KR20070096347A - Mutants having a producing ability of 4-hydroxybutyrate and method for preparing 4hb using the same - Google Patents
Mutants having a producing ability of 4-hydroxybutyrate and method for preparing 4hb using the same Download PDFInfo
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Abstract
Description
도 1은 숙신산으로부터 4HB를 생산하는 경로를 나타낸 모식도이다.1 is a schematic diagram showing a route for producing 4HB from succinic acid.
본 발명은 4HB(4-hydroxybutyrate) 생성능을 가지는 변이체 및 이를 이용한 4HB의 제조방법에 관한 것으로, 보다 구체적으로는, 숙신산 고생성능을 가지는 미생물에 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자가 도입되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자가 결실되어 있는 4HB 고생성능 변이체 및 상기 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 4HB(4-hydroxybutyrate)를 수득하는 것을 특징으로 하는 4HB의 제조방법에 관한 것이다.The present invention relates to a variant having 4HB (4-hydroxybutyrate) generating ability and a method for producing 4HB using the same, and more specifically, to convert succinic acid (succinate) to 4HB (4-hydroxybutyrate) to microorganisms having high succinic acid performance The 4HB high-performance variant in which the gene of the enzyme was introduced and the gene involved in converting succinate semialdehyde to succinic acid and the variant was deleted, and the variant was cultured in a medium containing carbohydrate, and then 4HB (from the culture medium). It relates to a method for producing 4HB, characterized in that 4-hydroxybutyrate) 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-butanediol, gammabutyrolactone (GBL) 및 THF 등의 다양한 탄소수 4개의 화학물질로 쉽게 전환될 수 있다. 상기, 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 화학생산공적의 대안으로 생물학적 공정이 제시되고 있다. In addition, 4HB can be easily converted to various 4 carbon atoms such as 1,4-butanediol, gammabutyrolactone (GBL) and THF. The various chemicals are used throughout the chemical industry as polymers, solvents, fine chemical intermediates, and the like. 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, biological processes have been proposed as an alternative to chemical production.
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 제10-2005-0076301호, 제10-2005-0076317호 및 제10-2005-0076348호에서는 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 No. 10-2004-60149 discloses a method for producing E. coli mutants capable of producing succinic acid at high concentration, and Korean Patent Application Nos. 10-2005-0076301, 10-2005-0076317, and 10-2005-0076348 discloses a method for producing succinic acid using a novel gene.
이에, 당업계에서는 상기 특허에 개시된 숙신산을 고농도로 생산할 수 있는 미생물을 이용하여, 화학산업 전반에 걸쳐 중요하게 사용되고 있는 탄소수 4개의 화학물질의 전구체인 4HB(4-hydroxybutyrate) 생성능을 가지는 변이체 및 이를 이용한 4HB의 생물학적 제조방법이 절실하게 요구되고 있는 실정이다. Thus, in the art using a microorganism capable of producing a high concentration of succinic acid disclosed in the patent, the variant having the ability to produce 4HB (4-hydroxybutyrate), which is a precursor of four carbon atoms chemically used throughout the chemical industry and this There is an urgent need for a method for biological preparation of 4HB.
이에 본 발명자들은 숙신산을 고농도로 생산할 수 있는 미생물을 이용하여 4-hydroxybutyrate(4HB)를 제조하고자 예의 노력한 결과, 클로스트리디움(Clostridium) 균주로부터 유래된 4HB 생합성 관련 유전자를 숙신산을 대량으로 생산할 수 있는 미생물에 도입시켜, 4HB 생성 미생물 변이체를 수득하고, 상기 미생물 변이체가 4HB를 효율적으로 생산하는 것을 확인하고 본 발명을 완성하게 되었 다. Therefore, the present inventors have made efforts to prepare 4-hydroxybutyrate (4HB) using microorganisms capable of producing succinic acid at high concentrations, and as a result, 4HB biosynthesis related genes derived from Clostridium strain can be produced in large quantities. It was introduced into the microorganisms to obtain 4HB producing microbial variants, which confirmed that the microbial variants produced 4HB efficiently and completed the present invention.
결국, 본 발명의 주된 목적은 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자가 도입 또는 증폭되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자가 결실되어 있는 것을 특징으로 하는 4HB 고생성능을 가지는 변이체를 제공하는데 있다.As a result, the main object of the present invention is that the gene of the enzyme converting succinate to 4HB (4-hydroxybutyrate) is introduced or amplified, and the gene involved in converting succinate semialdehyde to succinic acid is deleted. It is to provide a variant having 4HB high performance characterized in that.
본 발명의 다른 목적은 상기 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 4HG(4-hydroxybutyrate)를 수득하는 것을 특징으로 하는 4HB의 제조방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing 4HB, characterized in that after culturing the variant in a medium containing a carbohydrate, 4HG (4-hydroxybutyrate) from the culture solution.
상기 목적을 달성하기 위하여, 본 발명은 숙신산 고생성능을 가지는 미생물에서, 숙신산(succinate)을 4HB(4-hydroxybutyrate)로 전환하는 효소의 유전자가 도입 또는 증폭되어 있고, 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자가 결실되어 있는 것을 특징으로 하는 4HB 고생성능을 가지는 변이체를 제공한다. In order to achieve the above object, the present invention is a microorganism having a high performance of succinic acid, the gene of the enzyme for converting succinate (succinate) to 4HB (4-hydroxybutyrate) is introduced or amplified, succinate semialdehyde (succinate semialdehyde) Provided is a variant having 4HB high performance, characterized in that the gene involved in the conversion to succinic acid is deleted.
본 발명에 있어서, 상기 숙신산 고생성능을 가지는 변이체는 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 variant having high succinic acid performance may be selected from the group consisting of bacteria, yeast and fungi, wherein 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 a characteristic of producing succinic acid at a 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 is genus Mannheimia sp .) , Actinobacillus sp. and Anaerobiospirillum sp. may be selected from the group consisting of, but is not limited thereto. 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 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 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 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) may be selected from the group consisting of genes.
본 발명에 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 할 수 있고, 상기 숙신산 세미알데히드(succinate semialdehyde)를 숙신산으로 전환하는데 관여하는 유전자는 GabD(succinic semialdehyde dehydrogenase)를 코딩하는 유전자인 것을 특징으로 할 수 있으며, 상기 GabD를 코딩하는 유전자는 서열번호 5의 염기서열을 가 지는 것을 특징으로 할 수 있다. In the present invention, the gene encoding Cat1 is SEQ ID NO: 1, the gene encoding SucD is SEQ ID NO: 2, the gene encoding 4hbD is SEQ ID NO: 3 and the gene encoding GHB has the nucleotide sequence of SEQ ID NO: 4 The gene involved in converting succinate semialdehyde into succinic acid may be a gene encoding Succinic semialdehyde dehydrogenase (GabD), and the gene encoding GabD is a sequence. It may be characterized by having the nucleotide sequence of the number 5.
본 발명에 있어서, 숙신산의 운송에 관여하는 DctA(C4-dicarboxylate transport protein)를 코딩하는 유전자가 추가로 도입 또는 증폭되어 있는 것을 특징으로 할 수 있고, 상기 DctA를 코딩하는 유전자는 서열번호 6의 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, wherein the gene encoding the DctA is a base of SEQ ID NO: 6 It may be characterized by having a sequence.
본 발명은 또한, 숙신산 고생성능을 가지는 미생물에서 GabD를 코딩하는 유전자가 결실되어 있고, Cat1을 코딩하는 유전자, SucD를 코딩하는 유전자, 4hbD를 코딩하는 유전자 및 GHB를 코딩하는 유전자가 모두 도입 또는 증폭되어 있는 것을 특징으로 하는 4HB 고생성능을 가지는 미생물 변이체를 제공한다. In the present invention, a gene encoding GabD is deleted from a microorganism having high succinic acid performance, and a gene encoding Cat1, a gene encoding SucD, a gene encoding 4hbD, and a gene encoding GHB are all introduced or amplified. It provides a 4HB microbial variant having a high performance, characterized in that.
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 4HG(4-hydroxybutyrate)를 수득하는 것을 특징으로 하는 4HB의 제조방법을 제공한다.The present invention also provides a method for producing 4HB, wherein the variant is cultured in a medium containing carbohydrate, and 4HG (4-hydroxybutyrate) is obtained from the culture solution.
본 발명은 또한, 상기 변이체를 탄수화물을 포함하는 배지에서 배양한 다음, 상기 배양액으로부터 4HG(4-hydroxybutyrate)를 수득하는 것을 특징으로 하는 4HB의 제조방법을 제공한다.The present invention also provides a method for producing 4HB, wherein the variant is cultured in a medium containing carbohydrate, and 4HG (4-hydroxybutyrate) is obtained from the culture solution.
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 제10-2006-0011345에 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 microorganism of the present invention, E. coli was prepared according to the method disclosed in the applicant's applicant's domestic patent publication No. 10-2006-0011345. That is, the gene coding for glucose phophotransferase (ptsG) and pyruvate kinase in the W3110 strain transformed with the recombinant expression vector pTrcEBG expressing bacteriophage red operon (exo-beta-gam) 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 효소들이다. 물론 클로스트리디움 클루이베리가 4HB를 생산하는 것은 아니지만 상 기 균주에서 클로닝된 효소들이 4HB 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. Of course, Clostridium clui berry does not produce 4HB, but the enzymes cloned in the strains were found to play an important role in 4HB production.
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, as shown in FIG. 4-hydroxybutyrate dehydrogenase (GHB) and 4-hydroxybutyrate dehydrogenase (GHB), which convert semialdehyde dehydrogenase) and succinate semialdehyde into 4-hydroxybutyrate.
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) which is 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 유전 자를 결실시킨 숙신산 고농도 생성 변이균주 Mannheimia sp. LPK(KCTC 10558BP), LPK7 및 LPK4과 대장균 변이균주 W3110GFA를 이용하여, 4HB를 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 examples in the lumen of the bacteria, which produce a high concentration acid deletion mutant genetic characters from Mannheimia sp Mannheimia sp. Using only LPK (KCTC 10558BP), LPK7 and LPK4 and E. coli mutant strain W3110GFA, only 4HB was produced. It will be obvious to those of ordinary skill in the industry.
또한, 하기 실시예에서는 특정 배지와 배양방법 만을 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.
실시예Example 1: 숙신산 고생성능을 가지는 미생물의 제조방법 1: Manufacturing method of microorganism 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 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).
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 제10-2006-0011345에 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 Korean Patent Publication No. 10-2006-0011345 of the applicant. 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.
실시예Example 2: 4 2: 4 HBHB 전환 효소 Conversion enzyme 클로닝Cloning
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 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 cluiberry DSM 555. cat1 gene was amplified.
PCR 증폭실험에 사용한 프라이머는 다음과 같다. Primers used in the PCR amplification experiment are as follows.
[서열번호 7] cat1f-BspHI aaaaatcaat gagtaaaggg ataaagaatt cac [SEQ ID NO: 7] cat1f-BspHI aaaaatcaat gagtaaaggg ataaagaatt cac
[서열번호 8] cat1b-XbaI gctctagatt atttcatact accagttttt ataaa[SEQ ID NO: 8] 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 9] SucDf-BspHI aaaaatcaat gagtaatgaa gtatctataa aag [SEQ ID NO: 9] SucDf-BspHI aaaaatcaat gagtaatgaa gtatctataa aag
[서열번호 10] 4hbDb-XabI gctctagatt agataaaaaa gaggacattt cacaatatgg[SEQ ID NO: 10] 4hbDb-XabI gctctagatt agataaaaaa gaggacattt cacaatatgg
발현 벡터를 만들기 위해 상기 증폭된 sucD4hbD 유전자를 NcoI/XbaI 로 절단한pTrc99A (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 11] H16 GHBf-BspHI aaaaatcaat ggcgtttatc tactatctg[SEQ ID NO: 11] H16 GHBf-BspHI aaaaatcaat ggcgtttatc tactatctg
[서열번호 12] H16 GHBb-XbaI gctctagatt acatggactg ctcaagcata c[SEQ ID NO: 12] 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 DctA를 코딩하는 유전자의 클로닝을 위하여 대장균 W3110로부터 서열번호 6의 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 cloning the gene encoding DctA involved in the transport of succinic acid in Escherichia coli, an oligonucleotide primer synthesized based on a known gene sequence (NC_000913) was used to clone the gene dctA encoding the DctA of SEQ ID NO: 6 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 13] DctAf-EcoRI ggaattcatg aaaacctctc tgtttaaaag c[SEQ ID NO: 13] DctAf-EcoRI ggaattcatg aaaacctctc tgtttaaaag c
[서열번호 14] DctAb-XbaI gctctagatt aagaggataa ttcgtgcgtt ttgccSEQ ID NO: 14 DctAb-XbaI gctctagatt aagaggataa ttcgtgcgtt ttgcc
발현 벡터를 만들기 위해 상기 증폭된 dctA 유전자를 p10499A (Park et al ., FEMS Microbiol. Lett 214:217, 2002) 발현 벡터에 EcoRI/XbaI로 절단해 삽입함으로서 p10499DctA를 제조하였다. The amplified dctA to make an expression vector The gene p10499A (Park et al ., FEMS Microbiol. Lett 214: 217, 2002) p10499DctA was prepared by cutting and inserting into an Eco RI / Xba I expression vector.
실시예Example 3: 4 3: 4 HBHB 고생성능을 가지는 With high performance 변이체Variant 제작 making
3-1 숙신산 고생능 미생물에서 GabD 코딩하는 유전자 결실3-1 Deletion of GabD-coding Genes in Succinic 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 형질전환된 W3110GFA 균주에서 서열번호 5의 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, the E. coli mutant strain W3110GFA-1, which lost the GabD gene of SEQ ID NO: 5 in the W3110GFA strain transformed with the 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.
실시예Example 4: 4 4: 4 HBHB 수득 purchase
실시예 3에서 제조한 cat1, sucD, 4 hbD, dctA 유전자를 발현하는 재조합 플라스미드를 형질전환 시킨 W3110GFA-1를, 본 출원인의 출원인 특허 10-2006-0011345에 제시한 대로 배양을 실시하였다. 즉, 상기 실시예 3에서 제작된 대장균 변이균주(W3110GFA-1)를 CaCO3 과량 함유된 배지조건에서 호기적으로 배양하고, 이로부터 생산되는 산물을 분석하였다.W3110GFA-1 transformed with a recombinant plasmid expressing cat1 , sucD , 4 hbD , and dctA genes prepared in Example 3 was cultured as set forth in the applicant's patent 10-2006-0011345. That is, the Escherichia coli mutant strain (W3110GFA-1) prepared in Example 3 was cultured aerobically under medium conditions 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 맞춘 배지 100㎖을 함유한 250㎖ 플라스크에 대장균 W3110GFA 돌연변이주 5㎖을 접종하여, 30℃에서 120rpm으로 72시간동안 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 strains were inoculated into a 250 ml flask containing 100 mg of medium having a pH of 7.0 NaB 4 O 7 10H 2 O and adjusted to pH 7.0 with KOH, and incubated at 30 ° C. for 120 hours at 120 rpm. Then, 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, 화학산업 전반에 걸쳐 중요하게 사용되고 있는 탄소수 4개의 화학물질의 전구체인 4HB(4-hydroxybutyrate) 생성능을 가지는 변이체 및 이를 이용한 4HB의 생물학적 제조방법을 제공하는 효과가 있다. 또한, 본 발명에 의해 제조된 4HB는 1,4-BDO(1,4-butanediol), GBL(gammabutyrolactone) 및 THF(tetrahydrofuran) 등의 다양한 탄소수 4개의 화학물질로 쉽게 전환할 수 있다. As described in detail above, according to the present invention, there is provided a variant having 4HB (4-hydroxybutyrate) generating ability, which is a precursor of four carbon atoms, which is importantly used throughout the chemical industry, and a method of biologically manufacturing 4HB using the same. It works. In addition, 4HB prepared by the present invention can be easily converted to various 4 carbon atoms such as 1,4-BDO (1,4-butanediol), GBL (gammabutyrolactone) and THF (tetrahydrofuran).
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. 따 라서 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. Therefore, 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 4-Hydroxybutyrate and Method for Preparing 4HB Using the Same <130> P06-B024 <160> 14 <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> 1449 <212> DNA <213> Escherichia coli <400> 5 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> 6 <211> 1287 <212> DNA <213> Escherichia coli <400> 6 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> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aaaaatcaat gagtaaaggg ataaagaatt cac 33 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gctctagatt atttcatact accagttttt ataaa 35 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aaaaatcaat gagtaatgaa gtatctataa aag 33 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gctctagatt agataaaaaa gaggacattt cacaatatgg 40 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaaaatcaat ggcgtttatc tactatctg 29 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gctctagatt acatggactg ctcaagcata c 31 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggaattcatg aaaacctctc tgtttaaaag c 31 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctctagatt aagaggataa ttcgtgcgtt ttgcc 35 <110> LG Chem, Ltd. <120> Mutants Having a Producing Ability of 4-Hydroxybutyrate and Method for Preparing 4HB Using the Same <130> P06-B024 <160> 14 <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> 1449 <212> DNA <213> Escherichia coli <400> 5 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> 6 <211> 1287 <212> DNA <213> Escherichia coli <400> 6 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> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aaaaaatcaat gagtaaaggg ataaagaatt cac 33 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gctctagatt atttcatact accagttttt ataaa 35 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aaaaaatcaat gagtaatgaa gtatctataa aag 33 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gctctagatt agataaaaaa gaggacattt cacaatatgg 40 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaaaatcaat ggcgtttatc tactatctg 29 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gctctagatt acatggactg ctcaagcata c 31 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggaattcatg aaaacctctc tgtttaaaag c 31 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctctagatt aagaggataa ttcgtgcgtt ttgcc 35
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WO2012124945A2 (en) * | 2011-03-11 | 2012-09-20 | 한국화학연구원 | Method for producing gamma-aminobutyric acid using recombinant escherichia coli |
KR20130064037A (en) * | 2011-12-07 | 2013-06-17 | 한국과학기술원 | Mutants having a high producing ability of 4-hydroxybutyrate and preparing method for 4-hydroxybutyrate using the same |
US9447439B2 (en) | 2013-08-23 | 2016-09-20 | Samsung Electronics Co., Ltd. | Microorganism producing 4-hydroxybutyrate and a method for producing 4-hydroxybutyrate in anaerobic condition using the same |
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JP4672671B2 (en) | 2003-11-27 | 2011-04-20 | コリア アドバンスド インスティチュート オブ サイエンス アンド テクノロジィ | Novel rumen bacteria mutant and method for producing succinic acid using the same |
KR100656590B1 (en) * | 2004-07-30 | 2006-12-11 | 한국과학기술원 | Bacteria Variant and Method for Preparing Succinate and Amino Acids |
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WO2012124945A2 (en) * | 2011-03-11 | 2012-09-20 | 한국화학연구원 | Method for producing gamma-aminobutyric acid using recombinant escherichia coli |
WO2012124945A3 (en) * | 2011-03-11 | 2012-11-08 | 한국화학연구원 | Method for producing gamma-aminobutyric acid using recombinant escherichia coli |
KR20130064037A (en) * | 2011-12-07 | 2013-06-17 | 한국과학기술원 | Mutants having a high producing ability of 4-hydroxybutyrate and preparing method for 4-hydroxybutyrate using the same |
US9447439B2 (en) | 2013-08-23 | 2016-09-20 | Samsung Electronics Co., Ltd. | Microorganism producing 4-hydroxybutyrate and a method for producing 4-hydroxybutyrate in anaerobic condition using the same |
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