ttatttccaa tgaagtggcc ccgttcggcg gcatcaaagc ctcgggtctg 1380 ggtcgtgaag gttcgaagta tggcatcgaa gattacttag aaatcaaata tatgtgcatc 1440 ggtctttaa 1449 <210> 9 <211> 1287 <212> DNA <213> Escherichia coli <400> 9 atgaaaacct ctctgtttaa aagcctttac tttcaggtcc tgacagcgat agccattggt 60 attctccttg gccatttcta tcctgaaata ggcgagcaaa tgaaaccgct tggcgacggc 120 ttcgttaagc tcattaagat gatcatcgct cctgtcatct tttgtaccgt cgtaacgggc 180 attgcgggca tggaaagcat gaaggcggtc ggtcgtaccg gcgcagtcgc actgctttac 240 tttgaaattg tcagtaccat cgcgctgatt attggtctta tcatcgttaa cgtcgtgcag 300 cctggtgccg gaatgaacgt cgatccggca acgcttgatg cgaaagcggt agcggtttac 360 gccgatcagg cgaaagacca gggcattgtc gccttcatta tggatgtcat cccggcgagc 420 gtcattggcg catttgccag cggtaacatt ctgcaggtgc tgctgtttgc cgtactgttt 480 ggttttgcgc tccaccgtct gggcagcaaa ggccaactga tttttaacgt catcgaaagt 540 ttctcgcagg tcatcttcgg catcatcaat atgatcatgc gtctggcacc tattggtgcg 600 ttcggggcaa tggcgtttac catcggtaaa tacggcgtcg gcacactggt gcaactgggg 660 cagctgatta tctgtttcta cattacctgt atcctgtttg tggtgctggt attgggttca 720 atcgctaaag cgactggttt cagtatcttc aaatttatcc gctacatccg tgaagaactg 780 ctgattgtac tggggacttc atcttccgag tcggcgctgc cgcgtatgct cgacaagatg 840 gagaaactcg gctgccgtaa atcggtggtg gggctggtca tcccgacagg ctactcgttt 900 aaccttgatg gcacatcgat atacctgaca atggcggcgg tgtttatcgc ccaggccact 960 aacagtcaga tggatatcgt ccaccaaatc acgctgttaa tcgtgttgct gctttcttct 1020 aaaggggcgg caggggtaac gggtagtggc tttatcgtgc tggcggcgac gctctctgcg 1080 gtgggccatt tgccggtagc gggtctggcg ctgatcctcg gtatcgaccg ctttatgtca 1140 gaagctcgtg cgctgactaa cctggtcggt aacggcgtag cgaccattgt cgttgctaag 1200 tgggtgaaag aactggacca caaaaaactg gacgatgtgc tgaataatcg tgcgccggat 1260 ggcaaaacgc acgaattatc ctcttaa 1287 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aaaaaatcaat gagtaaaggg ataaagaatt cac 33 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gctctagatt atttcatact accagttttt ataaa 35 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aaaaaatcaat gagtaatgaa gtatctataa aag 33 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gctctagatt agataaaaaa gaggacattt cacaatatgg 40 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aaaaatcaat ggcgtttatc tactatctg 29 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gctctagatt acatggactg ctcaagcata c 31 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ggaattcatg aaaacctctc tgtttaaaag c 31 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gctctagatt aagaggataa ttcgtgcgtt ttgcc 35 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 aaacatgcca tggagtggga agagatatat aaagag 36 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cgggatcctt aaaatctctt tttaaattca ttcattaatg 40 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 aaaaatcaat gaaagttaca aatcaaaaag aac 33 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gctctagatt aaaatgcttt tatatagata tc 32 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 aaaaatcaat gaaagtcaca acagtaaagg 30 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gctctagatt aaggttgttt tttaaaacaa tt 32

Claims (30)

숙신산 고생성능을 가지는 미생물에서, 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자와 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자가 도입 또는 증폭되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자가 결실되어 있는 것을 특징으로 하는 1,4-BDO 고생성능을 가지는 변이체.In microorganisms with high succinic acid performance, the genes of enzymes that convert succinate to 4HB (4-hydroxybutyrate) and the enzymes that convert 4HB to 1,4-BDO (1,4-butanediol) are introduced or amplified. A variant having 1,4-BDO high performance, characterized in that a gene involved in converting succinate semialdehyde to succinic acid is deleted. 제1항에 있어서, 상기 숙신산 고생성능을 가지는 미생물은 박테리아, 효모 및 곰팡이로 구성된 군에서 선택되는 것을 특징으로 하는 변이체. The variant according to claim 1, wherein the microorganism having high succinic acid performance is selected from the group consisting of bacteria, yeast and mold. 제2항에 있어서, 상기 박테리아는 루멘박테리아, 코리네박테리움(Corynebacterium) 속, 브레비박테리움(Brevibacterium) 속 및 대장균으로 구성된 군에서 선택되는 것을 특징으로 하는 변이체. The variant according to claim 2, wherein the bacterium is selected from the group consisting of lumen bacteria, Corynebacterium genus, Brevibacterium genus and Escherichia coli. 제3항에 있어서, 상기 루멘박테리아는, 젖산 탈수소화효소를 코딩하는 유전자(ldhA)와 피루브산-개미산 분해효소를 코딩하는 유전자(pfl)가 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 하는 변이체.The method of claim 3, wherein the lumen bacteria, the gene encoding lactic acid dehydrogenase ( ldhA ) and the gene encoding pyruvate-formic acid degrading enzyme ( pfl ) is deleted, almost no other organic acid produced under anaerobic conditions Variant, characterized in that it has a characteristic of producing a high concentration of succinic acid without. 제3항에 있어서, 상기 루멘박테리아는, 젖산 탈수소화효소를 코딩하는 유전자(ldhA), 피루브산-개미산 분해효소를 코딩하는 유전자(pfl), 포스포트랜스아세틸화효소를 코딩하는 유전자(pta) 및 아세트산 키나제를 코딩하는 유전자(ackA)가 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 하는 변이체. The method of claim 3, wherein the lumen bacterium is a gene encoding lactic acid dehydrogenase ( ldhA ), a gene encoding pyruvate-formic acid degrading enzyme ( pfl ), a gene encoding phosphotransacetylacetylase ( pta ) and A variant, characterized in that the gene encoding acetic acid kinase ( ackA ) is deleted and that succinic acid is produced at high concentration with little generation of other organic acids under anaerobic conditions. 제3항에 있어서, 상기 루멘박테리아는, 젖산 탈수소화효소를 코딩하는 유전자(ldhA), 피루브산-개미산 분해효소를 코딩하는 유전자(pfl) 및 포스포피루브산 카르복실라제를 코딩하는 유전자(ppc)가 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 특징으로 하는 변이체. The method of claim 3, wherein the lumen bacterium is a gene encoding lactic acid dehydrogenase ( ldhA ), a gene encoding pyruvate-formic acid degrading enzyme ( pfl ) and a gene encoding phosphopyruvic acid carboxylase ( ppc ). A variant, characterized in that it has been deleted and has a characteristic of producing high concentrations of succinic acid in the anaerobic conditions with little generation of other organic acids. 제4항 내지 제6항 중 어느 한 항에 있어서, 상기 루멘 박테리아는 맨하이미아 속(Mannheimia sp.), 액티노바실러스 속(Actinobacillus sp.) 및 언에어로바이 오스피리륨 속(Anaerobiospirillum sp.)으로 구성된 군에서 선택되는 것을 특징으로 하는 변이체.The method of claim 4, wherein the lumen bacteria are of the genus Mannheimia sp. , Actinobacillus sp., And Anaerobiospirillum sp. Variant, characterized in that selected from the group consisting of. 제7항에 있어서, 상기 루멘 박테리아는 맨하이미아 속(Mannheimia sp .)인 것을 특징으로 하는 변이체. The method of claim 7, wherein the lumen bacteria is Mannheimia sp .) variant. 제8항에 있어서, 상기 루멘 박테리아는 맨하이미아 숙시니시프로듀슨스 MBEL55E(KCTC 0769BP), 맨하이미아 속 LPK(KCTC 10558BP), LPK4 및 LPK7(KCTC 10626BP)로 구성된 군에서 선택되는 것을 특징으로 하는 변이체. The method of claim 8, wherein the lumen bacteria are selected from the group consisting of S. nichia succinsis producers MBEL55E (KCTC 0769BP), the genus LPK (KCTC 10558BP), LPK4 and LPK7 (KCTC 10626BP) Variants. 제3항에 있어서, 상기 대장균은 포도당 인산전이효소를 코딩하는 유전자(ptsG) 및 피루베이트 키나아제를 코딩하는 유전자(pykA 및 pykF)가 모두 결실되어 있고, 혐기적 조건에서 다른 유기산은 거의 생성하지 않으면서 숙신산을 고농도로 생성하는 특성을 가지는 것을 하는 변이체. The method of claim 3, wherein the E. coli is deleted both the gene encoding the glucose phosphatase ( ptsG ) and the gene encoding pyruvate kinase ( pykA and pykF ), and rarely other organic acids are produced under anaerobic conditions A variant that has the characteristic of producing succinic acid at a high concentration. 제10항에 있어서, 대장균 변이체는 W3110GFA인 것을 특징으로 하는 변이체. The variant of claim 10, wherein the E. coli variant is W3110GFA. 제1항에 있어서, 상기 숙신산을 4HB로 전환하는 효소의 유전자와 상기 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자는 클로스트리디움 클루이베리(Clostridium kluyveri) 유래인 것을 특징으로 하는 변이체.According to claim 1, wherein the gene of the enzyme for converting the succinic acid to 4HB and the gene involved in the conversion of the succinate semialdehyde (succinate semialdehyde) to succinic acid variant, characterized in that derived from Clostridium kluyveri . 제1항에 있어서, 상기 숙신산을 4HB로 전환하는 효소의 유전자는 Cat1(succinyl-CoA transferase)를 코딩하는 유전자, SucD(succinate semialdehyde dehydrogenase)를 코딩하는 유전자, 4hbD(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자 및 GHB(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자로 구성된 군에서 선택되는 것을 특징으로 하는 변이체.According to claim 1, wherein the gene of the enzyme for converting succinic acid to 4HB is a gene encoding Cat1 (succinyl-CoA transferase), a gene encoding a succinate semialdehyde dehydrogenase (SucD), 4hbD (4-hydroxybutyrate dehydrogenase) And a gene selected from the group consisting of genes encoding 4-hydroxybutyrate dehydrogenase (GHB). 제13항에 있어서, 상기 Cat1를 코딩하는 유전자는 서열번호 1, SucD를 코딩하는 유전자는 서열번호 2, 4hbD를 코딩하는 유전자는 서열번호 3 및 GHB를 코딩하는 유전자는 서열번호 4의 염기서열을 가지는 것을 특징으로 하는 변이체.The gene encoding Cat1 according to claim 13, wherein the gene encoding Cat1 is SEQ ID NO: 1, the gene encoding SucD is SEQ ID NO: 2, 4hbD is a gene encoding SEQ ID NO: 3 and GHB is a nucleotide sequence of SEQ ID NO: 4 Variants characterized by having. 제1항에 있어서, 상기 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자는 클로스트리디움 아세토부틸리쿰(Clostridium acetobutylicum) 유래인 것을 특징으로 하는 변이체. According to claim 1, wherein the gene of the enzyme for converting 4HB to 1,4-BDO (1,4-butanediol) variant, characterized in that derived from Clostridium acetobutylicum ( Clostridium acetobutylicum ). 제1항에 있어서, 상기 4HB를 1,4-BDO(1,4-butanediol)로 전환하는 효소의 유전자는 Cat2(4-hydroxybutyrate-CoA transferase)를 코딩하는 유전자 또는 4HB-CoA를 환원하는 alcohol dehydrogenase를 코딩하는 유전자인 것을 특징으로 하는 변이체. According to claim 1, wherein the gene of the enzyme for converting 4HB to 1,4-BDO (1,4-butanediol) is a gene encoding Cat2 (4-hydroxybutyrate-CoA transferase) or alcohol dehydrogenase to reduce 4HB-CoA The variant, characterized in that the gene encoding. 제16항에 있어서, 상기 alcohol dehydrogenase는 클로스트리디움 아세토부틸리쿰(Clostridium acetobutylicum) 유래 butyl-CoA dehydrogenase인 것을 특징으로 하는 변이체. The variant according to claim 16, wherein the alcohol dehydrogenase is butyl-CoA dehydrogenase derived from Clostridium acetobutylicum . 제16항에 있어서, 상기 4-hydroxybutyrate-CoA transferase를 코딩하는 유전자는 서열번호 5의 염기서열을 가지는 것을 특징으로 하는 변이체. The variant according to claim 16, wherein the gene encoding 4-hydroxybutyrate-CoA transferase has a nucleotide sequence of SEQ ID NO: 5. 제17항에 있어서, 상기 butyl-CoA dehydrogenase를 코딩하는 유전자는 서열번호 6(CAP0035) 또는 서열번호 7(CAP0162)의 염기서열을 가지는 것을 특징으로 하는 변이체. The variant according to claim 17, wherein the gene encoding butyl-CoA dehydrogenase has a nucleotide sequence of SEQ ID NO: 6 (CAP0035) or SEQ ID NO: 7 (CAP0162). 제1항에 있어서, 상기 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자는 GabD(succinic semialdehyde dehydrogenase)를 코딩하는 유전자인 것을 특징으로 하는 변이체.The variant according to claim 1, wherein the gene involved in converting succinate semialdehyde to succinic acid is a gene encoding succinic semialdehyde dehydrogenase (GabD). 제20항에 있어서, 상기 GabD를 코딩하는 유전자는 서열번호 8의 염기서열을 가지는 것을 특징으로 하는 변이체.The variant according to claim 20, wherein the GabD encoding gene has a nucleotide sequence of SEQ ID NO: 8. 제1항 내지 제6항, 제10항 내지 제21항 중 어느 한 항에 있어서, 숙신산의 운송에 관여하는 DctA(C4-dicarboxylate transport protein)를 코딩하는 유전자가 추가로 도입 또는 증폭되어 있는 것을 특징으로 하는 변이체. The gene according to any one of claims 1 to 6 and 10 to 21, wherein the gene encoding DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid is further introduced or amplified. Variant. 제22항에 있어서, 상기 DctA를 코딩하는 유전자는 서열번호 9의 염기서열을 가지는 것을 특징으로 하는 변이체.The variant according to claim 22, wherein the gene encoding the DctA has a nucleotide sequence of SEQ ID NO: 9. 숙신산 고생성능을 가지는 미생물에서 GabD를 코딩하는 유전자가 결실되어 있고, Cat1을 코딩하는 유전자, SucD를 코딩하는 유전자, 4hbD를 코딩하는 유전자, GHB를 코딩하는 유전자, 4-hydroxybutyrate-CoA transferase를 코딩하는 유전자 ㅁ및 butyl-CoA dehydrogenase를 코딩하는 유전자가 모두 도입 또는 증폭되어 있는 것을 특징으로 하는 1,4-BDO 고생성능을 가지는 미생물 변이체. GabD-encoding genes are deleted from microorganisms with high succinic acid, genes encoding Cat1, genes encoding SucD, genes encoding 4hbD, genes encoding GHB, and 4-hydroxybutyrate-CoA transferase. A microbial variant having high 1,4-BDO performance, characterized in that both the genes encoding the gene K and the butyl-CoA dehydrogenase are introduced or amplified. 제24항에 있어서, 숙신산의 운송에 관여하는 DctA(C4-dicarboxylate transport protein)를 코딩하는 유전자가 추가로 도입 또는 증폭되어 있는 것을 특징으로 하는 변이체. The variant according to claim 24, wherein the gene encoding DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid is further introduced or amplified. 제1항 내지 제6항, 제10항 내지 제21항 중 어느 한 항의 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 1,4-BDO(1,4-butanediol)를 수득하는 것을 특징으로 하는 1,4-BDO의 제조방법.22. The culturing of the variant of any one of claims 1 to 6 and 10 to 21 in a medium containing a carbohydrate, and then obtaining 1,4-BDO (1,4-butanediol) from the culture solution 1,4-BDO manufacturing method characterized in that. 제24항 또는 제25항의 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 1,4-BDO(1,4-butanediol)를 수득하는 것을 특징으로 하는 1,4-BDO의 제조방법.A method for producing 1,4-BDO, comprising culturing the variant of claim 24 or 25 in a medium containing carbohydrate, and then obtaining 1,4-BDO (1,4-butanediol) from the culture solution. 서열번호 6의 염기서열을 가지는 CAP0035(butyl-CoA dehydrogenase) 유전자. CAP0035 (butyl-CoA dehydrogenase) gene having a nucleotide sequence of SEQ ID NO: 6. 서열번호 7의 염기서열을 가지는 CAP0162(butyl-CoA dehydrogenase) 유전자. CAP0162 (butyl-CoA dehydrogenase) gene having a nucleotide sequence of SEQ ID NO. 제28항 또는 제29항의 유전자를 함유하는 재조합벡터.A recombinant vector containing the gene of claim 28 or 29.
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