KR20190007403A

KR20190007403A - Method of producing 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutanoic acid

Info

Publication number: KR20190007403A
Application number: KR1020180081248A
Authority: KR
Inventors: 박성훈; 김용환; 이성국; 심증엽; 유태현; 라마 수만; 응웬화이남
Original assignee: 울산과학기술원
Priority date: 2017-07-12
Filing date: 2018-07-12
Publication date: 2019-01-22
Also published as: WO2019013573A3; KR102149044B1; WO2019013573A9; WO2019013573A2

Abstract

The present invention presents a newly proposed biosynthesis route having homoserine production for producing 2-hydroxy gamma butyrolactone (HGBL) and 2,4-dihydroxybutanoic acid, which is a precursor of 2-hydroxy gamma butyrolactone (HGBL) and a gene recombinant strain having the route. In the case of HGBL in which a hydroxyl group is substituted at the second position, the HGBL can be used as an important intermediate which can be used as a resin for photoresist, a raw material for medicines, and a material for coating a metal surface.

Description

2-히드록시 감마 부티로락톤 또는 2,4-디히드록시-부티레이트 의 제조 방법{Method of producing 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutanoic acid}Method for producing 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutanoic acid]

본 발명은 2-hydroxy gamma butyrolactone (HGBL) 및 이의 전구체인 2,4-dihydroxybutanoic acid 제조를 위하여 homoserine 생산을 중간단계로 하는 새롭게 제시된 생물합성 경로 및 이 경로를 가지는 유전자 재조합 균주를 제시한다. 2번 위치에 수산화기가 치환된 HGBL의 경우 포토레지스트를 위한 수지, 의약품 원료 및 금속 표면의 코팅을 위한 재료로 이용될 수 있는 중요한 중간체이다.The present invention provides a newly proposed biosynthetic pathway and homologous recombinant strains having homoserine production at an intermediate stage for the production of 2-hydroxy gamma butyrolactone (HGBL) and its precursor 2,4-dihydroxybutanoic acid. In the case of HGBL in which hydroxyl group is substituted at position 2, it is an important intermediate that can be used as resin for photoresist, material for pharmaceutical raw materials and material for coating metal surface.

미국특허 4,994,597 및 5,087,751 (Inone)은 3,4-디히드록시부티르산 유도체를 발표한다. 이러한 산 제조방법은 금속시안화물과 3,4-디히드록시 부틸 클로라이드의 반응과 가수분해가 관련된 본 발명과 구별된다. 상기 산은 3-히드록시부티로락톤의 중간물질이다.U.S. Pat. Nos. 4,994,597 and 5,087,751 (Inone) disclose 3,4-dihydroxybutyric acid derivatives. Such an acid production method is distinguished from the present invention in which the reaction of the metal cyanide with 3,4-dihydroxybutyl chloride is related to the hydrolysis. The acid is an intermediate of 3-hydroxybutyrolactone.

(S)-3-히드록시부티로락톤은 콜레스테롤 강하약, (S)-카르니틴, 안티 HIV 프로테아제 억제약, 광범위한 항생제를 포함한 다양한 약품의 중간물질 제조용 핵심 4-탄소 중간물질이다.(S) -3-hydroxybutyrolactone is a core 4-carbon intermediate for the production of intermediates for a variety of drugs including cholesterol lowering drugs, (S) -carnitine, anti-HIV protease inhibitors, and a wide range of antibiotics.

(R)-3-히드록시부티로락톤 또는 (R)-3,4-디히드록시 부티르산 감마 락톤은 다양한 약품 중간물질 제조용 핵심 4-탄소 중간물질이다. 또한 이것은 건강식품 첨가제 및 강장제 보충물, 다양한 신경 시스템 및 대사 장애의 치료를 포함한 여러 용도에 사용되는 성분 및 천연 발생 비타민인 1-카르니틴으로 전환될 수 있다. 카르니틴 시장은 수백톤 규모이다. 이것은 d 및 l 형태의 순수분리 제품으로 제조된다. 그러나 아직까지 상업적 가치가 있는 직접 합성루트는 없다.(R) -3-hydroxybutyrolactone or (R) -3,4-dihydroxybutyric acid gamma-lactone are the core 4-carbon intermediates for the preparation of various drug intermediates. It can also be converted to 1-carnitine, a naturally occurring vitamin and a component used in many applications, including health food additives and tonic supplement, a variety of nervous system and metabolic disorders. The market for carnitine is several hundred tons. It is produced in pure form in the d and l forms. However, there is no direct synthetic route that has commercial value yet.

(S)-3-히드록시부티로락톤은 Hollingsworth 공정으로 제조될 수 있다(미국특허 5,374,773). (R)-3-히드록시부티로락톤은 4-연결 L-헥소스를 갖는 출발물질을 사용할 필요가 있으므로 상기 공정으로 제조될 수 없다. 이러한 물질은 알려져 있지 않다.(S) -3-hydroxybutyrolactone can be prepared by the Hollingsworth process (U.S. Patent No. 5,374,773). (R) -3-hydroxybutyrolactone can not be prepared by this process since it is necessary to use a starting material having a 4-linked L-hexose. These substances are not known.

일반적인 락톤 제조방법은 다음 특허로 알려져 있다:A common lactone preparation process is known from the following patents:

U.S. Patent No. 3,024,250 to Klein et al., U.S.A. Patent No. 3,024,250 to Klein et al.,

U.S. Patent No. 3,868,370 to Smith, U.S.A. Patent No. 3,868,370 to Smith,

U.S. Patent No. 3,997,569 to Powell, U.S.A. Patent No. 3,997,569 to Powell,

U.S. Patent No. 4,105,674 to De Thomas et al., U.S.A. Patent No. 4,105, 674 to De Thomas et al.,

U.S. Patent No. 4,155,919 to Ramiouille et al., U.S.A. Patent No. 4,155, 919 to Ramiouille et al.,

U.S. Patent No. 4,772,729 to Rao, U.S.A. Patent No. 4,772,729 to Rao,

U.S. Patent No. 4,940,805 to Fisher et al., U.S.A. Patent No. 4,940, 805 to Fisher et al.,

U.S. Patent No. 5,292,939 to Hollingsworth, U.S.A. Patent No. 5,292,939 to Hollingsworth,

U.S. Patent No. 5,319,110 to Hollingsworth, U.S.A. Patent No. 5,319,110 to Hollingsworth,

U.S. Patent No. 5,374,773 to Hollingsworth, U.S.A. Patent No. 5,374,773 to Hollingsworth,

U.S. Patent No. 5,502,217 to Fuchikami et al.U.S.A. Patent No. No. 5,502,217 to Fuchikami et al.

이들 특허는 다양한 락톤 제조 공정을 발표한다. 그러나 이들 모두는 2-hydroxy 위치의 gammabutyrolactone의 제조 공정에 대해서는 언급하지 않고 있다.These patents announce various lactone manufacturing processes. However, all of them do not mention the manufacturing process of gammabutyrolactone at 2-hydroxy position.

본 발명은 글루코스를 탄소원으로 사용하여 2번 위치 탄소에 광학활성을 갖는 2,4-디히드록시-부틸레이트(2,4-dihydroxybutyrate) 또는 2-히드록시-감마-부티로락톤(2-hydroxy gamma butyrolactone)을 생산하는 미생물에 하기 (1) 내지 (4) 중 선택된 하나 이상의 단계를 수행하여 상기 미생물을 변이시키는 단계를 포함하는, 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤 생산 미생물 변이체의 제조 방법을 제공한다.The present invention relates to a process for preparing 2-hydroxy-2,4-dihydroxybutyrate (2,4-dihydroxybutyrate) or 2-hydroxy-gamma-butyrolactone dihydroxy-butyrate or 2-hydroxy-butyrolactone, which comprises the step of performing at least one step selected from the following (1) to (4) in a microorganism producing gamma butyrolactone, A method for producing gamma-butyrolactone-producing microorganism variants is provided.

(1) ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, 및 iclR 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실, 또는 acs, ppc, 및 metL 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계,(1) ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, and iclR deleting a gene of one or more genes selected from the group consisting of, or acs, ppc, and from the group consisting of metL gene at least one group selected Overexpressing the gene, or both,

(2) epd, dxs, 및 pdxj 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현시키는 단계,(2) overexpressing at least one gene selected from the group consisting of epd, dxs, and pdxj genes,

(3) lpd 및 ducA 중 하나 이상의 유전자의 과발현시키는 단계, 및(3) over-expressing one or more genes of lpd and ducA , and

(4) kgtP, dsdx, 및 actP 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실시키는 단계.(4) deletion of one or more genes selected from the group consisting of kgtP, dsdx, and actP genes.

또한, 본 발명은 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물의 게놈(genome)에 상기 (1) 내지 (4) 중 어느 하나 이상의 유전자 변이가 도입된, 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤 생산 미생물 변이체를 제공한다.The present invention also relates to a method for producing a mutant of any one of the above (1) to (4) in a genome of a microorganism producing 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone Dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone.

또한, 본 발명은 상기 미생물 변이체를 배양하는 단계를 포함하는, 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone) 또는 2,4-디히드록시 부틸레이트(2,4-dihydroxy butanoic acid)의 생산방법을 제공한다.The present invention also provides a method for producing a microorganism which comprises culturing a microorganism variant of the present invention comprising 2-hydroxy gamma butyrolactone or 2,4-dihydroxy butanoic acid, And a method for producing the same.

또한, 본 발명은 싱기 미생물 변이체 또는 그 배양물을 포함하는, 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone) 또는 2,4-디히드록시 부틸레이트(2,4-dihydroxy butanoic acid)의 생산용 조성물을 제공한다.The present invention also relates to a pharmaceutical composition comprising 2-hydroxy gamma butyrolactone or 2,4-dihydroxy butanoic acid, which comprises a microorganism strain or culture thereof, By weight based on the total weight of the composition.

또한, 본 발명은 서열번호 13의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소, 서열번호 14의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소, 및 서열번호 15의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소를 제공한다.The present invention also provides a transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 13, a transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 14, and a transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 15 .

또한, 본 발명은 서열번호 17의 아미노산 서열로 이루어진, L-히드록시-2-옥소-리덕테이즈 변이효소, 서열번호 18의 아미노산 서열로 이루어진, L-히드록시-2-옥소-리덕테이즈 변이효소를 제공한다.The present invention also relates to an L-hydroxy-2-oxo-reductase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 17, L-hydroxy-2-oxo-reductase comprising the amino acid sequence of SEQ ID NO: Provide mutated enzymes.

또한, 본 발명은 서열번호 20의 아미노산 서열로 이루어진, D-히드록시-2-옥소-리덕테이즈 변이효소, 서열번호 21의 아미노산 서열로 이루어진, D-히드록시-2-옥소-리덕테이즈 변이효소를 제공한다.The present invention also provides a D-hydroxy-2-oxo-reductase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 20, D-hydroxy-2-oxo-reductase comprising the amino acid sequence of SEQ ID NO: Provide mutated enzymes.

또한, 본 발명은 서열번호 22의 아미노산 서열로 이루어진, 락토네이즈 변이효소를 제공한다.Further, the present invention provides a lactonase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 22.

homoserine으로부터 생합성하는 경로를 통하여 광학활성을 갖는 순수 이성질체 혹은 2가지 이성질체가 혼합된 2-hydroxy gamma butyrolactone과 혹은 이의 전구체인 2,4-dihydroxy butanoic acid를 생산하는 방법을 제공한다. 상기 방법의 일 예가 도 1에 모식적으로 예시되어 있다.a method of producing 2-hydroxy gamma butyrolactone and its precursor, 2,4-dihydroxy butanoic acid, which is an optically active pure isomer or a mixture of two isomers through a pathway biosynthesized from homoserine. One example of the above method is illustrated schematically in Fig.

일 예에서, 상기 생산 방법은, 2-hydroxy gamma butyrolactone의 전구체인 2,4-dihydroxy butanoic acid를 광학 활성 순수 이성질체 혹은 이들의 혼합물인 상태로 생산하는 하는 경우 2,4-dihydroxy butanoic acid를 분리정제한 후 화학적인 방법으로 광학 활성 순수 이성질체 혹은 이들의 혼합물인 상태로 최종 2-hydroxy gamma butyrolactone를 생산하는 방법일 수 있다.In one example, 2,4-dihydroxy butanoic acid is isolated and purified when 2,4-dihydroxy butanoic acid, a precursor of 2-hydroxy gamma butyrolactone, is produced as an optically active pure isomer or a mixture thereof, Followed by a chemical method to produce the final 2-hydroxy gamma butyrolactone in the form of optically active pure isomers or mixtures thereof.

상기 생산 방법은 homoserine의 알파 위치 아민기를 제거하는 단계를 포함할 수 있으며, 상기 homoserine의 알파 위치 아민기 제거를 위해 사용되는 효소는 deaminase, dehydrogenase, transaminase로 이루어진 군에서 선택된 1종 이상일 수 있다.The production method may include a step of removing the alpha-position amine group of homoserine, and the enzyme used for removing the alpha-position amine group of homoserine may be at least one selected from the group consisting of deaminase, dehydrogenase, and transaminase.

일 예에서, 상기 생산 방법은 homoserine으로부터 amino기를 받는 amino acceptor로서 α-ketoglutarate (α-KG)를 사용하는 것을 특징으로 하는 transaminase를 사용하는 것일 수 있다. In one example, the production method may be to use a transaminase characterized in that α-ketoglutarate (α-KG) is used as an amino acceptor to receive an amino group from homoserine.

또한 상기 생산 방법은 상기 α-KG로부터 생산되는 glutamic acid로부터 amino acceptor인 α-KG를 재생함과 동시에 homoserine 생합성 전구체인 aspartic acid의 생산을 위해 aspartate transaminase를 동시에 사용하는 것을 특징으로 하는 경로를 포함할 수 있다.Also, the production method includes a pathway in which α-KG, which is an amino acceptor, is regenerated from glutamic acid produced from α-KG, and aspartate transaminase is simultaneously used for producing aspartic acid, a homoserine biosynthesis precursor .

다른 예에서, 상기 생산 방법은 도 1에 제시한 반응 경로 중 4-hydroxy-2-ox-butanoic acid의 2번 탄소에 결합된 산소를 stereo-specific 하게 혹은 non-specific 하게 환원시켜 hydroxy기로 전환하는 효소로는 L-lactate dehydrogenase와 D-lactate dehydrogenase, 혹은 이들의 혼합 효소를 사용하는 것을 특징으로 하는 경로를 포함할 수 있다.In another example, the production process is a process wherein the oxygen bound to the 2-carbon of 4-hydroxy-2-ox-butanoic acid in the reaction pathway shown in Figure 1 is converted to the hydroxy group by stereospecific or non- The enzyme may include a pathway that uses L-lactate dehydrogenase, D-lactate dehydrogenase, or a mixed enzyme thereof.

다른 예는, 도 1에 제시한 paraoxonase는 인간유래로 lactonase 활성을 갖는 유전자 (PON1)를 변이시켜 사용하며 homoserine으로부터 광학활성 2-hydroxy gamma butyrolactone을 생산하도록 제작된 균주의 periplasm에서 발현시키는 것을 특징으로 하는 균주를 제공한다. 상기 균주는 homoserine 과생산 특성을 갖는 homoserine 과생산 균주일 수 있다.Another example is that the paraoxonase shown in Fig. 1 is expressed in a periplasm of a strain produced to produce an optically active 2-hydroxy gamma butyrolactone from homoserine by using a mutant gene (PON1) having lactonase activity derived from a human. Lt; / RTI > The strain may be a homoserine and a producing strain and a homoserine producing strain.

다른 예는, 도 1에 보여주는 경로를 통해 homoserine으로부터 2번 위치 탄소에 광학활성을 갖는 2-hydroxy gamma butyrolactone 및 이의 유기산 전구체인 전구체인 2,4-dihydroxy butanoic acid를 효율적으로 생산하기 위하여, 도 4에 제시한 것과 같이, 당으로부터 homoserine을 효율적으로 생산하도록 제작된 유전자 변이 균주를 제공한다. 상기 균주는 homoserine 과생산 특성을 갖는 homoserine 과생산 균주일 수 있다.In another example, in order to efficiently produce 2-hydroxy gamma butyrolactone having optical activity at position 2 carbon from homoserine and 2,4-dihydroxy butanoic acid, which is a precursor of organic acid precursor thereof, through the route shown in FIG. 1, , Which provides a mutant strain produced so as to efficiently produce homoserine from sugar. The strain may be a homoserine and a producing strain and a homoserine producing strain.

상기 변이 균주를 제작하는 미생물로는 대장균, 효모, 코리네균 등으로 이루어진 군에서 선택된 1종 이상의 미생물이 사용될 수 있다. As the microorganism for producing the mutant strain, at least one microorganism selected from the group consisting of E. coli, yeast, Corynebacterium, etc. may be used.

상기 미생물은 다음과 같은 방법들 중에서 선택된 하나 이상을 개별적으로 혹은 조합하여 사용함으로써 개량된 것을 특징으로 하는 미생물일 수 있다:The microorganism may be a microorganism that is improved by using one or more of the following methods, individually or in combination:

1.phosphoenolpyruvate (PEP)를 oxaloacetic acid (OAA)로 효율적으로 전환하는 phosphoenolpyruvate carboxylase (ppc) 유전자의 과발현;Overexpression of phosphoenolpyruvate carboxylase (ppc) gene, which efficiently converts phosphoenolpyruvate (PEP) to oxaloacetic acid (OAA);

2. 발효 중 생성되는 acetate를 acetyl CoA로 전환하여 미생물이 다시 사용할 수 있도록 도와주는 acetyl-CoA synthase (acc) 유전자의 과발현;2. overexpression of acetyl-CoA synthase (acc) gene to help reuse microbes by converting acetate produced into fermentation into acetyl CoA;

3.glyoxylate shunt의 활성을 높이기 위해 glyoxylate shunt 유전자 발현을 억재하는 iclR 유전자의 제거;3. Removal of the iclR gene, which inhibits glyoxylate shunt gene expression to increase the activity of glyoxylate shunt;

4.Apspartic acid의 aspartyl phosphate 전환 효율을 높이기 위해 apspartate kinase의 활성을 향상시킴. apspartate kinase를 coding 하는 thrABC 유전자의 발현을 향상시키기 위해 isoleucine에 의해 작동하는 thrABC operon의 riboswitch를 제거하고 또한 promoter의 세기를 올려줌. 더 나아가 apspartate kinase 효소가 threonine과 lysine에 의해 feedback inhibition을 받는 것을 방지하기 위해 feedback inhibition을 받지 않도록 변이된 효소를 발현시키도록 함;4. Improve the activity of apspartate kinase to increase aspartyl phosphate conversion efficiency of appartic acid. To improve the expression of the thrABC gene coding for apspartate kinase, it removes the riboswitch of the thrABC operon, which is activated by isoleucine, and also increases the strength of the promoter. Furthermore, to prevent the feedback inhibition of apspartate kinase enzyme by threonine and lysine, it is necessary to express the mutated enzyme so that it does not undergo feedback inhibition;

5.lysine 생산을 제거하기 위해 diaminopimelate decarboxylase를 coding 하는 lysA을 제거;5. Elimination of lysA coding for diaminopimelate decarboxylase to eliminate lysine production;

6.methionine 생산을 제거하기 위해 homoserine succinyltransferase를 coding 하는 metA 유전자를 제거;6. Remove the metA gene encoding homoserine succinyltransferase to remove methionine production;

7. 생산된 homoserine이 homoserine phosphate로 전환되지 않도록 homoserine kinase를 coding하는 thrB 유전자를 제거. 7. Remove the thrB gene coding for homoserine kinase so that produced homoserine does not convert to homoserine phosphate.

상기 제작된 homoserine 과생산 균주에 앞서 설명한 생산 방법의 경로가 도입되어 당으로부터 2번 위치 탄소에 광학활성을 갖는 2-hydroxy gamma butyrolactone과 혹은 이의 전구체인 2,4-dihydroxy butanoic acid를 생산하도록 제작된 균주가 제공된다. 이 때, 상기 균주는 transaminase의 활성을 증대시키기 위하여 transaminase의 cofactor인 pyridoxal-5'-phosphate 의 생합성이 촉진되도록 vitamin B6 생합성 경로를 강화시킨 것을 특징으로 하는 것일 수 있다. The pathway of the production method described above was introduced into the produced homoserine and the production strain to produce 2-hydroxy gamma butyrolactone having optical activity at position 2 carbon and its precursor, 2,4-dihydroxy butanoic acid A strain is provided. In this case, the strain may be characterized in that the vitamin B6 biosynthetic pathway is enhanced so as to promote the biosynthesis of pyridoxal-5'-phosphate, which is a cofactor of transaminase, in order to increase the activity of transaminase.

상기 제작된 균주는 당으로부터 homoserine 을 거쳐 생합성된 2,4-dihydroxybutanoic acid를 세포 밖으로 빨리 분비시키기 위하여, 2,4-dihydroxybutanoic acid의 세포막 전달 단백질을 과발현시키는 것을 특징으로 하는 균주일 수 있다.The prepared strain may be a strain characterized by overexpressing the cell membrane transfer protein of 2,4-dihydroxybutanoic acid to rapidly release 2,4-dihydroxybutanoic acid biosynthesized from the sugar via homoserine to the outside of the cell.

또한, 앞서 설명한 균주를 배양하여 배양액으로부터 광학활성을 갖는 순수한 이성질체 혹은 2가지 이성질체가 혼합된 2-hydroxy gamma butyrolactone 및/또는 이의 전구체인 2,4-dihydroxy butanoic acid를 생산하는 방법이 제공된다.Also provided is a method for producing 2-hydroxy gamma butyrolactone and / or a precursor thereof, 2,4-dihydroxy butanoic acid, which is obtained by culturing the above-mentioned strain and mixing pure isomers or two isomers having optical activity from the culture solution.

상기 방법은, 2-hydroxy gamma butyrolactone 및/또는 이의 전구체인 2,4-dihydroxy butanoic acid를 효과적으로 생산하기 위하여, 질소원으로 yeast extract와 암모늄 염을 첨가하는 것은 물론 본 균주가 특별히 필요로 하는 아미노산, 즉 methionine, lysine, threonine, 그리고 isoleucine을 적절히 첨가하는 배지를 사용하여 배양하는 것을 특징으로 하는 것일 수 있다. In order to effectively produce 2-hydroxy gamma butyrolactone and / or 2,4-dihydroxy butanoic acid, which is a precursor thereof, yeast extract and ammonium salt are added to the nitrogen source, and the amino acid methionine, lysine, threonine, and isoleucine in a suitable medium.

상기 배양은 생물 반응기를 이용한 고농도 2-hydroxy gamma butyrolactone 및/또는 이의 전구체인 2,4-dihydroxy butanoic acid 생산을 위해 유가식으로 발효 중간에 당과, methionine, lysine, threonine, 및 isoleucine로 이루어진 군에서 선택된 1종 이상의 혼합물 (예컨대, methionine, lysine, threonine, 및 isoleucine의 혼합물)을 첨가하여 수행되는 것일 수 있다.The cultivation was carried out in a fermented medium for the production of high concentration 2-hydroxy gamma butyrolactone and / or its precursor 2,4-dihydroxy butanoic acid using a bioreactor, in the medium consisting of sugar, methionine, lysine, threonine and isoleucine Or a mixture of one or more selected ones (e.g., a mixture of methionine, lysine, threonine, and isoleucine).

본 발명은 글루코스를 탄소원으로 사용하여 광학적으로 순수한 2,4-디히드록시-부틸레이트(2,4-dihydroxybutyrate) 또는 2-히드록시-감마-부티로락톤(2-hydroxy gamma butyrolactone)을 생산하는 미생물에 하기 (1) 내지 (4) 중 선택된 하나 이상의 단계를 수행하여 상기 미생물을 변이시키는 단계를 포함하는, 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤 생산 미생물 변이체의 제조 방법을 제공한다.The present invention relates to the use of glucose as a carbon source to produce optically pure 2,4-dihydroxybutyrate or 2-hydroxy gamma butyrolactone A process for producing 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone, which comprises the step of carrying out at least one step selected from the following (1) to (4) Thereby producing a microorganism variant.

(1) ptsG, eda, adhE, pf1B, lysA, thrB, metA, LacI, ldhA, 및 iclR 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실, 또는 acs, ppc, 및 metL 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계,(1) ptsG, eda, adhE, pf1B, lysA, thrB, metA, LacI, ldhA, and iclR deleting a gene of one or more genes selected from the group consisting of, or acs, ppc, and from the group consisting of metL gene at least one group selected Overexpressing the gene, or both,

(2) epd, dxs, 및 pdx 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현시키는 단계,(2) overexpressing at least one gene selected from the group consisting of epd, dxs , and pdx genes,

상기 탄소원은 그 종류를 특별히 한정하지 않으나, 글루코스(Glucose)인 것이 적절하다.The kind of the carbon source is not particularly limited, but it is suitably glucose.

상기 "광학적으로 순수"란 용어는 (2S)-2,4-디히드록시-부틸레이트 또는 (2R)- 2,4-디히드록시-부틸레이트 또는 (2S)-2-히드록시 감마 부티로락톤 또는 (2R)-2-히드록시 감마 부티로락톤 각각의 광학 순도가 90% 내지 100%, 바람직하게는 95% 내지 100%, 더욱 바람직하게는 97% 내지 100%, 예를 들어 99% 내지 100%인 것을 의미한다. The term " optically pure " refers to (2S) -2,4-dihydroxy-butyrate or (2R) -2,4-dihydroxybutyrate or (2S) -2- The optical purity of each of lactone or (2R) -2-hydroxy-gamma butyrolactone is 90% to 100%, preferably 95% to 100%, more preferably 97% to 100% 100%.

본 발명의 일 예에 따르면, 상기 2,4-디히드록시-부틸레이트(2,4-dihydroxybutyrate) 또는 2-히드록시-감마-부티로락톤(2-hydroxy gamma butyrolactone)을 생산하는 미생물은 그 종류를 특별히 한정하지 않으나, 대장균(E. coli), 효모(Yeast), 및 코리네박테리움(Corynebacterium) 으로 이루어진 군에서 선택된 하나 이상일 수 있으며, 바람직하게는 대장균일 수 있다. According to one embodiment of the present invention, the microorganism producing the 2,4-dihydroxybutyrate or 2-hydroxy gamma butyrolactone is a microorganism producing the 2,4-dihydroxybutyrate or 2-hydroxy gamma-butyrolactone, The kind is not particularly limited, but may be one or more selected from the group consisting of E. coli , yeast, and Corynebacterium, preferably E. coli.

일 구체예에 따르면, 상기 미생물을 변이시키기 위해 (1) 내지 (4) 중 선택된 하나 이상의 단계를 수행하는 것은 동시 또는 순차적으로 수행할 수 있고, 반드시 각 단계를 시계열적 순서로 수행해야 하는 것은 아니며, (1) 내지 (4)의 변이 순서는 임의로 결정해 수행할 수 있고 두 개 이상의 단계를 동시에 수행하고 나머지 단계를 순차로 수행할 수도 있다.According to one embodiment, performing one or more steps selected from (1) to (4) to mutate the microorganism may be performed concurrently or sequentially, and it is not necessary to perform each step in a clockwise sequence , (1) to (4) may be arbitrarily determined and performed, and two or more steps may be simultaneously performed and the remaining steps may be sequentially performed.

바람직하게는, 상기 미생물을 변이시키는 단계는 (1) 단계, 및 (2) 내지 (4) 단계 중 선택된 하나 이상의 단계를 포함해 수행할 수 있으며, 가장 바람직하게는Preferably, the step of mutating the microorganism may be carried out by carrying out at least one step selected from the steps (1) and (2) to (4), and most preferably

(1) 내지 (4) 단계를 전부 포함하는 것일 수 있다.(1) to (4).

본 발명의 일 예에 따르면, 상기 (1) 단계에서 제거가능한 유전자 중 lysA, thrBC, metA는 미생물 내에서 중간체인 호모세린의 축적을 증가시키기 위해, 라이신, 메티오닌 및 트레오닌 생산 경로를 억제를 목적으로 제거하는 것이다. 구체적으로 각각 호모세린 산 염기성 효소를 코딩하는 유전자인 Diaminopimelate decboxylase를 코딩하는 lysA, Homoserinesuccinyl Transferase 를 코딩하는 metA, 그리고 Homoserine Kinase 및 Threonine Synthase를 코딩하는 thrBC 중 어느 하나 이상을 제거할 수 있다.According to one embodiment of the present invention, lysA, thrBC, and metA among the genes that can be removed in the step (1) are used for the purpose of inhibiting lysine, methionine, and threonine production pathway in order to increase the accumulation of homoserine, To remove it. Specifically, each of the homoserine met acid encoding a lysA, Homoserinesuccinyl Transferase encoding a gene encoding a basic Diaminopimelate decboxylase enzyme A, and thrBC encoding Homoserine Kinase and Threonine Synthase can be removed any more than one.

상기 (1) 단계에서 제거가능한 유전자 중 ldhA, adhE, pflB 유전자는 부산물인 젖산, 에탄올, 포름산 등의 생산을 막기 위해 제거하는 것일 수 있다. 상기 유전자들의 제거는 부산물 생성을 막고 homoserine으로 가는 탄소 flux를 증가시킨다.The genes ldhA, adhE and pflB among the genes that can be removed in step (1) may be removed to prevent the production of by-products such as lactic acid, ethanol, formic acid and the like. Elimination of these genes prevents byproduct formation and increases carbon flux to homoserine.

상기 (1) 단계에서 제거가능한 유전자 중 iclR 유전자는 아세테이트 재이용을 촉진하는 방법의 하나인 glyoxylate shunt의 활성을 제고하기 위하여 제거하는 것으로, 제거 방법은 통상적인 방법을 사용할 수 있으나, 바람직하게는 pop-in pop-out 방법으로 제거할 수 있다.Among the genes that can be removed in the above step (1), the iclR gene is removed in order to enhance the activity of the glyoxylate shunt, which is one of the methods for promoting acetal reuse. The removal method may be a conventional method, in pop-out method.

상기 (1) 단계에서 제거가능한 유전자 중 ptsG 유전자는 포도당 대사의 overflow metabolism을 제거하고 포도당의 세포막 전달에 phosphoenol pyruvate (PEP)가 사용되는 것을 막기 위해 제거하는 것일 수 있다. 이 경우 포도당은 GalP 등 다른 전달 단백질에 의해 세포내로 이송되며 Carbon Catabolite Repression의 방지, overflow metabolism에 의한 부산물 생성 방지 등을 기대할 수 있다. 상기 유전자의 제거 방법은 통상적인 방법을 사용할 수 있으나, 바람직하게는 MAGE 방법으로 제거할 수 있다.Among the genes that can be removed in the above step (1), the ptsG gene may be removed to eliminate the overflow metabolism of glucose metabolism and to prevent the use of phosphoenolpyruvate (PEP) for glucose cell membrane transport. In this case, glucose is transported into cells by other transfer proteins such as GalP, and prevention of Carbon Catabolite Repression and prevention of by-product formation by overflow metabolism can be expected. The gene may be removed by a conventional method, but preferably by the MAGE method.

상기 (1) 단계에서 제거가능한 유전자 중 eda 유전자는 KHG/KDG aldolase를 coding 하는 유전자로, 이 유전자가 제거될 경우 ED 경로가 제거되므로 EMP(Embden-Meyerhof-Parnas pathway) 경로의 이용이 촉진되고 또한 oxaloacetate 로의 탄소 flux를 올려줄 수 있다. 상기 유전자의 제거 방법은 통상적인 방법을 사용할 수 있으나, 바람직하게는 MAGE 방법을 사용할 수 있다.Among the genes that can be eliminated in step (1), the eda gene encodes KHG / KDG aldolase. When the gene is deleted, the ED pathway is eliminated and thus the use of the Embden-Meyerhof-Parnas pathway pathway is promoted. The carbon flux to oxaloacetate can be increased. As a method for removing the gene, a conventional method can be used, but the MAGE method can be preferably used.

상기 (1) 단계에서 제거가능한 유전자 중 lacI 유전자는 Lac 프로모터 활용(promoter utilization)을 위해 lacI 억제제를 제거하는 것으로 상기 유전자의 제거 방법은 통상적인 방법을 사용할 수 있으나, 바람직하게는 MAGE 방법을 사용할 수 있다.Among the genes that can be removed in step (1), the lacI gene removes the lacI inhibitor for the utilization of the Lac promoter (promoter utilization). The gene can be removed by a conventional method, preferably the MAGE method have.

상기 (1) 단계에서 유전자의 과발현은 통상의 방법을 사용할 수 있다. 예를들어 미생물 세포에 유전자가 포함된 벡터를 다량 도입하거나, 과발현 프로모터 치환 등의 방법을 사용할 수 있다.The overexpression of the gene in the step (1) may be performed by a conventional method. For example, a vector containing a gene in a microbial cell may be introduced in a large amount, or an over-expressing promoter may be substituted.

구체적으로, 상기 (1) 단계에서 acs 유전자의 과발현은 아세트산(acetic acid)의 생산을 최소화하기 위하여 acs 유전자의 발현을 증대시키는 것일 수 있다. pta-ack 및 poxB 유전자의 결실도 가능하지만 이들 유전자의 결실 특히 pta-ack 유전자의 결실이 세포성장에 나쁜 영향을 주는 경우가 많아 이를 배제하였다. Specifically, the above-mentioned (1) over-expression of the acs gene in step may be to increase the expression of the acs gene in order to minimize the production of acetic acid (acetic acid). The pTA-ack and poxB genes can also be deleted, but the deletion of these genes, especially the deletion of the pta-ack gene, often negatively affects cell growth.

acs 유전자의 발현을 증대시키는 방법은 관련업계에서 통상적으로 사용하는 방법을 제한없이 사용할 수 있지만, 바람직하게는 acs 발현 유전자의 프로모터를 유전자 과발현 프로모터로 바꾸어줄 수 있으며, lac 프로모터, tac 프로모터, trc 프로모터 등을 사용할 수 있으며, 바람직하게는 trc 프로모터로 치환하는 것이 적절하다. The method of increasing the expression of the acs gene can be used without limitation in a method commonly used in the related art. Preferably, the promoter of the acs expression gene can be converted into a gene overexpressing promoter, and the lac promoter, the tac promoter, the trc promoter Etc., and it is preferable to substitute with the trc promoter.

상기 (1) 단계에서 ppc 유전자의 과발현은 acetate 생성을 막고 호모세린으로의 탄소 flux를 향상시키기 위하여 anaplerotic 경로의 핵심 효소인 phosphoenol pyruvate carboxylase를 coding 하는 ppc 유전자의 발현을 증가시키기 위한 것으로, 통상적인 유전자 발현 증가방법을 사용할 수 있으며, ppc 유전자의 프로모터를 유전자 과발현 프로모터로 바꾸어줄 수 있고, 바람직하게는 기존 프로모터를 synthetic promoter 8 (서열번호: 23, TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA) 로 치환해 발현을 증가시킬 수 있다. 이를 위해 pop-in pop-out 방법을 사용할 수 있다.Overexpression of the ppc gene in step (1) is intended to increase the expression of the ppc gene coding for the phosphoenol pyruvate carboxylase, which is the key enzyme of the anaplorotic pathway, to inhibit acetate production and to improve the carbon flux to homoserine. Expression promoting method can be used and the promoter of the ppc gene can be converted into a gene overexpressing promoter and preferably the expression can be increased by replacing a conventional promoter with a synthetic promoter 8 (SEQ ID NO: 23, TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA). You can use the pop-in pop-out method to do this.

상기 (1) 단계에서 metL 유전자의 과발현은 호모세린 생산 경로의 bottleneck으로 알려진 aspartic acid 이후 경로를 최적화하기 위한 것이다. 호모세린 생산에서 3개의 aspartate kinase, 즉 AKI, AKII, AKIII 이 중요한 역할을 한다고 알려져 있으며, 이 중 AKII, AKIII 두 개의 효소만이 높은 homoserine 농도에서 좋은 활성을 보인다고 알려져 있고, AKII 및 AKIII 중에서 AKII는 aspartate kinase 와 serine dehydrogenase 활성을 동시에 가지고 있어 AKIII 보다는 좀 더 유리하다고 알려져 있다. 따라서 본 발명의 변이균주는 AKII 효소를 coding 하는 metL 유전자를 과발현 시킬 수 있다. Overexpression of the metL gene in step (1) is intended to optimize the pathway after the aspartic acid, known as the bottleneck of the homoserine production pathway. It is known that three aspartate kinases, AKI, AKII and AKIII, play important roles in homoserine production. Of these, only two enzymes, AKII and AKIII, are known to exhibit good activity at high homoserine concentrations. Among AKII and AKIII, Aspartate kinase and serine dehydrogenase activity are known to be more beneficial than AKIII. Therefore, the mutant strain of the present invention can overexpress the metL gene coding for the AKII enzyme.

상기 metL 유전자의 과발현 방법은 통상의 방법을 사용할 수 있으며, 바람직하게는 medium copy plasmid인 pUCPK plasmid를 이용하여 과발현시킬 수 있다. 이 때 metL의 발현 크기를 조절하기 위해 lac promoter 와 trc promoter 두 가지를 사용할 수 있다.The overexpression of the metL gene can be carried out by conventional methods, and can be over expressed using a medium copy plasmid, pUCPK plasmid. In this case, two lac promoters and trc promoters can be used to regulate metL expression.

상기 결실키기거나 과발현시키는 유전자를 표 1에 정리해 나타냈다.The genes for over-expressing or over-expressing the genes are summarized in Table 1.

본 발명의 일 구체예에 따르면, 상기 (1) 단계는 ptsG, eda, adhE, pf1B, lysA, thrB, metA, LacI, ldhA, 및 iclR 유전자 전부를 결실시키고, acs, ppc, 및 metL 유전자 전부를 과발현시키는 단계를 포함하는 것일 수 있다.According to one embodiment of the invention, the above-mentioned (1) comprises: ptsG, eda, adhE, pf1B, lysA, thrB, metA, LacI, ldhA, and iclR and deleting a gene altogether, acs, ppc, and the metL gene all And overexpressing the compound.

본 발명의 일 예에 따르면, 상기 (2) 단계는 Vitamin B6 생산 증가를 위해 수행하는 것으로, 대장균의 경우 PLP(Pyridoxal-5-Phosphate)는 DXP(1-deoxy-D-xylulose 5-phosphate) 의존 경로를 통해 생합성되며, PLP생합성 속도는 epd, dxs, pdxJ 유전자 등이 코딩하는 단백질에 의해 조절된다. 따라서, PLP 생합성 속도를 향상시키기 위하여 이 세 유전자중 하나 이상을 과발현 시킬 수 있다. 과발현 방법은 통상의 방법을 사용할 수 있으며, 바람직하게는 기존 프로모터를 합성 프로모터 9 (표 5)로 치환하는 것이 적절하며, Pop-in pop-out 방법을 사용할 수 있다. 이 때 사용가능한 프라이머(primer)를 표 14에 나타냈다.According to an embodiment of the present invention, the step (2) is carried out to increase the production of Vitamin B6. In the case of Escherichia coli, PLP (Pyridoxal-5-Phosphate) is dependent on 1-deoxy-D-xylulose 5-phosphate Pathway, and the rate of PLP biosynthesis is regulated by the proteins encoded by the epd, dxs, pdxJ genes and the like. Thus, one or more of these three genes may be overexpressed to improve the rate of PLP biosynthesis. As the over-expression method, a conventional method can be used, and it is preferable to replace the existing promoter with the synthetic promoter 9 (Table 5), and a pop-in pop-out method can be used. Table 14 shows the primers usable at this time.

바람직하게는, 상기 (2) 단계는 epd, dxs, 및 pdxj 유전자 전부를 과발현시키는 것이 바람직하다.Preferably, step (2) above preferably overexpresses all epd, dxs, and pdxj genes.

본 발명의 일 예에 있어서, 상기 (3) 단계에서 ldp, dcuA의 발현양 증가는 2,4-디히드록시-부틸레이트 (DHB)의 세포 외 이송속도를 증대시키기 위해 수행하는 것으로, 유기산 막 이송 단백질, 특히 lactic acid 와 저분자량의 carboxylic acid 이송 단백질 중 query로 사용하여 importer 및 exporter를 선정한 후, 이들 중 가장 가능성이 높은 2개의 exporter인 ldp 및/또는 dcuA 단백질의 발현양을 증가시키는 것이다.In one embodiment of the present invention, the increase in the expression level of ldp and dcuA in step (3) is performed in order to increase the extracellular transport rate of 2,4-dihydroxy-butyrate (DHB) After selecting importers and exporters as a query among transport proteins, especially lactic acid and low molecular weight carboxylic acid transfer proteins, the expression levels of ldp and / or dcuA proteins, two of the most likely exporters, .

이들 단백질의 발현 증가 방법은 통상의 방법을 사용할 수 있으며, 바람직하게는 프로모터(promoter)를 치환할 수 있고, 막 단백질의 과량 발현은 세포성장에 방해가 되므로 중간 세기의 합성 프로모터인 SP5(Synthetic promoter 5) 등을 사용하는 것이 바람직하다.As a method for increasing the expression of these proteins, a conventional method can be used, preferably a promoter can be substituted, and overexpression of a membrane protein interferes with cell growth, so that a synthetic promoter of intermediate intensity, SP5 (Synthetic promoter 5) or the like is preferably used.

바람직하게는, 상기 (3) 단계는 lpd, 및 ducA 유전자 전부를 과발현시킬 수 있다.Preferably, step (3) above can overexpress all of the lpd and ducA genes.

본 발명의 일 예에 있어서, 상기 (4) 단계는 2,4-디히드록시-부틸레이트 (DHB)의 세포 내 재도입을 억제하기 위해 수행하는 것으로, importer 로 가장 가능성이 높을 것으로 예상되는 3개의 막 단백질을 암호화하는 유전자인 kgtP, dsdx 및 actP 중 어느 하나 이상을 제거할 수 있다. 이 때 사용할 수 있는 프라이머(primer) 서열을 표 18에 나타냈다.In one embodiment of the present invention, step (4) is performed to inhibit intracellular re-introduction of 2,4-dihydroxy-butyrate (DHB) Any one or more of the genes coding for the membrane proteins, kgtP, dsdx and actP , can be removed. The primer sequences that can be used at this time are shown in Table 18. < tb >< TABLE >

바람직하게는, 상기 (4) 단계는 kgtP, dsdx, 및 actP 유전자 전부를 제거시킬 수 있다.Preferably, step (4) may remove all of the kgtP, dsdx , and actP genes.

상기 (1)단계를 사용해 제조한 변이균주를 표 2(EcW1 내지 EcW13)에, 상기 (1)단계 및 (2)단계까지 수행해 제조한 변이균주를 표 15(EcW13 내지 EcW16)에, 상기 (1)단계, (2)단계, (3)단계 또는/및 (4)단계까지 수행해 제조한 변이균주를 표 19(EcW16 내지 EcW20)에 정리해 나타냈다. The mutant strains prepared by carrying out the mutation strains prepared in the above step (1) to the strains of Table 2 (EcW1 to EcW13), the strains obtained in the above steps (1) and (2) are shown in Table 15 (EcW13 to EcW16) The strains prepared by performing the steps of (1), (2), (3) or / or (4) were listed in Table 19 (EcW16 to EcW20).

본 발명의 일 예에 따르면, 상기 미생물 변이체 제조방법은 호모세린으로부터 4-히드록시-2-옥소-부틸레이트(4-hydroxy-2-oxo-butyrate)의 전환을 촉진하는 단계를 추가로 포함하는 것일 수 있다.According to one embodiment of the present invention, the method for producing a microorganism variant further comprises the step of promoting the conversion of 4-hydroxy-2-oxo-butyrate from homoserine Lt; / RTI >

상기 전환을 촉진하는 단계란, 호모세린(homoserine)의 알파 위치 아민기를 제거해 4-히드록시-2-옥소-부틸레이트의 전환을 촉진하는 것을 의미하며, 구체적으로, 트랜스아미네이즈(transaminase)를 처리하여 호모세린(homoserine)의 알파 위치 아민기를 제거하는 단계를 포함하는 것일 수 있다. 상기 트랜스아미네이즈는 이를 암호화하는 유전자가 포함된 벡터를 미생물에 다량 도입해 형질전환하거나, 트랜스아미네이즈를 암호화하는 유전자의 프로모터를 과발현 프로모터로 치환시키는 방법 등을 사용할 수 있다.The step of promoting the conversion means that the conversion of 4-hydroxy-2-oxo-butylate is promoted by removing the alpha-position amine group of homoserine. Specifically, transaminase is treated To remove the alpha-position amine group of homoserine. The transaminase may be transformed by introducing a vector containing the gene encoding the gene into a microorganism in a large amount, or a method of replacing the promoter of the gene encoding the transaminase with an over-expressing promoter.

상기 트랜스아미네이즈는 그 종류를 특별히 한정하지 않으나, 피루브산(pyruvate)을 아미노기 어셉터(acceptor)로 사용하는 효소일 수 있으며, 구체적으로 서열번호 12의 아미노산 서열로 이루어진 효소, 서열번호 13의 아미노산 서열로 이루어진 효소, 서열번호 14의 아미노산 서열로 이루어진 효소, 및 서열번호 15의 아미노산 서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상을 사용할 수 있고, 바람직하게는 서열번호 13의 아미노산 서열로 이루어진 효소 또는 서열번호 14의 아미노산 서열로 이루어진 효소 또는 서열번호 15의 아미노산 서열로 이루어진 효소를 사용하는 것이 바람직하다.The transaminase may be an enzyme using pyruvate as an amino acceptor, specifically, an enzyme consisting of the amino acid sequence of SEQ ID NO: 12, an amino acid sequence of SEQ ID NO: 13 An enzyme consisting of the amino acid sequence of SEQ ID NO: 14, and an enzyme consisting of the amino acid sequence of SEQ ID NO: 15, preferably an enzyme consisting of the amino acid sequence of SEQ ID NO: An enzyme consisting of the amino acid sequence of SEQ ID NO: 14 or an enzyme consisting of the amino acid sequence of SEQ ID NO: 15 is preferably used.

상기 서열번호 12의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는서열번호 1의 염기서열로 이루어진 것일 수 있으며, 서열번호 13의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 2의 염기서열로 이루어진 것일 수 있고, 서열번호 14의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 3의 염기서열로 이루어진 것일 수 있고, 서열번호 15의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 4의 염기서열로 이루어진 것일 수 있다. The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 12 may be composed of the nucleotide sequence of SEQ ID NO: 1, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 13 may be the nucleotide sequence of SEQ ID NO: The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 14 may be composed of the nucleotide sequence of SEQ ID NO: 3, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 15 may be the nucleotide sequence of SEQ ID NO: .

본 발명의 일 예에 따르면, 상기 미생물 변이체 제조방법은 4-히드록시-2-옥소-부틸레이트를 2,4-디히드록시-부틸레이트로 전환을 촉진하는 단계를 추가로 포함하는 것일 수 있다.According to one embodiment of the present invention, the method for producing a microorganism variant may further comprise the step of promoting conversion of 4-hydroxy-2-oxo-butyrate to 2,4-dihydroxy-butyrate .

상기 2,4-디히드록시-부틸레이트로의 전환을 촉진하는 단계는 4-히드록시-2-옥소-부틸레이트의 2번 탄소에 위치한 케톤(ketone)기의 환원을 촉진시키는 것을 의미한다. 4-히드록시-2-옥소-부틸레이트를 환원시키는 리덕테이즈를 암호화하는 유전자가 포함된 벡터를 미생물에 다량 도입해 형질전환하거나, 리덕테이즈를 암호화하는 유전자의 프로모터를 과발현 프로모터로 치환시키는 방법 등을 사용할 수 있다.The step of promoting the conversion to 2,4-dihydroxy-butyrate means promoting the reduction of the ketone group located at the 2-carbon of 4-hydroxy-2-oxo-butyrate. A vector containing a gene encoding a reductase that reduces 4-hydroxy-2-oxo-butylate is introduced into a microorganism in a large amount and transformed, or a promoter of a gene encoding reductase is replaced with an over-expression promoter Method or the like can be used.

본 발명의 일 예에 따르면, 상기 환원반응에 의해 제조되는 2,4-디히드록시-부틸레이트는 순수 광학이성질체인 (2S)-2,4-디히드록시-부틸레이트 및 (2R)-2,4-디히드록시-부틸레이트로 이루어진 군에서 선택된 하나이상일 수 있고, 라세미체, 광학이성질체 혼합물을 제조할 수도 있으며, (2S)-2,4-디히드록시-부틸레이트 또는 (2R)-2,4-디히드록시-부틸레이트 각각의 100% 순수한 단일 화합물을 제조할 수 있다. 상기 2,4-디히드록시-부틸레이트의 광학이성질체 종류는 화합물의 활용용도에 따라 적의 선택해 제조할 수 있다.According to one example of the present invention, the 2,4-dihydroxy-butyrate produced by the reduction reaction is (2S) -2,4-dihydroxy-butyrate, which is a pure optical isomer, and (2S) -2,4-dihydroxy-butyrate or (2R) -2,4-dihydroxy-butyrate may be used, -2, < / RTI > 4-dihydroxy-butyrate < / RTI > The kind of the optical isomer of 2,4-dihydroxy-butyrate may be appropriately selected depending on the application of the compound.

(2S)-2,4-디히드록시-부틸레이트의 전환을 촉진하기 위해서 L-히드록시-2-옥소-리덕테이즈(L-hydroxy-2-oxo-reductase) 를 과발현하고, (2R)-2,4-디히드록시-부틸레이트로의 전환을 촉진하기 위해 D-히드록시-2-옥소-리덕테이즈(D-hydroxy-2-oxo-reductase)를 과발현할 수 있다.Over-expression of L-hydroxy-2-oxo-reductase to promote the conversion of (2S) -2,4-dihydroxy- D-hydroxy-2-oxo-reductase may be overexpressed to promote the conversion of D-hydroxy-2-oxo-reductase to dihydroxy-butylate.

본 발명의 바람직한 일 예에 따르면, 상기 L-히드록시-2-옥소-리덕테이즈는 서열번호 16의 아미노산 서열로 이루어진 효소, 서열번호 17의 아미노산 서열로 이루어진 효소 및 서열번호 18의 아미노산 서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상을 포함하는 것일 수 있으며, 바람직하게는 서열번호 17의 아미노산 서열로 이루어진 효소 또는/및 서열번호 18의 아미노산 서열로 이루어진 효소일 수 있다. 상기 서열번호 16의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 5의 염기서열로 이루어진 것일 수 있으며, 상기 서열번호 17의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 6의 염기서열로 이루어진 것일 수 있고, 상기 서열번호 18의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 7의 염기서열로 이루어진 것일 수 있다.According to a preferred embodiment of the present invention, the L-hydroxy-2-oxo-reductase comprises an enzyme consisting of the amino acid sequence of SEQ ID NO: 16, an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence of SEQ ID NO: An enzyme consisting of the amino acid sequence of SEQ ID NO: 17, and / or an enzyme consisting of the amino acid sequence of SEQ ID NO: 18. The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 16 may be composed of the nucleotide sequence of SEQ ID NO: 5, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 17 may be the nucleotide sequence of SEQ ID NO: And the gene coding for the enzyme consisting of the amino acid sequence of SEQ ID NO: 18 may be composed of the nucleotide sequence of SEQ ID NO:

본 발명의 또다른 일 예에 따르면, 상기 D-히드록시-2-옥소-리덕테이즈는 서열번호 19의 아미노산 서열로 이루어진 효소, 서열번호 20의 아미노산 서열로 이루어진 효소, 및 서열번호 21의 아미노산 서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상을 포함할 수 있으며, 바람직하게는 서열번호 20의 아미노산 서열로 이루어진 효소, 또는/및 서열번호 21의 아미노산 서열로 이루어진 효소를 포함할 수 있다. 상기 서열번호 19의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 8의 염기서열로 이루어진 것일 수 있으며, 상기 서열번호 20의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 9의 염기서열로 이루어진 것일 수 있고, 상기 서열번호 21의 아미노산 서열로 이루어진 효소를 암호화하는 유전자는 서열번호 10의 염기서열로 이루어진 것일 수 있다.According to another embodiment of the present invention, the D-hydroxy-2-oxo-reductase is an enzyme consisting of the amino acid sequence of SEQ ID NO: 19, an enzyme consisting of the amino acid sequence of SEQ ID NO: 20, An enzyme consisting of the amino acid sequence of SEQ ID NO: 20, and / or an enzyme consisting of the amino acid sequence of SEQ ID NO: 21. The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 19 may be composed of the nucleotide sequence of SEQ ID NO: 8, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 20 may be the nucleotide sequence of SEQ ID NO: And the gene coding for the enzyme consisting of the amino acid sequence of SEQ ID NO: 21 may be the nucleotide sequence of SEQ ID NO:

본 발명의 일 예에 따르면, 상기 방법은 2,4-디히드록시-부틸레이트에 락톤화를 촉진하여 2-히드록시-감마-부티로락톤의 생산을 촉진하는 단계를 추가로 포함하는 것일 수 있다. 상기 락톤화의 촉진은 락토네이즈(lactonase)의 발현을 촉진하여 수행되는 것일 수 있으며, 상기 발현 촉진 방법은 락토네이즈 유전자를 포함하는 벡터를 과량 세포에 도입해 락토네이즈 양을 증가시키거나 락토네이즈 유전자를 발현하는 프로모터를 과발현 프로모터를 치환하는 등 다양한 방법을 사용할 수 있다.According to one embodiment of the present invention, the method may further comprise the step of promoting lactonization to 2,4-dihydroxy-butyrate to promote the production of 2-hydroxy-gamma-butyrolactone have. The lactoneization promoting may be carried out by promoting the expression of lactonase. The expression promoting method may be carried out by introducing a vector containing a lactonase gene into an excess cell to increase the amount of lactonase, May be replaced with an over-expressing promoter.

바람직하게는, 상기 락톤화의 촉진은 서열번호 22의 아미노산 서열로 이루어진 락토네이즈(lactonase)의 발현을 촉진하여 수행되는 것일 수 있으며, 상기 서열번호 22의 아미노산 서열로 이루어진 락토네이즈를 암호화하는 유전자는 서열번호 11의 염기서열로 이루어진 것일 수 있다.Preferably, the promoting of the lactonization may be performed by promoting the expression of lactonase comprising the amino acid sequence of SEQ ID NO: 22, and the gene encoding the lactonase comprising the amino acid sequence of SEQ ID NO: 11 < / RTI >

상기 생성되는 2-히드록시-감마-부티로락톤은 순수한 광학적 이성질체인(2S)-2-히드록시 감마 부티로락톤 및 (2R)-2-히드록시 감마 부티로락톤으로 이루어진 군에서 선택된 하나 이상일 수 있으며, 바람직하게는 (2S)-2-히드록시 감마 부티로락톤 또는 (2R)-2-히드록시 감마 부티로락톤일 수 있다.The resulting 2-hydroxy-gamma-butyrolactone may be at least one selected from the group consisting of the pure optical isomers (2S) -2-hydroxy gamma butyrolactone and (2R) -2-hydroxy gamma butyrolactone And preferably (2S) -2-hydroxy gamma butyrolactone or (2R) -2-hydroxy gamma butyrolactone.

상기 방법에 의해 생산된 (2S)-2-히드록시 감마 부티로락톤 또는 (2R)-2-히드록시 감마 부티로락톤은 광학 순도가 90% 내지 100%, 바람직하게는 95% 내지 100%, 더욱 바람직하게는 99% 내지 100%일 수 있다.The (2S) -2-hydroxy gamma butyrolactone or (2R) -2-hydroxy gamma butyrolactone produced by the above method has an optical purity of 90% to 100%, preferably 95% to 100% And more preferably 99% to 100%.

본 발명의 일 예에 따르면, 상기 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물의 게놈(genome)에 (1) 내지 (4) 중 어느 하나 이상의 유전자 변이가 도입된, 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤 생산 미생물 변이체를 제공할 수 있다.According to one embodiment of the present invention, the genome of the microorganism producing the 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone is added to any one of (1) to (4) 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variant into which the above-mentioned gene mutation has been introduced.

(1) ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, 및 iclR 유전자로 이루어진 군에서 선택된 하나 이상의 결실, 또는 acs, ppc, 및 metL 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계; (1) ptsG, eda, adhE, pf1B, lysA, thrB C, metA, LacI, ldhA, and iclR deletions one selected from the group consisting of genes or more, or one or more genes selected from the acs, ppc, and the group consisting of metL gene Overexpressing, or both;

(2) epd, dxs, 및 pdxj 유전자로 이루어진 군에서 선택된 하나 이상의 유전자의 과발현,(2) overexpression of one or more genes selected from the group consisting of epd , dxs , and pdx j genes,

(3) ldp 및 ducA 중 하나 이상의 유전자의 과발현,(3) overexpression of at least one of ldp and ducA ,

(4) kgtP, dsdx, 및 actP 유전자로 이루어진 군에서 선택된 하나 이상의 유전자의 결실.(4) deletion of one or more genes selected from the group consisting of kgtP , dsdx , and actP genes.

본 발의 일 예에 따르면, 상기 미생물 변이체는 바람직하게는 pts, eda, lacI, thrB, metA, lysA, adhE, pflB, ldhA, 및 iclR 유전자가 결실되고, acs 유전자를 과발현, 및 ppc 유전자가 과별현된 미생물 변이체를 제조할 수 있다. 상기 미생물 변이체는 표 15에 나타낸 EcW13 균주일 수 있으며, 바람직하게는 기탁번호 KCCM12281P의 균주일 수 있다.According to one embodiment of the present invention, the microorganism variant is preferably selected from the group consisting of pts, eda, lacl, thrB, metA, lysA, adhE, pflB, ldhA and iclR genes are deleted and overexpressed acs gene, Microorganism variants can be produced. The microorganism variant may be the EcW13 strain shown in Table 15, preferably the strain of Accession No. KCCM12281P.

본 발의 또다른 일 예에 따르면, 상기 미생물 변이체는 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물의 게놈(genome)에서, ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, iclR, kgtP, dsd, 및 actP 유전자가 결실되고, acs, ppc, metL, epd, dxs, pdxj, ldp, 및 ducA 유전자가 과발현된, 미생물 변이체를 제조할 수 있으면, 상기 미생물 변이체는 하기 실시예에서 실험한 EcW20 균주로 기탁번호 KCCM12282P 의 균주일 수 있다.According to another embodiment of the present invention, the microorganism variant is selected from the group consisting of ptsG, eda, adhE (2-hydroxy-2-hydroxy- , pf1B, lysA, thrBC, metA, LacI, ldhA, iclR, kgtP, dsd, and actP genes and overexpressing acs, ppc, metL, epd, dxs, pdxj, ldp and ducA genes If it can be produced, the microorganism variant may be a strain of EcT20, which was tested in the following Examples, and the strain of Accession No. KCCM12282P.

본 발명의 일 예에 따르면, 상기 미생물 변이체는 하기 (1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 벡터를 추가로 포함할 수 있다.According to one embodiment of the present invention, the microorganism variant may further comprise one or more genes selected from the following (1) to (4) or a recombinant vector comprising the same.

(1) 서열번호 13의 아미노산서열로 이루어진 효소를 암호화하는 유전자, 서열번호 14의 아미노산서열로 이루어진 효소를 암호화하는 유전자, 및 서열번호 15의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 트랜스아미네이즈 변이효소 암호화 유전자, (1) a gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 13, a gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 14, and a gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 15 One or more transaminase mutant enzyme encoding genes,

(2) 서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 L-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자,(2) at least one L-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: Enzyme-encoding genes,

(3) 서열번호 20의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 21의 아미노산열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 D-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자, 및(3) at least one D-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 20 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: An enzyme-encoding gene, and

(4) 서열번호 22의 아미노산서열로 이루어진 락톤네이즈 효소를 암호화하는 유전자.(4) A gene encoding the lactonase enzyme comprising the amino acid sequence of SEQ ID NO: 22.

서열번호 13의 아미노산서열로 이루어진 효소를 암호화하는 유전자는 서열번호 2의 염기서열로 이루어진 것일 수 있으며, 서열번호 14의 아미노산서열로 이루어진 효소를 암호화하는 유전자는 서열번호 3의 염기서열로 이루어진 것일 수 있고, 서열번호 15의 아미노산서열로 이루어진 효소를 암호화하는 유전자는 서열번호 4의 염기서열로 이루어진 것일 수 있으며, The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 13 may be composed of the nucleotide sequence of SEQ ID NO: 2, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 14 may be composed of the nucleotide sequence of SEQ ID NO: The gene coding for the enzyme consisting of the amino acid sequence of SEQ ID NO: 15 may be composed of the nucleotide sequence of SEQ ID NO: 4,

서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자는 서열번호 6의 염기서열로 이루어진 것일 수 있고, 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자는 서열번호 7의 염기서열로 이루어진 것일 수 있으며, The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 17 may be composed of the nucleotide sequence of SEQ ID NO: 6, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 18 may be composed of the nucleotide sequence of SEQ ID NO: In addition,

서열번호 19의 아미노산열로 이루어진 효소를 암호화하는 유전자는 서열번호 9의 염기서열로 이루어진 것일 수 있고, 서열번호 20의 아미노산서열로 이루어진 효소를 암호화하는 유전자는 서열번호 10의 염기서열로 이루어진 것일 수 있다.The gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 19 may be composed of the nucleotide sequence of SEQ ID NO: 9, and the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 20 may be the nucleotide sequence of SEQ ID NO: have.

본 발명의 일 구체예에 따르면, 상기 미생물 변이체는 호모세린, 4-히드록시-2-옥소 부티레이트, 2,4-디히드록시-부틸레이트, 및 2-히드록시-감마-부티로락톤로 이루어진 군에서 선택된 하나 이상을 과량 생산하는 것인, 미생물 변이체일 수 있다.According to one embodiment of the present invention, the microorganism variant is selected from the group consisting of homoserine, 4-hydroxy-2-oxobutyrate, 2,4-dihydroxy-butyrate, and 2-hydroxy-gamma-butyrolactone Lt; RTI ID = 0.0 > 1, < / RTI >

본 발명의 일 예에 따르면, 앞서 설명한 상기 미생물 변이체를 배양하는 단계를 포함하는, 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone) 또는 2,4-디히드록시 부틸레이트(2,4-dihydroxy butanoic acid)의 생산방법을 제공한다.According to one embodiment of the present invention, there is provided a method for producing a microorganism which comprises culturing the aforementioned microorganism variant as described above, wherein 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutyrate (2,4 dihydroxy butanoic acid. < / RTI >

상기 생산방법에 사용하는 상기 미생물 변이체는 하기 (1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 벡터를 추가로 포함하는 것일 수 있다.The microorganism variant used in the production method may further comprise one or more genes selected from the following (1) to (4) or a recombinant vector comprising the same.

(1) 서열번호 13의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 14의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 트랜스아미네이즈 변이효소 암호화 유전자,(1) at least one transaminase mutant encoding gene selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 13 and an enzyme encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 14,

(2) 서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 L-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자, (2) at least one L-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: Enzyme-encoding genes,

(4) 서열번호 22의 아미노산서열로 이루어진 효소를 암호화하는 유전자.(4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.

본 발명의 일 구체예에 따르면, 상기 생산방법에서 배양하는 단계는 질소원으로 효모 추출물 및 암모늄 염을 포함하는 배지에서 수행하는 것일 수 있다.According to one embodiment of the present invention, the step of culturing in the production method may be performed in a culture medium containing yeast extract and ammonium salt as a nitrogen source.

또 다른 일 예에 따르면, 상기 배양하는 단계 이후에, 당과 메티오닌(methionine), 라이신(lysine), 트레오닌(threonine), 및 이소류신(isoleucine)으로 이루어진 군에서 선택된 하나 이상의 아미노산을 첨가하여 유가식으로 발효하는 단계를 추가로 포함할 수 있으며, 이에 더 나아가 발효단계에서 순수 광학 이성질체인 (2S)-2,4-디히드록시 부틸레이트 또는 (2R)-2,4-디히드록시 부틸레이트를 생산한 후, pH 1.0 내지 3.0, 바람직하게는 pH 1.0 내지 2.0 범위로 낮추는 화학적 변화단계를 거쳐 순수한 (2S)-2-히드록시-감마-부티로락톤 또는 (2R)-2-히드록시-감마-부티로락톤으로 전환할 수 있다. According to another embodiment, after the step of culturing, at least one amino acid selected from the group consisting of a sugar and methionine, lysine, threonine, and isoleucine is added, (2S) -2,4-dihydroxybutyrate or (2R) -2,4-dihydroxybutyrate, which is a pure optical isomer, in the fermentation step (2S) -2-hydroxy-gamma-butyrolactone or (2R) -2-hydroxy-gamma-butyrolactone through a chemical modification step which is followed by lowering the pH to a range of 1.0 to 3.0, Butyrolactone. &Lt; / RTI >

예를 들어, (2S)-2,4-디히드록시 부틸레이트를 생산한 후, pH를 1.0 내지 3.0으로 낮추어 광학순도가 95% 내지 100%, 바람직하게는 97% 내지 100%인 (2S)-2-히드록시-감마-부티로락톤를 얻을 수 있으며, (2R)-2,4-디히드록시 부틸레이트를 생산한 후 pH를 1.0 내지 3.0으로 낮추어 광학순도가 95% 내지 100%, 바람직하게는 97% 내지 100%인 (2R)-2-히드록시-감마-부티로락톤을 얻을 수 있다.For example, after producing (2S) -2,4-dihydroxybutyrate, the pH is lowered to 1.0 to 3.0 to obtain (2S) -2,4-dihydroxybutyrate having an optical purity of 95% to 100%, preferably 97% Hydroxy-gamma-butyrolactone can be obtained. After the production of (2R) -2,4-dihydroxybutyrate, the pH is lowered to 1.0 to 3.0 to obtain an optical purity of 95% to 100% (2R) -2-hydroxy-gamma-butyrolactone, which is 97% to 100%.

본 발명의 일 예는, 상기에서 설명한 미생물 변이체 또는 그 배양물을 포함하는, 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone) 또는 2,4-디히드록시 부틸레이트(2,4-dihydroxy butanoic acid)의 생산용 조성물을 제공한다.One example of the present invention is a pharmaceutical composition comprising 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutyrate (2,4-dihydroxybutyrate), including the above- dihydroxy butanoic acid).

상기 미생물 변이체는 하기 (1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 벡터를 추가로 포함하는 것일 수 있다.The microorganism variant may further comprise one or more genes selected from the following (1) to (4) or a recombinant vector comprising the same.

(1) 서열번호 13의 아미노산으로 이루어진 효소를 암호화하는 염기서열로 이루어진 유전자 및 서열번호 14의 아미노산으로 이루어진 효소를 암호화하는 염기서열로 이루어진 유전자로 이루어진 군에서 선택된 하나 이상의 트랜스아미네이즈 변이효소 암호화 유전자,(1) a gene consisting of a nucleotide sequence encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 13 and a gene consisting of a nucleotide sequence encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 14; and at least one transaminase mutant enzyme encoding gene ,

(2) 서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 L-히드록시-2-옥소-리덕테이즈 변이효소암호화 유전자, (2) at least one L-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: Enzyme-encoding genes,

(4) 서열번호 22의 아미노산서열로 이루어진 락토네이즈 효소를 암호화하는 유전자.(4) A gene encoding the lactonase enzyme comprising the amino acid sequence of SEQ ID NO: 22.

본 발명의 일 예는 서열번호 13의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소를 제공한다. 상기 트랜스아미네이즈 변이효소를 암호화하는 유전자는 서열번호 2의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides a transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: The gene encoding the transaminase mutant enzyme may be one comprising the nucleotide sequence of SEQ ID NO: 2.

본 발명의 일 예는 서열번호 14의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소를 제공한다. 상기 트랜스아미네이즈 변이효소를 암호화하는 유전자는 서열번호 3의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides a transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 14. The gene coding for the transaminase mutant enzyme may be composed of the nucleotide sequence of SEQ ID NO: 3.

본 발명의 일 예는 서열번호 15의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소를 제공한다. 상기 트랜스아미네이즈 변이효소를 암호화하는 유전자는 서열번호 4의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides a transaminase mutant enzyme consisting of the amino acid sequence of SEQ ID NO: The gene encoding the transaminase mutant enzyme may be one comprising the nucleotide sequence of SEQ ID NO: 4.

본 발명의 일 예는 서열번호 17의 아미노산 서열로 이루어진 L-히드록시-2-옥소-리덕테이즈 변이효소를 제공한다. 상기 L-히드록시-2-옥소-리덕테이즈 변이효소를 암호화하는 유전자는 서열번호 6의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides an L-hydroxy-2-oxo-lyudatease mutant enzyme consisting of the amino acid sequence of SEQ ID NO: The gene coding for the L-hydroxy-2-oxo-lyudatease mutant enzyme may comprise the nucleotide sequence of SEQ ID NO: 6.

본 발명의 일 예는 서열번호 18의 아미노산 서열로 이루어진 L-히드록시-2-옥소-리덕테이즈 변이효소를 제공한다. 상기 L-히드록시-2-옥소-리덕테이즈 변이효소를 암호화하는 유전자는 서열번호 7의 염기서열로 이루어진 것일 수 있다.One example of the present invention provides an L-hydroxy-2-oxo-lyudatease mutant enzyme consisting of the amino acid sequence of SEQ ID NO: 18. The gene coding for the L-hydroxy-2-oxo-reductase mutant may be a nucleotide sequence of SEQ ID NO: 7.

본 발명의 일 예는 서열번호 20의 아미노산 서열로 이루어진 D-히드록시-2-옥소-리덕테이즈 변이효소를 제공한다. 상기 D-히드록시-2-옥소-리덕테이즈 변이효소를 암호화하는 유전자는 서열번호 9의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides a D-hydroxy-2-oxo-lyudatease mutant enzyme consisting of the amino acid sequence of SEQ ID NO: 20. The gene encoding the D-hydroxy-2-oxo-reductase mutant enzyme may be one comprising the nucleotide sequence of SEQ ID NO:

본 발명의 일 예는 서열번호 21의 아미노산 서열로 이루어진 D-히드록시-2-옥소-리덕테이즈 변이효소를 제공한다. 상기 D-히드록시-2-옥소-리덕테이즈 변이효소를 암호화하는 유전자는 서열번호 10의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides a D-hydroxy-2-oxo-lyudatease mutant enzyme consisting of the amino acid sequence of SEQ ID NO: 21. The gene coding for the D-hydroxy-2-oxo-reductase mutant enzyme may be composed of the nucleotide sequence of SEQ ID NO: 10.

본 발명의 일 예는 서열번호 22의 아미노산 서열로 이루어진 락토네이즈 변이효소를 제공한다. 상기 락토네이즈 변이효소를 암호화하는 유전자는 서열번호 11의 염기서열로 이루어진 것일 수 있다.An example of the present invention provides a lactonase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 22. The gene coding for the lactonase mutation enzyme may comprise the nucleotide sequence of SEQ ID NO: 11.

본 발명은 2-hydroxy gamma butyrolactone (HGBL) 및 이의 전구체인 2,4-dihydroxybutanoic acid 를 과발현하는 변이 균주 및 이를 이용한 2-hydroxy gamma butyrolactone (HGBL) 및 이의 전구체인 2,4-dihydroxybutanoic acid을 다량 생산하는 방법을 제공한다. 또한, 본 발명의 변이체와 변이효소를 이용할 경우, 2-히드록시-감마-부티로락톤을 (R) 또는 (S) 형태의 순수한 광학이성질체로 제조할 수 있다. 상기 이성질체로서 이들 화합물은 제약, 농약, 조미료 및 향료의 중간물질 외에 특히 반도체용 포토레지스트, 금속 표면의 코팅재료의 중간체로서 특히 유용하게 응용될 수 있다.The present invention relates to a mutant strain that overexpresses 2-hydroxy gamma butyrolactone (HGBL) and its precursor, 2,4-dihydroxybutanoic acid, and mass production of 2-hydroxy gamma butyrolactone (HGBL) and its precursor 2,4-dihydroxybutanoic acid . &Lt; / RTI > In addition, when the mutant and the mutant enzyme of the present invention are used, 2-hydroxy-gamma-butyrolactone can be made into a pure optical isomer of (R) or (S) form. As the above isomers, these compounds can be particularly usefully applied as intermediates for pharmaceuticals, pesticides, seasonings and flavors as well as intermediates for photoresists for semiconductors and coating materials for metal surfaces.

도 1은 homoserine 으로부터 광학적으로 순수한 2-hydroxy gamma butyrolactone 및 이의 유기산 전구체인 2,4-dihydroxybutanoic acid를 생합성하는 신규 생합성 경로를 제시하고 있다.
도 2는 homoserine 으로부터 homoserine dehydrogenase를 이용하여 4-hydroxy-2-oxo-butanoate를 생합성하는 경로를 제시하고 있다.
도 3은 homoserine 으로부터 homoserine transaminase 및 aspartate transaminase를 이용하여 4-hydroxy-2-oxo-butanoate를 생합성하는 경로를 제시하고 있다.
도 4는 포도당을 이용하여 homoserine을 대량으로 생산하기 위한 균주 개량 전략, 그리고 이로부터 2-hydroxy gamma butyrolactone 및 이의 전구체인 2,4-dihydroxybutanoic acid를 생합성하는 전략을 제시하고 있다.
도 5는 표 5에 기재된 유전자 발현 향상이나 조절을 위해 합성한 합성 프로모터(synthetic promoter)의 상대 활성을 GFP를 이용해 측정한 결과를 나타낸다.
도 6a는 TA1, TA2, TA3, TA6 효소를 생산하는 균주 파쇄액 전체를 denaturing 조건에서 전기영동한 결과로, TA1, TA2, TA3, 및 TA6 4 종류는 insoluble 한 형태로만 효소가 생성됨을 나타낸다.
도 6b는 TA4, TA5, TA7, YA8, TA9, TA10, TA11, TA12, TA13, TA14 및 TA15 등 총 11종 효소를 생산하는 균주의 파쇄액을 원심분리하여 상등액을 얻고 상등액 내 존재하는 단백질을 denaturing 조건에서 전기영동한 결과로 TA4, TA5, TA7, YA8, TA9, TA10, TA11, TA12, TA13, TA14 및 TA15 효소가 soluble 한 상태로 얻어진다는 것을 보여준다.
도 7a는 soluble 한 형태로 발현된 11종의 트랜스아미네이즈 효소 중, TA4, TA5, TA10, TA11, TA15 등 5개의 효소만이 호모세린(homoserine)에 대하여 활성을 나타내었고 이 중 TA4가 가장 높은 활성을 가진다는 것을 나타낸다.
도 7b는 호모세린에 대하여 가장 높은 활성을 나타낸 TA4 효소를 affinity chromatography로 순수분리하여 정제한 결과를 전기영동한 결과이다.
도 8은 Transaminase 스크리닝을 위한 reporter 균주의 성능 확인 결과를 보여준다. E. coli BL21 (DE3) 야생균주와 pET-TA4 유전자 재조합 플라스미드를 갖는 reporter 균주를 각각 소량(0.5 mM)의 메티오닌과 트레오닌 그리고 10 mM의 homoserine이 포함된 배지에서 배양하였다. TA4 효소 발현 플라스미드를 갖는 reporter 균주가 플라스미드를 갖지않는 야생균주에 비해 빠른 성장을 보였고 IPTG 첨가량이 높은 경우, 즉 0.5 mM을 첨가한 경우보다 1.0 mM을 첨가했을 때 균주 성장 속도가 증가하였다. 즉 TA4 효소가 존재할 때 homoserine이 세포성장을 위한 질소원으로 사용된다는 것을 보여준다.
도 9는 대장균 내 존재하는 아스파르트산 아미노산 전이효소(PDB ID: 1ASM)와 TA4 효소의 아미노산 서열을 비교한 결과이다.
도 10은 대장균 아스파르트산 아미노산 전이효소(PDB ID: 1ASM)의 크리스탈 구조를 주형으로 사용하여, 상동성 모델링을 통해 TA4의 3차원 구조를 구축하고, PYMOL뷰어(http://www.pymol.org)를 사용하여 확인한 단백질 구조를 나타낸다.
도 11은 대장균 아스파르트산 아미노산 전이효소 (1ASM) 구조에서 말레산의 카르복실산(아스파르트산 잔기의 카르복실산)과 상호작용을 하고 있는 4개의 아미노산(Ile17, Gly38, Asn194, Arg386)을 확인하고, 이와 비교한 TA4 모델 구조를 통해서 TA4 효소 내 아스파르트산 잔기의 카르복실산과 상호작용을 할 것으로 예상되는 4개의 아미노산(Lys14, Gly40, Asn178, Try364)을 선정해 나타낸 것이다.
도 12는 TA4-1 내지 TA4-6의 변이 효소의 활성을 나타낸다. 이중 TA4-1은 Y364Q, TA4-2는 N174D의 아미노산 서열 변이를 갖고 있으며 높은 활성을 보여 주었다. TA4-6는 TA4-1 및 TA4-2가 갖는 변이를 모두 갖는 효소로 가장 높은 활성인 20 U/mg protein의 활성을 나타내었다.
도 13은 표 9에 나타낸 8가지의 LDH 효소의 효소활성을 pyruvate 와 OHB를 기질로 사용하여 분석한 결과를 나타낸다.
도 14는 대장균 락트산 탈수소효소의 크리스탈 구조(PDB ID: 2G8Y)를 주형으로 사용하여, 상동성 모델링을 통해 Ae_ldhA의 3차원 구조를 구축한 결과로, Ae_ldhA 모델은 MOE(Molecular Operating Environment)를 사용하여 제작했으며, PROCHECK 및 ProSA 온라인 구조 분석을 통해 평가 및PYMOL뷰어(http://www.pymol.org)를 사용하여 확인한 단백질 구조를 나타낸 것이다.
도 15a는 Ae_ldhA 변이체 디자인을 위해 피부르산과 HOB 구조(Pubchem Database)의 구조를 Ae_ldhA1의 효소 활성 위치에 Triangular Matching 방법을 사용하여 도킹 시뮬레이션(docking simulation)을 한 후, 이를 이용하여 효소의 아미노산 잔기와 피부르산과의 상호작용을 검토한 결과를 나타낸다.
도 15b는 도킹 시뮬레이션(docking simulation)을 이용하여 효소의 아미노산 잔기와 HOB와의 상호작용을 검토한 결과를 나타낸다.
도 16은 site-directed mutagenesis로 얻은 Ae_ldhA 변이체 효소의 활성을 NADH의 산화 정도를 340nm의 흡광도에서 관찰해 측정한 결과를 나타낸다. 변이 효소의 활성을 야생 효소의 활성과 비교하여 상대 활성으로 나타내었다. 효소의 활성(specific activity; 1U)은 1분 동안 1 μmol의 NADH를 NAD로 산화시키는데 필요한 효소의 양으로 정의하였다.
도 17은 표 12에 나타낸 9가지의 균주 내 (D)-lactate dehydrogenase 효소활성을 pyruvate 와 OHB를 기질로 사용하여 분석한 결과를 나타낸다.
도 18은 PDB databank에 나와있는 Lb-LDH 효소의 결정구조를 이용하여 engineering 해야 할 아미노산 잔기를 나타낸 것이다.
도 19는 OHB D-reductase 효소의 변이체 효소의 활성을 NADH의 산화 정도를 340nm의 흡광도에서 관찰해 측정한 결과를 나타내며, 효소의 활성(specific activity; 1U)은 1분 동안 1 μmol의 NADH를 NAD+로 산화시키는데 필요한 효소의 양으로 정의하였다. 변이 효소의 활성을 야생 효소의 활성과 비교하여 상대 활성으로 나타내었다.
도 20a는 대장균에서 PLP가 생합성되는 DXP 의존 경로에 작용하는 생합성 유전자의 구조를 나타낸다.
도 20b는 대장균에서 PLP가 생합성되는 DXP 의존 경로의 생합성 경로를 나타낸다. 대사산물의 약어는 다음과 같다. G6P; glusoe-6-phosphate; E4P, erythrose-4-phosphate; GA3P, glyceraldehyde-3-phosphate; 4PE, 4-phospho-D-erythronate; 3P4K, 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate; 4PT, 4-phosphohydroxy-L-threonine; 2A3B, 3-hydroxy-1-amino-acetone phosphate; DXS, deoxyxylulose-5-phosphate. 이탤릭체는 효소이름을 나타낸다: EPD, erythrose-4-phosphate dehydrogenase; PdxB, 4-phospho-D-erythronate; SerC, 3-phosphoserine aminotransferas; PdxA, 4-phosphohydroxy-L-threonine dehydrogenase; PdxJ, PNP synthase; Dxs, 1-deoxyxylulose 5-phosphate synthase; PdhH, PNP oxidase.
도 21은 L-form DHB를 생산하는데 사용된 플라스미드 지도로 트랜스아미네이즈로는 TA4-1을, 그리고 락테이즈 디하드로지네이즈로는 ldh-2와 ldh-8을 각각 사용한 경우를 나타낸 것이다.
도 22는 플라스크 배양을 통한 L-DHB 생산 결과로, 유전자 재조합 pBAD_TA4-1_LDH2 (pDHB-L)플라스미드가 도입된 EcW20(pDHB-L) 균주를 다양한 arabinose 농도에서 배양한 후 생성된 DHB의 농도(g/L)를 측정했다.‘Control’은 pBAD_TA4-1_LDH2 (pDHB-L)플라스미드를 갖지 않는 EcW20 균주를 나타낸다.
도 23은 플라스크 배양을 통한 D-DHB 생산 결과로, 유전자 재조합 pBAD_TA4-1_LDH8 (pDHB-D)플라스미드가 도입된 EcW20(pDHB-D) 균주를 다양한 arabinose 농도에서 배양한 후 생성된 DNB의 농도(g/L)를 측정했다. ‘Control’은 pBAD_TA4-1_LDH8 (pDHB-D)플라스미드를 갖지 않는 EcW20 균주를 나타낸다.
도 24a는 L-form 2,4-dihydroxybutyric acid 생산을 위한 EcW20(pDHB-L) 균주의 유가식 생물반응기 실험결과를 나타낸다.
도 24b는 D-form 2,4-dihydroxybutyric acid 생산을 위한 EcW20(pDHB-D) 균주의 유가식 생물반응기 실험결과를 나타낸다.
도 25는 2,4-dihydroxybutyric acid를 HGBL로 전환하는데 필요한 락토네이즈를 발현하는 pACYC_Pon1 플라스미드 맵을 보여준다. pon1(G3C9) 유전자는 tac promoter 에 의해 발현되고 생산된 단백질이 세포막으로 이동하는데 필요한 lead sequence를 갖도록 제작되었다.
도 26a는 L-DHB 와 HGBL 생산을 위하여 생물반응기 2단 배양을 실시한 결과로, Glucose를 기질로 사용했을 때, 시간에 따라 생성되는 L-form의 2,4-DHB와 HGBL의 생산량 및 Biomass를 나타낸 것이다.
도 26b는 D-DHB 와 HGBL 생산을 위하여 생물반응기 2단 배양을 실시한 결과로, Glucose를 기질로 사용했을 때, 시간에 따라 생성되는 D-form의 2,4-DHB와 HGBL의 생산량 및 Biomass를 나타낸 것이다. D-HGBL의 생산량은 0.1 g/L 이하로 매우 낮게 나타났으며, 이는 Pon1 효소의 활성이 2,4-DHB에 대하여 매우 낮았기 때문이다.Figure 1 shows a novel biosynthetic pathway for biosynthesis of optically pure 2-hydroxy gamma butyrolactone and its organic acid precursor 2,4-dihydroxybutanoic acid from homoserine.
Figure 2 shows the pathway for the biosynthesis of 4-hydroxy-2-oxo-butanoate from homoserine using homoserine dehydrogenase.
FIG. 3 shows a pathway for biosynthesis of 4-hydroxy-2-oxo-butanoate using homoserine transaminase and aspartate transaminase from homoserine.
FIG. 4 shows a strategy for the mass production of homoserine using glucose and a strategy for biosynthesis of 2-hydroxy gamma butyrolactone and its precursor 2,4-dihydroxybutanoic acid.
FIG. 5 shows the results of measurement of the relative activity of a synthetic promoter synthesized for improving or controlling gene expression shown in Table 5 using GFP.
FIG. 6A shows the results of electrophoresis of the whole strain-producing lysate producing TA1, TA2, TA3 and TA6 enzymes under denaturing conditions, indicating that four types of TA1, TA2, TA3 and TA6 are enzymatically produced only in insoluble form.
6B shows the results of centrifugation of a strain producing strains of 11 enzymes including TA4, TA5, TA7, YA8, TA9, TA10, TA11, TA12, TA13, TA14 and TA15 to obtain a supernatant, And TA9, TA9, TA10, TA11, TA12, TA13, TA14 and TA15 enzymes were obtained in a soluble state.
FIG. 7a shows that among the eleven transaminase enzymes expressed in soluble form, only five enzymes such as TA4, TA5, TA10, TA11, and TA15 were active against homoserine, and TA4 was the highest Activity.
FIG. 7B shows the results of electrophoresis of purified TA4 enzyme, which exhibits the highest activity for homoserine by affinity chromatography.
FIG. 8 shows the results of confirming the performance of a reporter strain for transaminase screening. Reporter strains harboring E. coli BL21 (DE3) wild-type strains and pET-TA4 recombinant plasmids were cultured in medium containing small amounts (0.5 mM) of methionine, threonine and 10 mM homoserine, respectively. The reporter strain with the TA4 enzyme - expressing plasmid showed faster growth than the wild - type strain without the plasmid, and the growth rate of the strain was increased when 1.0 mM was added in the case of high IPTG addition. That is, when TA4 enzyme is present, homoserine is used as a nitrogen source for cell growth.
9 is a result of comparing the amino acid sequence of the TA4 enzyme with the aspartic acid amino acid transferase (PDB ID: 1 ASM) present in E. coli.
10 shows the construction of a three-dimensional structure of TA4 through homology modeling using the crystal structure of E. coli aspartic acid amino acid transferase (PDB ID: 1 ASM) as a template, and a PYMOL viewer (http://www.pymol.org ). &Lt; / RTI >
Figure 11 identifies four amino acids (Ile17, Gly38, Asn194, Arg386) interacting with the carboxylic acid (carboxylic acid of the aspartic acid residue) of the maleic acid in the E. coli aspartic acid amino acid transferase (1ASM) , And four amino acids (Lys14, Gly40, Asn178, and Try364) expected to interact with carboxylic acid residues of aspartic acid residues in the TA4 enzyme through the TA4 model structure.
Fig. 12 shows the activity of mutant enzymes TA4-1 to TA4-6. TA4-1 had Y364Q, TA4-2 had N174D amino acid sequence variation and showed high activity. TA4-6 was the enzyme with TA4-1 and TA4-2 mutations and showed the highest activity of 20 U / mg protein.
FIG. 13 shows the results of analysis of enzymatic activities of the eight LDH enzymes shown in Table 9 using pyruvate and OHB as substrates.
14 shows the result of constructing the three-dimensional structure of Ae_ldhA by homology modeling using the crystal structure (PDB ID: 2G8Y) of E. coli lactate dehydrogenase as a template, and the Ae_ldhA model was obtained using MOE (Molecular Operating Environment) And evaluated through PROCHECK and ProSA online structure analysis. The protein structure confirmed using the PYMOL viewer (http://www.pymol.org) is shown.
FIG. 15a shows the structure of the skin leucine and HOB structure (Pubchem Database) for the Ae_ldhA mutant design by docking simulation using the triangular matching method at the enzyme active site of Ae_ldhA1, The results of reviewing the interaction of skin with acid.
FIG. 15B shows the result of examining the interaction between amino acid residues of the enzyme and HOB using a docking simulation.
16 shows the activity of the Ae_ldhA mutant enzyme obtained by site-directed mutagenesis and the results of measurement of the degree of oxidation of NADH observed at 340 nm absorbance. The activity of the mutant enzyme was shown to be relative to the activity of the wild enzyme. The specific activity (1 U) of the enzyme was defined as the amount of enzyme required to oxidize 1 μmol of NADH to NAD for 1 min.
FIG. 17 shows the results of analysis of (D) -lactate dehydrogenase enzyme activities in nine strains shown in Table 12 using pyruvate and OHB as substrates.
FIG. 18 shows the amino acid residues to be engineered using the crystal structure of the Lb-LDH enzyme shown in the PDB databank.
19 shows the activity of the mutant enzyme of the OHB D-reductase enzyme measured by measuring the degree of oxidation of NADH at an absorbance of 340 nm. The activity of the enzyme (1 U) was determined by adding 1 μmol of NADH to NAD + As the amount of enzyme required to oxidize. The activity of the mutant enzyme was shown to be relative to the activity of the wild enzyme.
20A shows the structure of a biosynthetic gene acting on a DXP-dependent pathway in which PLP is biosynthesized in E. coli.
20B shows a biosynthetic pathway of DXP-dependent pathway in which PLP is biosynthesized in E. coli. Abbreviations for metabolites are as follows. G6P; glucose-6-phosphate; E4P, erythrose-4-phosphate; GA3P, glyceraldehyde-3-phosphate; 4PE, 4-phospho-D-erythronate; 3P4K, 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate; 4PT, 4-phosphohydroxy-L-threonine; 2A3B, 3-hydroxy-1-amino-acetone phosphate; DXS, deoxyxylulose-5-phosphate. Italic indicates enzyme name: EPD, erythrose-4-phosphate dehydrogenase; PdxB, 4-phospho-D-erythronate; SerC, 3-phosphoserine aminotransferase; PdxA, 4-phosphohydroxy-L-threonine dehydrogenase; PdxJ, PNP synthase; Dxs, 1-deoxyxylulose 5-phosphate synthase; PdhH, PNP oxidase.
Fig. 21 shows the plasmid map used for producing L-form DHB, TA4-1 as transaminase, and ldh-2 and ldh-8 as lactase dehydrogenase, respectively.
FIG. 22 shows the results of the production of L-DHB by flask culture and the concentration of DHB produced after incubation of EcW20 (pDHB-L) strain in which recombinant pBAD_TA4-1_LDH2 (pDHB-L) plasmid was introduced at various arabinose concentrations / L). &Quot; Control " represents an EcW20 strain having no pBAD_TA4-1_LDH2 (pDHB-L) plasmid.
23 shows the results of D-DHB production through flask culture, showing the concentration of DNB produced after incubation of EcW20 (pDHB-D) strain in which recombinant pBAD_TA4-1_LDH8 (pDHB-D) plasmid was introduced at various arabinose concentrations / L) was measured. 'Control' represents the EcW20 strain which does not have the pBAD_TA4-1_LDH8 (pDHB-D) plasmid.
24A shows the results of a bioreactor bioreactor experiment of an EcW20 (pDHB-L) strain for the production of L-form 2,4-dihydroxybutyric acid.
FIG. 24B shows the results of an experimental bioreactor experiment of EcW20 (pDHB-D) strain for the production of D-form 2,4-dihydroxybutyric acid.
25 shows the plasmid map of pACYC_Pon1 expressing lactonase necessary to convert 2,4-dihydroxybutyric acid to HGBL. The pon1 (G3C9) gene is expressed by the tac promoter and the produced protein is designed to have the necessary lead sequence to move to the cell membrane.
FIG. 26A shows the results of the two-stage bioreactor culture for the production of L-DHB and HGBL. As a result, the production of 2,4-DHB and HGBL and the biomass production of L- .
FIG. 26B shows the results of the two-stage bioreactor culture for the production of D-DHB and HGBL. When glucose was used as a substrate, the amount of 2,4-DHB and HGBL produced in D- . The yield of D-HGBL was very low, less than 0.1 g / L, because the activity of Pon1 enzyme was very low for 2,4-DHB.

호모세린(homoserine)으로부터 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone)을 생합성하기 총 3개의 단계로 이루어진 신규 생합성 경로를 도 1에서 제안하였다. 구체적으로 각 단계의 반응은 다음과 같다. A novel biosynthetic pathway consisting of three steps of biosynthesis of 2-hydroxy gamma butyrolactone from homoserine was proposed in FIG. Specifically, the reaction of each step is as follows.

Step 1: 호모세린 내 알파 위치 아민 제거 반응 Step 1: Alpha-position amine removal reaction in homoserine

Step 2: 환원 반응 (2번 탄소에 결합된 산소 환원) Step 2: Reduction reaction (oxygen reduction bound to carbon # 2)

Step 3: 락톤화 반응 (Lactonization)Step 3: Lactonization

상기 첫번째 반응은 호모세린 디아미네이즈(homoserine deaminase) 또는 호모세린 트랜스아미네이즈(homoserine transaminase) 효소에 의해 촉매될 수 있으며, 호모세린 디아미네이즈에 의한 반응은 도 2에 나타냈으며, 호모세린 트랜스아미네이즈에 의한 반응은 도 3에 각각 나타냈다.The first reaction can be catalyzed by homoserine deaminase or homoserine transaminase, and the reaction by homoserine diamine is shown in Fig. 2, and homoserine transaminase The reactions by Nath are shown in Fig. 3, respectively.

도 2에 제시한 아미노산의 알파 위치 아민기를 제거하는 효소로는 글루타메이트(glutamate)에 높은 활성을 보이는 글루타메이트 디아미네이즈(glutamate deaminase; dehydrogenase), 그리고 세린(serine)에 높은 활성을 보이는 세린 디아미네이즈(serine deaminase), 그리고 활성이 매우 낮지만 다른 많은 아미노산들에 조금씩 활성을 보이는 아미노산 산화효소 (amino acid oxidase)를 사용할 수 있으며 도 2의 반응에는 이 효소들을 직접 사용하거나 또는 높은 활성을 갖도록 변이시킨 후 사용할 수 있다. Examples of the enzyme that removes the alpha-position amine group of the amino acid shown in Fig. 2 include glutamate deaminase (dehydrogenase), which exhibits high activity on glutamate, and serine diaminase (amino acid oxidase), which is very active but slightly active in many other amino acids, can be used. In the reaction of FIG. 2, these enzymes are directly used or mutated to have high activity You can use it after.

도 3에 제시한 transamination 반응에는 변이를 통해 homoserine에 대해 활성을 갖도록 제작된 변이 alanine aminotransferase를 비롯하여 이와 유사한 amino transferase를 사용할 수 있다. 또한 homoserine으로부터 amino기를 받는 amino acceptor로는 α-ketoglutarate (α-KG)를 사용할 수 있으며 (transaminase 반응에서 α-KG/glutamic acid pair가 이 목적으로 가장 널리 사용됨), α-KG로부터 생산되는 glutamic acid로부터 amino acceptor인 α-KG를 재생함과 동시에 homoserine 생합성 전구체인 aspartic acid의 생산을 위해 aspartate transaminase를 동시에 사용하는 것이 바람직하다.In the transamination reaction shown in FIG. 3, a mutant alanine aminotransferase designed to have activity against homoserine through mutation, and a similar amino transferase can be used. Also, α-ketoglutarate (α-KG) can be used as the amino acceptor to receive amino groups from homoserine (α-KG / glutamic acid pair is most widely used for this purpose in transaminase reaction) and glutamic acid produced from α-KG it is preferable to use aspartate transaminase for the production of aspartic acid, which is a homoserine biosynthesis precursor, while regenerating the amino acceptor α-KG.

도 1에 제시한 두 번째 단계 반응의 효소로는 L-lactate dehydrogenase와 D-lactate dehydrogenase를 사용할 수 있다. 사용하는 효소에 따라 히드록시기는 (R) 혹은 (S) 형태로 2번 위치 탄소에 결합하게 되고 이에 따라 최종 얻어지는 2-hydroxy gamma butyrolactone의 구조도 (R) 혹은 (S) 형태의 isomer가 얻어지게 된다.L-lactate dehydrogenase and D-lactate dehydrogenase can be used as the enzymes of the second step reaction shown in FIG. Depending on the enzyme used, the hydroxy group is bonded to the 2-position carbon in the form of (R) or (S), resulting in the isomer of the resulting (R) or (S) form of the 2-hydroxy gamma butyrolactone .

도 1에 제시한 paraoxonase는 인간유래로 lactonase 활성을 갖는 유전자 (PON1)를 변이시켜 사용할 수 있으며 PON1의 반응이 가역적이고 산성 pH에서만 lactone을 생산할 수 있으므로 세포질이 아닌 periplasm에서 발현시키는 것이 바람직하다.The paraoxonase shown in FIG. 1 can be used by mutating a gene having a lactonase activity (PON1) derived from a human. Since the reaction of PON1 is reversible and lactone can be produced only at an acidic pH, it is preferable to express it in a periplasm rather than a cytoplasm.

도 1에 보여주는 경로를 통해 homoserine 으로부터 2-hydroxy gamma butyrolactone 및 이의 유기산 전구체를 효율적으로 생산하려면 도면 4에 제시한 것과 같이 당으로부터 homoserine을 효율적으로 생산하는 유전자 변이 미생물을 개발하여야 한다. 이 미생물로는 대장균을 우선적으로 이용할 수 있으나, 그 외에 효모, 코리네균 등 다양한 미생물을 이용할 수 있다. 이 숙주 세포는 다음과 같은 방법을 개별적으로 혹은 조합하여 사용함으로써 개량될 수 있다.In order to efficiently produce 2-hydroxy gamma butyrolactone and its organic acid precursor from homoserine through the route shown in FIG. 1, it is necessary to develop a gene mutant microorganism that efficiently produces homoserine from sugar as shown in FIG. As this microorganism, Escherichia coli can be preferentially used, but various microorganisms such as yeast, Corynebacterium can be used. These host cells can be modified by using the following methods individually or in combination.

1. phosphoenolpyruvate (PEP)를 oxaloacetic acid (OAA)로 효율적으로 전환하는 phosphoenolpyruvate carboxylase (ppc) 유전자의 과발현;Overexpression of the phosphoenolpyruvate carboxylase ( ppc ) gene, which efficiently converts phosphoenolpyruvate (PEP) to oxaloacetic acid (OAA);

2. 발효 중 생성되는 acetate를 acetyl CoA로 전환하여 미생물이 다시 사용할 수 있도록 도와주는 acetyl-CoA synthase (acc) 유전자의 과발현;2. overexpression of acetyl-CoA synthase ( acc ) gene to help reuse microbes by converting acetate produced into fermentation into acetyl CoA;

3. glyoxylate shunt의 활성을 높이기 위해 glyoxylate shunt 유전자 발현을 억재하는 iclR 유전자의 제거;3. Removal of the iclR gene, which inhibits glyoxylate shunt gene expression to increase glyoxylate shunt activity;

4. Oxaloacetate에서 asprtic acid로의 전환 효율을 높여주기 위하여 aspartate transaminase의 과발현;Overexpression of aspartate transaminase to increase the conversion efficiency of oxaloacetate to asprtic acid;

5. Apspartic acid의 aspartyl phosphate 전환 효율을 높이기 위해 apspartate kinase의 활성을 향상시킴. apspartate kinase를 coding 하는 thrABC 유전자의 발현을 향상시키기 위해 isoleucine에 의해 작동하는 thrABC operon의 riboswitch를 제거하고 또한 promoter의 세기를 올려줌. 더 나아가 apspartate kinase 효소가 threonine과 lysine에 의해 feedback inhibition을 받는 것을 방지하기 위해 변이된 효소를 발현시키도록 함;5. Improve the activity of apspartate kinase to increase aspartyl phosphate conversion efficiency of apspartic acid. To improve the expression of the thrABC gene coding for apspartate kinase, it removes the riboswitch of the thrABC operon, which is activated by isoleucine, and also increases the strength of the promoter. Further, the apspartate kinase enzyme is allowed to express the mutated enzyme to prevent feedback inhibition by threonine and lysine;

6. lysine 생산을 제거하기 위해 diaminopimelate dcarboxylase를 coding 하는 lysA을 제거;6. Elimination of lysA coding for diaminopimelate dcarboxylase to remove lysine production;

7. methionine 생산을 제거하기 위해 homoserine succinyltransferase를 coding 하는 metA 유전자를 제거;7. Remove the metA gene encoding homoserine succinyltransferase to remove methionine production;

8. 생산된 homoserine이 homoserine phosphate로 전환되지 않도록 homoserine kinase를 coding하는 thrB 유전자를 제거. 8. Remove the thrB gene coding for homoserine kinase so that produced homoserine does not convert to homoserine phosphate.

Homoserine의 생산이 활성화 되면 homoserine을 4-hydroxy-2-oxo-butanoic acid (HOB)로 전환하기 위하여 도면 2에 제시된 deaminase 효소 또는 도면 3에 제시된 transaminase 효소를 coding 하는 유전자를 발현시킨다. 더 나아가 transaminase의 cofactor인 pyridoxal-5'-phosphate (vitamin B6의 일종임)의 생합성을 촉진하기 위해 vitamine B6 생합성 경로를 강화시킨 균주를 사용하는 것이 좋다. In order to convert homoserine to 4-hydroxy-2-oxo-butanoic acid (HOB) when homoserine production is activated, the deaminase enzyme shown in FIG. 2 or the gene encoding the transaminase enzyme shown in FIG. 3 is expressed. Furthermore, it is recommended to use a strain that has enhanced the vitamine B6 biosynthetic pathway to promote the biosynthesis of pyridoxal-5'-phosphate (a kind of vitamin B6), a cofactor of transaminase.

더 나아가 homoserine으로부터 HOB가 효율적으로 생합성 되도록 조작한 균주는 추가적으로 homoserine dehydrogenase 및 혹은 PON1을 발현시켜 2,4-dihydroxy-butanoic acid (dHBA)와 2-hydroxy gamma butyrolactone (HGBL)을 생산할 수 있다. 일반적으로 PON1을 사용하여 dHBA를 HGBL로 전환하는 생물학적 반응은 효율이 낮으므로 dHBA를 생물학적으로 생산한 후 분리 정제하여 화학적으로 HGBL로 전환할 수도 있다. 이 경우 미생물 내에서 생산된 dHBA를 세포 밖으로 빨리 분비시키기 위하여 dHBA의 막 전달 단백질을 과발현 시키는 것이 바람직하다. Furthermore, strains engineered to efficiently biosynthesize HOB from homoserine can produce 2,4-dihydroxy-butanoic acid (dHBA) and 2-hydroxy gamma butyrolactone (HGBL) by further expressing homoserine dehydrogenase and / or PON1. In general, the biological reaction to convert dHBA to HGBL using PON1 is less efficient, so dHBA can be produced biologically and then separated and purified to chemically convert to HGBL. In this case, it is preferable to overexpress the membrane transfer protein of dHBA in order to rapidly secrete the dHBA produced in the microorganism out of the cell.

이렇게 도 4에서 제안하는 homoserine 생합성 경로가 강화된 균주에 도 1에서 제안한 homoserine의 전환을 통해 dHBA나 HGBL이 생산되도록 조작된 미생물을 당을 주된 탄소원으로 하여 배양하면 목적산물인 dHBA와 HGBL을 생산할 수 있다. 이때 미생물의 효율적인 배양과 목적 산물의 효율적인 생산을 위해 배지 내에 질소원으로 yeast extract와 암모늄 염을 첨가하는 것은 물론 본 균주가 특별히 필요로 하는 아미노산, 즉 methionine, lysine, threonine, 그리고 isoleucine을 적절히 첨가할 필요가 있다. 또한 생물 반응기를 이용한 고농도 생산에서는 유가식으로 발효 중간 중간에 당과 아미노산 혼합물을 적절히 첨가해 주는 것이 바람직하다. Thus, the microorganisms that have been engineered to produce dHBA or HGBL through the conversion of the homoserine suggested in FIG. 1 into a strain enhanced with the homoserine biosynthetic pathway suggested in FIG. 4 can be produced by culturing the strain as a main carbon source to produce the desired products dHBA and HGBL have. In order to efficiently cultivate the microorganisms and efficiently produce the desired products, it is necessary to add yeast extract and ammonium salt as a nitrogen source in the culture medium, as well as to appropriately add amino acids such as methionine, lysine, threonine and isoleucine . In addition, it is desirable to add sugar and amino acid mixture properly during fermentation in a high yield production using a bioreactor.

도 4는 포도당을 이용하여 homoserine을 대량으로 생산하기 위한 균주 개량 전략, 그리고 이로부터 2-hydroxy gamma butyrolactone 및 이의 전구체인 2,4-dihydroxybutanoic acid를 생합성하는 전략을 모식적으로 보여준다. 도 4에서, 녹색 화살표는 강화해야하는 반응, 붉은색 화살표는 효소의 생산이나 효소 활성을 억제하는 기작, 살구색 X표는 억제 반응의 제거 혹은 유전자 제거의 표시, 이탤릭체 작은 글씨는 조작 대상 유전자를 각각 나타낸다.FIG. 4 schematically shows a strategy for the mass production of homoserine using glucose and a strategy for biosynthesis of 2-hydroxy gamma butyrolactone and its precursor, 2,4-dihydroxybutanoic acid. In Fig. 4, the green arrow indicates the reaction to be enhanced, the red arrow indicates the mechanism of inhibiting enzyme production or enzymatic activity, the apricot X mark indicates the elimination of the inhibitory reaction or the display of gene deletion, .

이하 구체적인 실시예를 설명한다. 본 발명이 하기 실시예에 의해 한정되는 것은 아니다. Hereinafter, specific examples will be described. The present invention is not limited by the following examples.

실시예 1: 호모세린 (homoserine) 생산균주의 제작Example 1: Production of homoserine-producing strain

(1) 호모세린(Homoserine) 경로는 aspartate 계열의 아미노산을 합성하는 것으로 잘 알려져 있다. 호모세린의 축적을 증가시키기 위해, 라이신(lys), 메티오닌(met) 및 트레오닌(thr) 생산 경로를 제거하였다. 구체적으로 라이신 생합성의 마지막 단계 효소, 즉, 디아미노피멜레이트 디카르복실라제(diaminopimelate decarboxylase)를 코딩하는 lysA, 호모세린숙시닐트랜스퍼라제(homoserinesuccinyl transferase)를 코딩하는 metA, 그리고 호모세린 카이네이즈(homoserine kinase) 및 트레오닌 합성효소(threonine synthase)를 코딩하는 thrBC를 제거하였다 (표 1). (1) The homoserine pathway is known to synthesize aspartate amino acids. To increase the accumulation of homoserine, lysine, methionine (met) and threonine (thr) production pathways were removed. Specifically, the last step of lysine biosynthesis, namely, lysA encoding diaminopimelate decarboxylase, metA encoding homoserinesuccinyl transferase, and homoserine kinase (homoserine kinase) ) And thrBC coding for threonine synthase (Table 1).

(2) 또한 부산물인 젖산, 에탄올, 포름산 등의 생산을 막기 위해 ldhA, adhE, pflB 유전자를 제거하였다. 이들 유전자를 제거함으로써, 부산물의 생성을 막고 호모세린으로 가는 탄소 flux를 증가시키고자 하였다. (2) The ldhA , adhE and pflB genes were also removed to prevent the production of by-products such as lactic acid, ethanol, and formic acid. By removing these genes, we intended to increase the carbon flux to homoserine by preventing the formation of by-products.

(3) 다음으로, 아세트산의 생산을 최소화하기 위하여 acs 유전자의 발현을 증대시켰다. Acs의 발현을 증대시키기 위해, acs 발현 유전자 프로모터를 trc 프로모터로 바꾸었다. (3) Next, expression of the acs gene was increased to minimize the production of acetic acid. To increase the expression of Acs, the acs expression gene promoter was changed to the trc promoter.

(4) 아세테이트 재이용을 촉진하는 방법의 하나로 glyoxylate shunt의 활성을 제고하기 위하여 negative regulator인 iclR 유전자를 pop-in pop-out 방법으로 제거하였다.(4) As a method to promote acetyl reuse, the negative regulator iclR gene was removed by pop-in pop-out method to enhance the activity of glyoxylate shunt.

(5) acetate 생성을 막고 homoserine으로의 탄소 flux를 향상시키기 위하여 anaplerotic 경로의 핵심 효소인 phosphoenol pyruvate carboxylase를 coding 하는 ppc 유전자의 발현을 합성프로모터 8(Synthetic promoter 8: SP8) (서열번호 23: TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA)를 사용하여 증가시켰다. 이를 위해 ppc 유전자의 promoter를 pop-in pop-out 방법으로 synthetic promoter 8로 치환하였다. (5) Expression of the ppc gene coding for phosphoenol pyruvate carboxylase, which is the key enzyme of the anaplorotic pathway, to inhibit acetate formation and improve the carbon flux to homoserine was examined by using synthetic promoter 8 (SP8) (SEQ ID NO: 23: TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA) Lt; / RTI > For this purpose, the promoter of the ppc gene was replaced with a synthetic promoter 8 by a pop-in pop-out method.

(6) 포도당 대사의 overflow metabolism을 제거하고 포도당의 세포막 전달에 phosphoenol pyruvate (PEP)가 사용되는 것을 막기 위해, ptsG 유전자를 MAGE 방법으로 제거하였다. 이 경우 포도당은 GalP 등 다른 전달 단백질에 의해 세포내로 이송되며 Carbon Catabolite Repression의 방지, overflow metabolism에 의한 부산물 생성 방지 등의 효과를 기대할 수 있다. (6) The ptsG gene was removed by the MAGE method in order to eliminate the overflow metabolism of glucose metabolism and to prevent the use of phosphoenol pyruvate (PEP) for glucose cell membrane transport. In this case, glucose is transported into cells by other transfer proteins such as GalP, and prevention of Carbon Catabolite Repression and prevention of by-product formation by overflow metabolism can be expected.

(7) EMP 경로를 이용하는 해당경로에서 oxaloacetate 로의 탄소 flux를 올려 주기 위하여 KHG/KDG aldolase를 coding 하는 eda 유전자를 결실시켜 ED 경로를 제거하였다. MAGE 방법을 사용하였다.(7) In order to raise the carbon flux to oxaloacetate in the corresponding pathway using the EMP pathway, the ED pathway was deleted by deletion of the eda gene coding for KHG / KDG aldolase. MAGE method was used.

(8) 마지막으로, 호모세린 생산 경로의 bottleneck으로 알려진 aspartic acid 이후 경로를 최적화하였다. 호모세린 생산에서 3개의 aspartate kinase, 즉 AKI, AKII, AKIII 가 중요한 역할을 한다고 알려져 있으며, 이중 AKII, AKIII 두개의 효소만이 높은 호모세린 농도에서 우수한 활성을 보인다. 또한, AKII 및 AKIII 중에서 AKII는 aspartate kinase 와 serine dehydrogenase 활성을 동시에 가지고 있어 AKIII 보다는 좀더 유리하다고 알려져 있다. 따라서 이번 균주 개발에서는 AKII 효소를 coding하는 metL 유전자를 medium copy plasmid인 pUCPK plasmid를 이용하여 과발현시켰다. 이 때 metL의 발현 크기를 조절하기 위해 lac promoter 와 trc promoter 두 가지를 사용하였다. (8) Finally, we optimized the pathway after aspartic acid, known as the bottleneck of the homoserine production pathway. Three aspartate kinases, AKI, AKII, and AKIII, are known to play an important role in homoserine production. Only two enzymes, AKII and AKIII, exhibit superior activity at high homoserine concentrations. In addition, among AKII and AKIII, AKII is known to be more advantageous than AKIII because it has both aspartate kinase and serine dehydrogenase activity. Therefore, the metL gene encoding the AKII enzyme was overexpressed using the medium copy plasmid pUCPK plasmid. We used both lac promoter and trc promoter to regulate metL expression.

아래 표 1에 호모세린을 과발현시키기 위해, 제거(deletion), 치환(substitution) 또는 과발현(overexpression)한 경쟁경로 유전자와 제거 목적, 제거 방법을 나타냈다.In order to overexpress homoserine, Table 1 below shows a competitive pathway gene for deletion, substitution or overexpression, and the purpose of elimination and the elimination method.

Deletion (Chromosome)Deletion (Chromosome) 　PurposePurpose MethodMethod 1One Δpts? Pts Increase carbon flux toward　　 HomoserineIncrease carbon flux toward Homoserine MAGEMAGE 22 ΔedaΔeda 33 ΔadhEΔadhE Deletion of　 Ethanol production pathwayDeletion of Ethanol production pathway 44 ΔpflBΔpflB Deletion of　 Formate production pathwayDeletion of Formate production pathway 55 ΔlysAΔlyse Deletion of　 Lysine production pathwayDeletion of Lysine production pathway 66 ΔthrBΔthrB Deletion of　 Threonine production pathwayDeletion of Threonine production pathway 77 ΔmetAΔmetA Deletion of　 Methionine production pathwayDeletion of Methionine production pathway 88 ΔlacIΔlacI Remove of　 lacI repressor for promoter utilizationRemove of lac I repressor for promoter utilization 99 ΔldhAΔldhA Deletion of Lactate production pathwayDeletion of Lactate production pathway Pop in-pop-outPop in-pop-out 1010 ΔiclRΔiclR Deletion of repression in glyoylate shuntDeletion of repression in glyoylate shunt Substitution (Chromosome)Substitution (Chromosome) 　PurposePurpose MethodMethod 1111 ΔPacs::PtrcΔPacs :: Ptrc Reduce acetate formation and increase carbon flux to homoserineReduce acetate formation and increase carbon flux to homoserine Pop in-Pop outPop in-Pop out 1212 ΔPppc::Psyn8ΔPppc :: Psyn8 Increase anaplerotic flux (PEP top OAA)Increase anaplerotic flux (PEP top OAA) Pop in-Pop outPop in-Pop out Overexpression (Plasmid)Overexpression (Plasmid) 　PurposePurpose MethodMethod 1313 pUCPK-Ptrc-metL pUCPK-P trc - metL Remove the bottleneck of homoserine productionRemove the bottleneck of homoserine production Plasmid　
cloningPlasmid
cloning

대장균인 W3110 균주 및 BL21(DE3)은 Korean Collection for Type Cultures (KCTC)에서 구입하였다. 대장균 TOP10 균주는 플라스미드의 cloning 과 유지에 사용하였다. 유전자 제거에는 multiplex automated genome engineering (MAGE) 기법과 pop-in pop-out 방법을 사용하였다. Escherichia coli W3110 and BL21 (DE3) were purchased from Korean Collection for Type Cultures (KCTC). E. coli TOP10 strain was used for cloning and maintenance of the plasmid. We used multiplex automated genome engineering (MAGE) and pop-in pop-out methods for gene deletion.

본 발명자들이 개발한 Homoserine 과생산 변이 대장균주의 종류를 아래 표 2에 나타냈다.　Homoserine and production variant E. coli strains developed by the present inventors are shown in Table 2 below.

StrainsStrains DescriptionDescription SourceSource E. coli TOP10 E. coli TOP10 WTWT DSM, GermanyDSM, Germany E. coli W3110 E. coli W3110 WTWT DSM, Germany DSM, Germany WTWT E. coli W3110 E. coli W3110 DSM, GermanyDSM, Germany EcW1EcW1 Δpts? Pts This studyThis study EcW2EcW2 Δpts ΔedaΔpts Δeda This studyThis study EcW3EcW3 Δpts Δeda ΔlacIΔpts Δeda ΔlacI This studyThis study EcW4EcW4 Δpts Δeda ΔlacI ΔthrBΔpts Δeda ΔlacI ΔthrB This studyThis study EcW5EcW5 Δpts Δeda ΔlacI ΔthrB ΔmetAΔpts Δeda ΔlacI ΔthrB ΔmetA This studyThis study EcW6EcW6 Δpts Δeda ΔlacI ΔthrB ΔmetA ΔlysAΔpts Δeda ΔlacI ΔthrB ΔmetA Δlyse This studyThis study EcW7EcW7 Δpts Δeda ΔlacI ΔthrB ΔmetA ΔlysA ΔadhEΔpts Δeda ΔlacI ΔthrB ΔmetA Δlyse ΔadhE This studyThis study EcW8EcW8 Δpts Δeda ΔlacI ΔthrB ΔmetA ΔlysA ΔadhE ΔpflBΔpts Δeda ΔlacI ΔthrB ΔmetA Δlyse ΔadhE ΔpflB This studyThis study EcW9EcW9 Δpts Δeda ΔlacI ΔthrB ΔmetA ΔlysA ΔadhE ΔpflB ΔldhAΔpts Δeda ΔlacI ΔthrB ΔmetA Δlyse ΔadhE ΔpflB ΔldhA This studyThis study EcW10EcW10 Δpts Δeda ΔlacI ΔthrB ΔmetA ΔlysA ΔadhE ΔpflB ΔldhA ΔiclRΔpts Δeda ΔlacI ΔthrB ΔmetA ΔlysA ΔadhE ΔpflB ΔldhA ΔiclR This studyThis study EcW11EcW11 Ec10, replacing acs native promoter by Ptrc promoterEc10, replacing acs native promoter by P trc promoter This studyThis study EcW12EcW12 Ec11, replacing ppc native promoter by synthetic promoter 8Ec11, replacing ppc native promoter by synthetic promoter 8 This studyThis study EcW13EcW13 Ec12, pUCPK-Ptrc-metL Ec12, pUCPK-P trc - metL This studyThis study

유전체 분리 kit는 Promega (Madison, WI, USA)에서 구매하였다. High-fidelity pfu-a 중합효소는 Invitrogen (Seoul, Korea) 에서 구입하였다. DNA 절단효소 및 DNA 개량효소는 New England Bio-LAb (Beverly, MA, USA)에서 구매하였다. Miniprep 및 DNA gel extraction kit은 Cosmotech (Seoul, Korea)에서 구입하였다. 유전자 증폭 등에 사용된 primer 는 Macrogene (Seoul, Korea)에서 합성하였다. 효모추출물, 트립톤, 트립케이스 soy broth, 펩톤 등은 Difco (Becton-Dickinson, NJ, USA)사에서 구입하였다. 그 외 시약과 효소는 모두 Sigma-Aldrich (St. Louis, MO, USA)사에서 구입하였다.Dielectric isolation kit was purchased from Promega (Madison, WI, USA). High-fidelity pfu-a polymerase was purchased from Invitrogen (Seoul, Korea). DNA cleavage enzymes and DNA modification enzymes were purchased from New England Bio-LAb (Beverly, MA, USA). Miniprep and DNA gel extraction kit were purchased from Cosmotech (Seoul, Korea). The primers used for gene amplification were synthesized in Macrogene (Seoul, Korea). Yeast extract, tryptone, trip case soy broth, and peptone were purchased from Difco (Becton-Dickinson, NJ, USA). All other reagents and enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA).

1-1 MAGE 방법을 이용한 유전자 결실 및 치환1-1 Gene deletion and substitution using MAGE method

MAGE (Multiplex Automated Genome Engineering) 방법은 in vivo genome editing에 매우 강력한 tool이다. 이 방법에서는 target이 되는 염색체에 해당하는 single-stranded DNA (ssDNA)를 바로 engineering 하려는 균주에 도입하게 된다. 여러 site를 대상으로 할 경우, ssDNA 여러 종류를 일시에 또는 순차적으로 도입할 수도 있다. The Multiplex Automated Genome Engineering (MAGE) method is a very powerful tool for in vivo genome editing. In this method, single-stranded DNA (ssDNA) corresponding to the target chromosome is directly introduced into the strain to be engineered. If you are targeting multiple sites, you can introduce several types of ssDNA either simultaneously or sequentially.

본 실험에서는 recombinase 역할을 하는 beta-단백질을 pSIM5 플라스미드에 도입하고 이를 electroporation 방법으로 대장균에 넣어 주었다. 합성 ssDNA 올리고는 제거하려는 염색체 target 유전자 서열과 겹치는 homology arm, 즉 5‘-terminal homolgy arm과 3’terminal homology arm이 존재하도록 제작하였다 (표 3). 세포 내에서 염색체와 homology를 갖는 oligo 들은 염색체 복제기간에 target 유전자의 lagging strand와 결합하고 homologous recombination을 통해 target 유전자에 변이를 일으킨다. In this experiment, beta- protein, which plays a role of recombinase, was introduced into pSIM5 plasmid and inserted into E. coli by electroporation method. The synthetic ssDNA oligo was constructed to have a homology arm, 5'-terminal homolgy arm and 3'terminal homology arm, overlapping the target gene sequence of the chromosome to be deleted (Table 3). Oligos with chromosomal homology within the cell bind to the lagging strand of the target gene during chromosome replication and cause mutations in the target gene through homologous recombination.

MAGE 실험을 위해 한천 고체 배지에서 자라는 single colony를 따서 섭씨 30도, LB+Cm 배지에서 12시간 배양하였다. 이후 이 배양액 100 microliter를 새로운 LB+Cm 배지에 옮기고 섭씨 30도에서 0.6 OD (600 nm)가 될 때까지 배양하였다. 이후 culture tube를 15분간 섭씨 42도에서 방치하였다가 30 분간 얼음물 속에 넣었다. 이 후 준비된 competent cell을 4℃, 5000 rpm에서 10분간 원심분리하고 차가운 증류수로 3차례 세척하였다. 그리고 세포 pellet을 10% glycerol 용액 100 mL에 현탁시켰다. 이후 ssDNA oligo 50 uM을 세포현탁액과 혼합하고 이를 elctro cuvette에 넣은 후 electroporation 시켰다. 이 후 이 혼합액을 배양 tube에 옮기고 5 mL의 LB를 첨가하고 섭씨 30도에서 3시간 배양하였다 (1 차 cycle). 이러한 과정을 6-10 회 반복한 후 (6-10 cycles) 100 uL의 세포배양액을 agar plate에 도말하고 밤새 배양하였다. 생성된 colony 들을 PCR 방법으로 스크리닝하고 유전자가 결실된 변이주들을 확인, 확보하였다.For MAGE experiments, single colonies were grown in agar solid medium and cultured for 12 hours in LB + Cm medium at 30 ° C. After that, 100 microliter of this culture was transferred to new LB + Cm medium and cultured at 30 ° C. until it reached 0.6 OD (600 nm). The culture tube was then left at 42 ° C for 15 minutes and then placed in ice water for 30 minutes. The competent cells were centrifuged at 5000 rpm for 10 min at 4 ° C and washed three times with cold distilled water. The cell pellet was suspended in 100 mL of 10% glycerol solution. Then 50 μM ssDNA oligo was mixed with the cell suspension and placed in an elctro cuvette for electroporation. After this, the mixture was transferred to a culture tube, and 5 mL of LB was added and cultured at 30 ° C. for 3 hours (first cycle). After repeating this process 6-10 times (6-10 cycles), 100 uL of cell culture was plated on agar plate and cultured overnight. The generated colonies were screened by PCR method and mutant strains deficient in the genes were identified and secured.

아래 표 3에는 유전자 제거 또는 클로닝에 사용한 프라이머의 종류를 나타냈다.Table 3 below shows the types of primers used for gene removal or cloning.

PrimerPrimer DescriptionDescription 서열번호SEQ ID NO: Primer for gene　　 deletion using MAGEPrimer for gene deletion using MAGE pts_RPpts_RP cggaaccgcctgcttctgccataacatgcgatacaacggcggTTAcagcagaatacctgcgataggcagtacggataccggcagcatccggaaccgcctgcttctgccataacatgcgatacaacggcggTTAcagcagaatacctgcgataggcagtacggataccggcagcatc 2424 pts_seq_FPpts_seq_FP GgcgtcggttccgcgaatttcGgcgtcggttccgcgaatttc 2525 pts_seq_RPpts_seq_RP GgtgcagaccaaacggtaccGgtgcagaccaaacggtacc 2626 eda_FPeda_FP caactgcacactcggtacgcagagtcacttccagaacgcgcaTTAcaccgcgtgttccagtttttttaccacgataaccggtacaacccaactgcacactcggtacgcagagtcacttccagaacgcgcaTTAcaccgcgtgttccagtttttttaccacgataaccggtacaacc 2727 eda_RPeda_RP ggttgtaccggttatcgtggtaaaaaaactggaacacgcggtgTAAtgcgcgttctggaagtgactctgcgtaccgagtgtgcagttgggttgtaccggttatcgtggtaaaaaaactggaacacgcggtgTAAtgcgcgttctggaagtgactctgcgtaccgagtgtgcagttg 2828 eda_seq_FPeda_seq_FP CcgatggcaaaagcgttggCcgatggcaaaagcgttgg 2929 eda_seq_RPeda_seq_RP CgcgccagcttagtaatgcCgcgccagcttagtaatgc 3030 poxB_RPpoxB_RP ggcaggggtgaaacgcatctggggagtcacaggcgactctctgaacTAAccatcgagtggatgtccacccgccacgaagaagtggcggccggcaggggtgaaacgcatctggggagtcacaggcgactctctgaacTAAccatcgagtggatgtccacccgccacgaagaagtggcggcc 3131 poxB_seq_FPpoxB_seq_FP TggaataacgcagcagttgTggaataacgcagcagttg 3232 poxB_seq_RPpoxB_seq_RP GgtcttagtgacagtcttaatcGgtcttagtgacagtcttaatc 3333 pta_FPpta_FP gaccagcgtcagccttggcgtgatccgtgcaatggaacgcaaaTAActcagccgcgtaccggtggcgatgcgcccgatcagactacggaccagcgtcagccttggcgtgatccgtgcaatggaacgcaaaTAActcagccgcgtaccggtggcgatgcgcccgatcagactacg 3434 pta_RPpta_RP cgtagtctgatcgggcgcatcgccaccggtacgcggctgagTTAtttgcgttccattgcacggatcacgccaaggctgacgctggtccgtagtctgatcgggcgcatcgccaccggtacgcggctgagTTAtttgcgttccattgcacggatcacgccaaggctgacgctggtc 3535 pta_seq_FPpta_seq_FP GgcgttcgtctgagcgttttcGgcgttcgtctgagcgttttc 3636 pta_seq_RPpta_seq_RP GgattcttgcagcttcgccGgattcttgcagcttcgcc 3737 ackA_FPackA_FP gaaatttgccatcatcgatgcagtaaatggtgaagagtaccttTGAaagcacgtatcaaatggaaaatggacggcaataaacaggaagclol 3838 ackA_RPackA_RP gcttcctgtttattgccgtccattttccatttgatacgtgcttTCAaaggtactcttcaccatttactgcatcgatgatggcaaatttcgcttcctgtttattgccgtccattttccatttgatacgtgcttTCAaaggtactcttcaccatttactgcatcgatgatggcaaatttc 3939 ackA_seq_FPackA_seq_FP TctggtttagccgaatgtttccTctggtttagccgaatgtttcc 4040 ackA_seq_RPackA_seq_RP AtgttcagttcttctaccggAtgttcagttcttctaccgg 4141 ldhA_RPldhA_RP gcaacaggtgaacgagtcctttggctttgagctggaattttttTAActgccaatggctgcgaagcggtatgtattttcgtaaacgatggcaacaggtgaacgagtcctttggctttgagctggaattttttTAActgccaatggctgcgaagcggtatgtattttcgtaaacgatg 4242 ldhA_seq_FPldhA_seq_FP TtttccggtgtcagcgggTtttccggtgtcagcggg 4343 ldhA_seq_RPldhA_seq_RP GactttctgctgacggGactttctgctgacgg 4444 adhE_RPadhE_RP gtaaaaaaagcccagcgtgaatatgccagtttcactcaagagcaaTAActgctgcagatgctcgaatcccactcgcgaaaatggccgttggtaaaaaaagcccagcgtgaatatgccagtttcactcaagagcaaTAActgctgcagatgctcgaatcccactcgcgaaaatggccgttg 4545 adhE_seq_FPadhE_seq_FP GgagctgtatgcggctttaacGgagctgtatgcggctttaac 4646 adhE_seq_RPadhE_seq_RP GtagacaaaatcttccgcgccgGtagacaaaatcttccgcgccg 4747 pflB_RPpflB_RP ccaaaggtgactggcagaatgaagtaaacgtccgtgacttcattTAAagtccttcctggctggcgctactgaagcgaccaccaccctgccaaaggtgactggcagaatgaagtaaacgtccgtgacttcattTAAagtccttcctggctggcgctactgaagcgaccaccaccctg 4848 pflB_seq_FPpflB_seq_FP TgcagagaagtgaactgtgccTgcagagaagtgaactgtgcc 4949 pflB_seq_RPpflB_seq_RP CagaaaaactacactccgtacgCagaaaaactacactccgtacg 5050 iclR_FPiclR_FP CccggcttgttgcgccagttccgtgagtgccacactgccattcagccacgcgttaaagactgaacctgtccagtcgctggtgcggtggcCccggcttgttgcgccagttccgtgagtgccacactgccattcagccacgcgttaaagactgaacctgtccagtcgctggtgcggtggc 5151 iclR_RPiclR_RP GccaccgcaccagcgactggacaggttcagtctttaacgcgtggctgaatggcagtgtggcactcacggaactggcgcaacaagccgggGccaccgcaccagcgactggacaggttcagtctttaacgcgtggctgaatggcagtgtggcactcacggaactggcgcaacaagccggg 5252 iclR_seq_FPiclR_seq_FP GcgcgtttgggcgagatcGcgcgtttgggcgagatc 5353 iclR_seq_RPiclR_seq_RP CtgaaattactggagtggCtgaaattactggagtgg 5454 lysA_FPlysA_FP ggctttctgtgcaaagcgcaccacatcaaactgtttcagcgctgTTAgacccacaccgggcagccaaattcagcgggcaaacgcagcagggctttctgtgcaaagcgcaccacatcaaactgtttcagcgctgTTAgacccacaccgggcagccaaattcagcgggcaaacgcagcag 5555 lysA_RPlysA_RP ctgctgcgtttgcccgctgaatttggctgcccggtgtgggtcTAAcagcgctgaaacagtttgatgtggtgcgctttgcacagaaagccctgctgcgtttgcccgctgaatttggctgcccggtgtgggtcTAAcagcgctgaaacagtttgatgtggtgcgctttgcacagaaagcc 5656 lysA_seq_FPlysA_seq_FP CctgttccagatgggcataatcCctgttccagatgggcataatc 5757 lysA_seq_RPlysA_seq_RP GggtctacgatgcgcaaattattcgGggtctacgatgcgcaaattattcg 5858 thrB_RPthrB_RP cttatcggcaaagcgtccgaggttgttgagactgaatgtctctgTTAtgcaccatcaacaggtgtcaccgccgccccgagcacatcaaaccttatcggcaaagcgtccgaggttgttgagactgaatgtctctgTTAtgcaccatcaacaggtgtcaccgccgccccgagcacatcaaac 5959 thrB_seq_FPthrB_seq_FP TtgctcggagatgtagtcacggTtgctcggagatgtagtcacgg 6060 thrB_seq_RPthrB_seq_RP GcgatactgcgccggtaaaatagGcgatactgcgccggtaaaatag 6161 metA_FPmetA_FP GaagaaaacgtctttgtgatgacaacttctcgtgcgtctggttaattaacctgatgccgaagaagattgaaactgaaaatcagtttctgGaagaaaacgtctttgtgatgacaacttctcgtgcgtctggttaattaacctgatgccgaagaagattgaaactgaaaatcagtttctg 6262 metA_RPmetA_RP CagaaactgattttcagtttcaatcttcttcggcatcaggttaattaaccagacgcacgagaagttgtcatcacaaagacgttttcttcCagaaactgattttcagtttcaatcttcttcggcatcaggttaattaaccagacgcacgagaagttgtcatcacaaagacgttttcttc 6363 metA_seq_FPmetA_seq_FP CtggtcaggaaattcgtccacCtggtcaggaaattcgtccac 6464 metA_seq_RPmetA_seq_RP CgaatcaacgctgccggaaagCgaatcaacgctgccggaaag 6565 lacI_RPlacI_RP cttatcagaccgtttcccgcgtggtgaaccaggccagccacgttTGAcgatggcggagctgaattacattcccaaccgcgtggcacaaccttatcagaccgtttcccgcgtggtgaaccaggccagccacgttTGAcgatggcggagctgaattacattcccaaccgcgtggcacaac 6666 lacI_seq_FPlacI_seq_FP GatatttatgccagccagccGatatttatgccagccagcc 6767 lacI_seq_RPlacI_seq_RP CcacgtttctgcgaaaacgcgggCcacgtttctgcgaaaacgcggg 6868 Primer for gene　　 deletion using Pop-in Pop-out methodPrimer for gene deletion using Pop-in Pop-out method US-Ptrc-acs-FPUS-Ptrc-acs-FP gtttaatcggtacccggggatcgcggccgccgcgcccttcctgccagtcaattttcgtttaatcggtacccggggatcgcggccgccgcgcccttcctgccagtcaattttc 6969 US-Ptrc-acs-RPUS-Ptrc-acs-RP CttttaagaaggagatatacatatgagccaaattcacaaacacaccCttttaagaaggagatatacatatgagccaaattcacaaacacacc 7070 Ptrc-acs-FPPtrc-acs-FP GgtgtgtttgtgaatttggctcatatgtatatctccttcttaaaagGgtgtgtttgtgaatttggctcatatgtatatctccttcttaaaag 7171 Ptrc-acs-RPPtrc-acs-RP CggcgtgcgtttatttttatccttgtcatcgactgcacggtgcaccaatgcCggcgtgcgtttatttttatccttgtcatcgactgcacggtgcaccaatgc 7272 DS-Ptrc-acs-FPDS-Ptrc-acs-FP GcattggtgcaccgtgcagtcgatgacaaggataaaaataaacgcacgccgGcattggtgcaccgtgcagtcgatgacaaggataaaaataaacgcacgccg 7373 DS-Ptrc-acs-RPDS-Ptrc-acs-RP GggcgcgcgccattctccggtcgactctagatcatgccgtcatcccacGggcgcgcgccattctccggtcgactctagatcatgccgtcatcccac 7474 US-ldhA-FPUS-ldhA-FP AtcgcggccgctgtctgttttgcggtcAtcgcggccgctgtctgttttgcggtc 7575 US-ldhA-RPUS-IdhA-RP CtggagaaagtcttatgtaatcttgccgctccccCtggagaaagtcttatgtaatcttgccgctcccc 7676 DS-ldhA-FPDS-ldhA-FP GgggagcggcaagattacataagactttctccagGgggagcggcaagattacataagactttctccag 7777 DS-ldhA-RPDS-ldhA-RP CtagaggatcccaagcagaatcaagttctaccgCtagaggatcccaagcagaatcaagttctaccg 7878 Gn-ldhA-FPGn-ldhA-FP GatggtacggcgattgggatgGatggtacggcgattgggatg 7979 Gn-ldhA-RPGn-ldhA-RP GccagggagaaaaaatcagGccagggagaaaaaatcag 8080 US-iclR-FPUS-iclR-FP CtgcgcggacgctgaggatcccggtgatcccgtcctctcacgCtgcgcggacgctgaggatcccggtgatcccgtcctctcacg 8181 US-iclR-RPUS-iclR-RP TtcgaaccccagagtcccgcctgccgctcgtaggtcctgTtcgaaccccagagtcccgcctgccgctcgtaggtcctg 8282 DS-iclR-FPDS-iclR-FP GcaggacctacgagcggcaggcgggactctggggttcgaaatgGcaggacctacgagcggcaggcgggactctggggttcgaaatg 8383 DS-iclR-RPDS-iclR-RP TgcctgcaggtcgactctagattatttgttaactgttaattgtccttgttcTgcctgcaggtcgactctagattatttgttaactgttaattgtccttgttc 8484 Gn-iclR-FPGn-iclR-FP CtgaacagcaggtcgtccCtgaacagcaggtcgtcc 8585 Gn-iclR-RPGn-iclR-RP GcgtcgaaaccttcgatgGcgtcgaaaccttcgatg 8686

1-2. Pop-in pop-out 방법에 의한 유전자 결실 또는 유전자 치환1-2. Gene deletion or gene replacement by pop-in pop-out method

ldhA 및 iclR 유전자를 제거하기 위해 sacB-Km 카세트를 갖는 pKOV 플라스미드를 사용하였다 (표 4). 구체적으로, target 유전자의 upstream과 downstream region 600-700 bp 를 갖는 fragment를 E. coli W3110 염색체로부터 해당 primer를 사용하여 PCR 증폭하였다(표 3). 이 fragment의 sequence를 확인한 후 Gene Art Seamless Cloning and Assembly Kit (Invitrogen, USA)를 이용하여 pKOV plasmid에 삽입하였다. 이 후 E. coli 3110 에 recombinant pKOV plasmid 를 도입하고 homology recombination을 통해 해당 유전자를 결실시켰으며, 설탕 배지 배양을 통해 pKOV plasmid를 제거하였다. 최종적으로 PCR 방법으로 스크리닝하고 유전자가 결실된 변이주들을 확인, 확보하였다.　 A pKOV plasmid with the sacB-Km cassette was used to remove the ldhA and iclR genes (Table 4). Specifically, a fragment with an upstream and downstream region of target gene of 600-700 bp was PCR amplified from the E. coli W3110 chromosome using the corresponding primers (Table 3). The sequence of this fragment was confirmed and inserted into pKOV plasmid using Gene Art Seamless Cloning and Assembly Kit (Invitrogen, USA). Then, the recombinant pKOV plasmid was introduced into E. coli 3110, and the corresponding gene was deleted by homology recombination. The pKOV plasmid was removed through sugar culture. Finally, we screened by PCR method and identified mutant strains in which genes were deleted.

1-3. 유전자 조작에 사용된 플라스미드 제작1-3. Plasmid production used in gene manipulation

Pop-in pop-out 혹은 MAGE 방법에 의한 유전자 결실, 프로모터 치환, 유전자 과발현 등에 사용한 플라스미드를 아래 표 4에 나타냈다. 유전자 재조합 플라스미드를 제작하기 위하여 먼저 target 유전자의 open reading frame (ORF)를 PCR 증폭하고 적절한 primer (표 3)를 사용하여 overlap 증폭하였다. 이렇게 하여 얻어진 DNA fragment를 제한 효소로 자르고 해당 플라스미드, 즉 pUCPK, pET-T7p, pBbA1k, pTrc-99a 등에 cloning 하였다. 이 후 이 plasmid를 해당 host 균주에 도입하였다.Plasmids used for gene deletion, promoter substitution, gene overexpression by pop-in pop-out or MAGE method are shown in Table 4 below. In order to construct a recombinant plasmid, open reading frame (ORF) of the target gene was first PCR amplified and overlap amplified using appropriate primers (Table 3). The resulting DNA fragment was cut with restriction enzymes and cloned into the corresponding plasmids, namely pUCPK, pET-T7p, pBbA1k, pTrc-99a and the like. This plasmid was then introduced into the corresponding host strain.

PlasmidPlasmid DescriptionDescription SourceSource pSIM5pSIM5 MAGE, β recombinant protein, Cm25MAGE, beta recombinant protein, Cm25 Addgene, USAAddgene, USA pUCPKpUCPK Overexpression, lac promoter, medium copy, K50Overexpression, lac promoter, medium copy, K50 Addgene, USAAddgene, USA pETpET Overexpression, T7 promoter, high copy, K50Overexpression, T7 promoter, high copy, K50 Addgene, USAAddgene, USA pBbA1kpBbA1k Overexpression, trc promoter, medium copy, K50Overexpression, trc promoter, medium copy, K50 Addgene, USAAddgene, USA pTrc-99apTrc-99a Overexpression, trc promoter, low copy, K50Overexpression, trc promoter, low copy, K50 Addgene, USAAddgene, USA pKOVpKOV Pop in-Pop out, Cm25Pop in-Pop out, Cm25 Addgene, USAAddgene, USA pUCPK-metL pUCPK- metL Ptrc-metL Ptrc- metL This studyThis study

1-4. 유전자 발현에 사용되는 합성 프로모터 라이브러리 제작 및 활성측정 1-4. Production and activity measurement of synthetic promoter library used for gene expression

유전자 발현 향상이나 조절을 위해 다수의 프로모터를 사용하였다. 동일한 서열을 갖는 프로모터의 경우 유전자 불안정성을 크게 증가시키므로 서로 다른 서열의 합성 프로모터(synthetic promoters) 라이브러리를 구축하였다(표 5). 또한 이들의 상대 활성을 GFP를 이용하여 측정하였다 (도 5). 총 9개의 프로모터를 합성하고 선정하였으며, 이들의 활성은 대략 4배 정도 범위를 가지고 있었다.A number of promoters were used to enhance or control gene expression. In the case of the promoters having the same sequence, synthetic promoters of different sequences were constructed because the gene instability was greatly increased (Table 5). Their relative activities were also measured using GFP (Figure 5). A total of 9 promoters were synthesized and selected, and their activities ranged from about 4 times.

프로모터Promoter 염기서열Base sequence 서열번호SEQ ID NO: SP1SP1 AAAAAGAGTATTGACTTCGCATCTTTTTGTACCTATAATGTGTGGAAAAAAGAGTATTGACTTCGCATCTTTTTGTACCTATAATGTGTGGA 8787 SP2SP2 AAAAAATTTATTTGCTTATTAATTCATCCGGCTCGTATATGTGTGGAAAAAAATTTATTTGCTTATTAATTCATCCGGCTCGTATATGTGTGGA 8888 SP3SP3 TTGACATCAGGAAAATTTTTCTGTATAATGTGTGGATTGACATCAGGAAAATTTTTCTGTATAATGTGTGGA 8989 SP4SP4 TTGACAATTAATCATCCGGCTCGTAATTTATGTGGATTGACAATTAATCATCCGGCTCGTAATTTATGTGGA 9090 SP5SP5 AAAAAATTTATTTGCTTTCGCATCTTTTTGTACCTATAAGTGTGGAAAAAAATTTATTTGCTTTCGCATCTTTTTGTACCTATAAGTGTGGA 9191 SP6SP6 AAAAAATTTATTTGCTTTCGCATCTTTTTGTACCTGTAATGTGTGGAAAAAAATTTATTTGCTTTCGCATCTTTTTGTACCTGTAATGTGTGGA 9292 SP7SP7 TTCACTTTTAATCATCCGGCTCGTATAATGTGTGGATTCACTTTTAATCATCCGGCTCGTATAATGTGTGGA 9393 SP8SP8 TTTCAATTTAATCATCCGGCTCGTATAATGTGTGGATTTCAATTTAATCATCCGGCTCGTATAATGTGTGGA 9494 SP9SP9 TTCCCTATTAATCATCCGGCTCGTATAATGTGTGGATTCCCTATTAATCATCCGGCTCGTATAATGTGTGGA 9595

실시예Example 2: 호모세린의 아미노기 제거를 위한 2: for the removal of amino groups of homoserine 트랜스아미네이즈Transaminate ( ( TATA ) 효소의 스크리닝 및 변이효소 제작Screening of enzymes and production of mutant enzymes

2-1. 트랜스아미네이즈(TA) 효소의 스크리닝2-1. Screening of transaminate (TA) enzymes

효소의 확보Securing enzyme

트랜스아미네이즈(TA) 효소는 생명체 내에서 중요한 역할을 담당하고 오랫동안 다양하게 연구되어 왔지만 homoserine에 특이적인 TA 효소는 생체 내 역할이 없기 때문에 아직 밝혀지지 않았다. 일반적으로 많은 효소들이 생물학적 기질과 유사한 화합물에 대해 활성을 가지고 있으므로 이미 알려진 TA 효소 중 aspatate transaminase, alanine transaminase, branched chain transaminase and aromatic transaminase 중에서 17개를 선택하여 호모세린에 대한 활성을 조사했다(표 6). 이들 효소는 E. coli, Enterobacter spp., Bacillus subtilis, Mesorhizobium loti, Agrobacterium tumefaciens, Pseudomonas denitrificans, P. putida and P. fluorescens 등으로부터 확보하였다:Transaminate (TA) enzymes play an important role in life and have been studied for a long time, but the TA enzyme specific for homoserine has not yet been revealed in vivo. In general, many enzymes have activity on compounds similar to biological substrates, so 17 of the known TA enzymes, aspartate transaminase, alanine transaminase, branched chain transaminase and aromatic transaminase, were selected and investigated for homoserine activity ). These enzymes were obtained from E. coli , Enterobacter spp ., Bacillus subtilis , Mesorhizobium loti , Agrobacterium tumefaciens , Pseudomonas denitrificans, P. putida and P. fluorescens .

GeneGene EnzymeEnzyme SourceSource Acc. No.Acc. No. PrimerPrimer 서열번호SEQ ID NO: aspATaspAT Aspatate transaminaseAspatate transaminase M.　　 lotiM. loti ANN59010.1ANN59010.1 F: gaccatggaattcatggaagagtttcacaaggtcF: gaccatggaattcatggaagagtttcacaaggtc 9696 R: acagccaagcttttaccggtgggcggacagcR: acagccaagcttttaccggtgggcggacagc 9797 aspATaspAT Aspatate transaminaseAspatate transaminase M.　　 lotiM. loti ANN57047.1ANN57047.1 F: gaccatggaattcatgctccacacgatttcgF: gaccatggaattcatgctccacacgatttcg 9898 R: agtttggatcctcacttggcgaggaacgR: agtttggatcctcacttggcgaggaacg 9999 aspATaspAT Aspatate transaminaseAspatate transaminase A.　　 tumefaciensA. tumefaciens ACM26415.1ACM26415.1 F: gaccatggaattcatggaagagtttcataaagF: gaccatggaattcatggaagagtttcataaag 100100 R: agtttggatccttaagaacgatgagcgR: agtttggatccttaagaacgatgagcg 101101 aspATaspAT Aspatate transaminaseAspatate transaminase B. subtilisB. subtilis AQR85543.1AQR85543.1 F: ttcagaattcaaaagatcttttaagaaggagatatacatatgcagagcaaaggcgF: ttcagaattcaaaagatcttttaagaaggagatatacatatgcagagcaaaggcg 102102 R: agtttggatccttaatggtgatggtgatggtgR: agtttggatccttaatggtgatggtgatggtg 103103 aspATaspAT Aspatate transaminaseAspatate transaminase E. coliE. coli AVI55449.1AVI55449.1 F: gaccatggaattcatgactgaaccccgttcagF: gaccatggaattcatgactgaaccccgttcag 104104 R: acagccaagcttttagtccgccttcgccggcR: acagccaagcttttagtccgccttcgccggc 105105 bcATbcAT branched chain transaminasebranched chain transaminase D. mccartyiD. McCartyi AQY72500.1AQY72500.1 F: gaaggagatatacatatggctagcatgacF: gaaggagatatacatatggctagcatgac 106106 R: ctttgttagcagccggatctcagtggtggtggtggR: ctttgttagcagccggatctcagtggtggtggtgg 107107 bcATbcAT branched chain transaminasebranched chain transaminase E. coliE. coli AVI53929.1AVI53929.1 F: aagaaggagatatacatatggctagcatgactggF: aagaaggagatatacatatggctagcatgactgg 108108 R: ccggatctcagtggtggtggtggtggtgctcgR: ccggatctcagtggtggtggtggtggtgctcg 109109 bcATbcAT branched chain transaminasebranched chain transaminase E. sppE. spp AOL13801.1AOL13801.1 F: taagaaggagatatacatatggctagcatgactggF: taagaaggagatatacatatggctagcatgactgg 110110 R: gtgctcgagttgattaacttgatctaaccagccgtatR: gtgctcgagttgattaacttgatctaaccagccgtat 111111 aroATaroAT acromatic　　 transaminaseacromatic transaminase E. coliE. coli AUV21492.1AUV21492.1 F: taagaaggagatatacatatgtttcaaaaagttgF: taagaaggagatatacatatgtttcaaaaagttg 112112 R: gtgctcgagttgattaagcagataatcgR: gtgctcgagttgattaagcagataatcg 113113 aroATaroAT acromatic　　 transaminaseacromatic transaminase E. coliE. coli AVI54196.1
　AVI54196.1
F: gaccatggaattcatggctgacactcgccctgF: gaccatggaattcatggctgacactcgccctg 114114 R: aacagccaagcttttattccgcgttttcgtgR: aacagccaagcttttattccgcgttttcgtg 115115 alaATalaAT alanine transaminasealanine transaminase P. denitrificansP. denitrificans AGI22743.1AGI22743.1 F: cagaattcaaaagatcttttaagaaggagatatacatatgactgaaccccgF: cagaattcaaaagatcttttaagaaggagatatacatatgactgaaccccg 116116 R: agatccttactcgagtttggatccttagtccgccttcgccR: agatccttactcgagtttggatccttagtccgccttcgcc 117117 alaATalaAT alanine transaminasealanine transaminase P. putidaP. putida AE015451.2AE015451.2 F: cagaattcaaaagatcttttaagaaggagatatacatatggccaacccaggttcgF: cagaattcaaaagatcttttaagaaggagatatacatatggccaacccaggttcg 118118 R: ctcgagtttggatccttacttacgagtcaggccR: ctcgagtttggatccttacttacgagtcaggcc 119119 alaCalaC alanine transaminasealanine transaminase E. coliE. coli AAN66442.1AAN66442.1 F: TtcagaattcaaaagatcttttaagaaggagatatacatatggctgacactcgcF: Ttcagaattcaaaagatcttttaagaaggagatatacatatggctgacactcgc 120120 R: cgagtttggatccttattccgcgttttcgtgR: cgagtttggatccttattccgcgttttcgtg 121121 alaCalaC alanine transaminase alanine transaminase E. coliE. coli AAN66442.1
A140P-Y275DAAN66442.1
A140P-Y275D F: ttcagaattcaaaagatcttttaagaaggagatatacatatggctgacactcgcF: ttcagaattcaaaagatcttttaagaaggagatatacatatggctgacactcgc 122122 R: cgagtttggatccttattccgcgttttcgtgR: cgagtttggatccttattccgcgttttcgtg 123123 aspATaspAT aspatate transaminaseaspatate transaminase P. denitrificansP. denitrificans AGI22891.1AGI22891.1 F: ttcagaattcaaaagatcttttaagaaggagatatacatatgctcggacccggcgF: ttcagaattcaaaagatcttttaagaaggagatatacatatgctcggacccggcg 124124 tcgagtttggatcctcagcgattctggtgcgcgtcgagtttggatcctcagcgattctggtgcgcg 125125 aroATaroAT acromatic transaminaseacromatic transaminase P. denitrificansP. denitrificans AGI22971.1AGI22971.1 F: gtggtggtggtggtggtgctcgagcagtttcaggcgcagcgcF: gtggtggtggtggtggtgctcgagcagtttcaggcgcagcgc 126126 R: aactttaagaaggagatatacatatgatgagcaagttctggagtcccR: aactttaagaaggagatatacatatgatgagcaagttctggagtccc 127127 aroATaroAT acromatic transaminaseacromatic transaminase P. fluorescensP. fluorescens AUM68313.1AUM68313.1 F: gtggtggtggtggtggtgctcgaggagttcgccgagggcF: gtggtggtggtggtggtgctcgaggagttcgccgagggc 128128 R: actttaagaaggagatatacatatgatgagtaaattctggagcccgR: actttaagaaggagatatacatatgatgagtaaattctggagcccg 129129

효소의 생산 Production of enzymes

E. coli BL21(DE3) 숙주에서 표 6의 효소를 발현하였다. LB medium을 사용하였고 plasmid의 유지와 보존을 위해 kanamycin 50 mg/L 를 사용하였다. 발현 벡터로는 pET 그리고 프로모터로 T7을 사용하였다. TA 유전자의 cloning을 위해　coding sequence는 해당 미생물의 genomic DNA로부터 증폭하였고 pET vector에 His-tag (C 말단)를 포함하도록 하여 seamless cloning 및 assembly kit (Invitrogen)을 사용하여 E. coli Top10 균주에 cloning 하였다. 얻어진 TA 유전자 종류에 따라 plasmid는 pET/TAi (i = 1 ~ 17) 로 명명하였고 sequence를 확인한 후 (Macrogen, Seoul Korea) 효소 생산 균주인 E. coli BL21 (DE3)에 도입하였다.The enzyme of Table 6 was expressed in E. coli BL21 (DE3) host. LB medium was used and kanamycin 50 mg / L was used for the maintenance and preservation of plasmids. PET as the expression vector and T7 as the promoter. For the TA gene cloning, the coding sequence was amplified from the genomic DNA of the microorganism and cloned into the E. coli Top 10 strain using the seamless cloning and assembly kit (Invitrogen) containing the His-tag (C terminus) in the pET vector . The plasmids were named pET / TAi (i = 1-17) according to the type of TA gene obtained. Sequence was confirmed (Macrogen, Seoul Korea) and introduced into E. coli BL21 (DE3), an enzyme producing strain.

GeneGene EnzymeEnzyme SourceSource Acc. No.Acc. No. pET/TAipET / TAi aspATaspAT Aspatate transaminaseAspatate transaminase M.　　 lotiM. loti ANN59010.1ANN59010.1 TA1TA1 aspATaspAT Aspatate transaminaseAspatate transaminase M.　　 lotiM. loti ANN57047.1ANN57047.1 TA2TA2 aspATaspAT Aspatate transaminaseAspatate transaminase A.　　 tumefaciensA. tumefaciens ACM26415.1ACM26415.1 TA3TA3 aspATaspAT Aspatate transaminaseAspatate transaminase B. subtilisB. subtilis AQR85543.1AQR85543.1 TA4TA4 aspATaspAT Aspatate transaminaseAspatate transaminase E. coliE. coli AVI55449.1AVI55449.1 TA5TA5 bcATbcAT branched chain transaminasebranched chain transaminase D. mccartyiD. McCartyi AQY72500.1AQY72500.1 TA6TA6 bcATbcAT branched chain transaminasebranched chain transaminase E. coliE. coli AVI53929.1AVI53929.1 TA7TA7 bcATbcAT branched chain transaminasebranched chain transaminase E. sppE. spp AOL13801.1AOL13801.1 TA8TA8 aroATaroAT acromatic　　 transaminaseacromatic transaminase E. coliE. coli AUV21492.1AUV21492.1 TA9TA9 aroATaroAT acromatic　　 transaminaseacromatic transaminase E. coliE. coli AVI54196.1
　AVI54196.1
TA10TA10 alaATalaAT alanine transaminasealanine transaminase P. denitrificansP. denitrificans AGI22743.1AGI22743.1 TA11TA11 alaATalaAT alanine transaminasealanine transaminase P. putidaP. putida AE015451.2AE015451.2 TA12TA12 alaCalaC alanine transaminasealanine transaminase E. coliE. coli AAN66442.1AAN66442.1 TA13TA13 alaCalaC alanine transaminase alanine transaminase E. coliE. coli AAN66442.1
A140P-Y275DAAN66442.1
A140P-Y275D TA14TA14 aspATaspAT aspatate transaminaseaspatate transaminase P. denitrificansP. denitrificans AGI22891.1AGI22891.1 TA15TA15 aroATaroAT acromatic transaminaseacromatic transaminase P. denitrificansP. denitrificans AGI22971.1AGI22971.1 TA16TA16 aroATaroAT acromatic transaminaseacromatic transaminase P. fluorescensP. fluorescens AUM68313.1AUM68313.1 TA17TA17

효소생산을 위해 TA를 생산하는 재조합 균주는 50 mg/L kanamycin을 포함하는 LB 배지에서 배양하였다. 액체부피 20 mL, 250 mL 플라스크를 사용하였고 20℃, 200 rpm 조건에서 배양하였다. 세포농도가 0.6 OD (600 nm)에 도달했을 때 0.1 mM의 IPTG를 inducer로 첨가하였고 그 이후 10시간 동안 추가로 배양하였다. 그 이후 세포를 원심분리 (10,000 g, 10 분)하고 100 mM 인산 완충용액 (pH 7.0)으로 세척하고 binding 완충용액 (20 mM 인산 완충용액, 0.5 M NaCl, 20 mM imidazole)에 현탁하였다. 이 후 세포를 Sonicator로 파쇄하고 원심분리하여 미파쇄부분과 고형분을 제거하고 용액부분만 모아서 세포활성과 SDS-PAGE 를 이용한 단백질 분석에 이용하였다.Recombinant strains producing TA for enzyme production were cultured in LB medium containing 50 mg / L kanamycin. Liquid volume 20 mL, 250 mL flasks were used and cultured at 20 ° C and 200 rpm. When the cell concentration reached 0.6 OD (600 nm), 0.1 mM IPTG was added to the inducer and then further cultured for 10 hours. The cells were then centrifuged (10,000 g, 10 min), washed with 100 mM phosphate buffer (pH 7.0) and suspended in binding buffer (20 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole). The cells were disrupted with a sonicator and centrifuged to remove the unfragmented portion and solids, and the collected solution was used for protein analysis using cell activity and SDS-PAGE.

효소의 순수분리는 Ni-NTA-HP resin 충진컬럼 (17-5248-01; GE Healthcare, Sweden)을을 이용하여 이루어졌다. 앞서 얻은 미생물 파쇄 용액부분을 모아 컬럼에 넣어 정제한 후 효소 용액을 얻었다. 이후 용액에 포함된 salt를 제거하기 위해 10 kDa cut-off membrane을 사용하여 dialysis 시켰다. 얻어진 효소는 denaturing 혹은 non-naturing 조건에서 전기 영동하였다 (도 6a 및 6b). 그리고 glycerol 이 20% 되도록 첨가한 후 - 80℃에 보관하였다.　 The pure separation of the enzyme was done using a Ni-NTA-HP resin packing column (17-5248-01; GE Healthcare, Sweden). The fraction of the microbial crushing solution obtained was collected and purified in a column to obtain an enzyme solution. After that, dialysis was performed using a 10 kDa cut-off membrane to remove salts contained in the solution. The resulting enzyme was electrophoresed under denaturing or non-naturing conditions (FIGS. 6A and 6B). Then, glycerol was added at 20% and stored at -80 ℃.

효소의 활성측정Enzyme activity measurement

호모세린에 대한 활성은 각각 pyruvate 와 2-oxoglutarate를 amine 받게로써 사용하여 측정하였다: Activity against homoserine was measured using pyruvate and 2-oxoglutarate as amine acceptors, respectively:

(i) Homoserine + pyruvate　 ↔　 2-oxo-4-hydroxybutyric acid + alanine(i) Homoserine + pyruvate ↔ 2-oxo-4-hydroxybutyric acid + domain

(ii)Homoserine + 2-oxoglutarate　 ↔　 2-oxo-4-hydroxybutyric acid + glutamate(ii) Homoserine + 2-oxoglutarate ↔ 2-oxo-4-hydroxybutyric acid + glutamate

생성물인 알라닌(alanine) 또는 글루타메이트(glutamate)의 생산이나, 반응물인 피루빈산(pyruvate), 2-옥소글루타레이트(2-oxoglutarate)의 소모를 통해 효소활성을 측정할 수 있었다. 모든 효소에서 2-oxoglutarate를 받게로 사용한 경우 효소 활성이 관찰되지 않았기에 (i)의 반응, 즉 pyruvate를 받게로 하는 반응을 이용해 TA 활성을 조사하였다. 생성물인 alanine은 OPA로 수식한 후 HPLC로 측정하였다.Enzyme activity could be measured by production of alanine or glutamate as a product or consumption of pyruvate, 2-oxoglutarate as a reactant. When all enzymes were used to receive 2-oxoglutarate, no enzyme activity was observed, so TA activity was investigated using the reaction (i), that is, the reaction to receive pyruvate. The product alanine was measured by HPLC after modification with OPA.

TA 활성 측정을 위해 50 mM 인산 완충용액(pH 7.0), 0.1 mM cofactor pyridoxal phosphate (PLP), 10 mM homoserine, 그리고 적당량의 TA 효소가 포함되도록 반응 용액을 제조하였다. 이 용액을 약 5분간 37℃에서 incubation 한 후 10 mM의 pyruvate를 첨가하여 반응을 시작하였다. 반응 10분 후 12 mM의 과염소산(perchloric acid)을 첨가하여 반응을 중단시키고 10,000g에서 5분간 원심분리하였다. 이후 5 μL sample을 취하여 12.5 μL의 OPA 용액 (25 mg OPA, 50 μL 2-mercaptoethanol, pH 9.5 0.5 mM 포화 sodium borate 용액, 4.5 mL methanol)과 혼합하고 상온에서 incubation 한 후 filter 정제하였다. OPA 에 의해 변형된 alanine은 Zorbax eclipse XBD-C18 column을 장착한 HPLC로 정량 분석하였다. OPA로 수식된 Alanine derivative는 10.5 분의 retention time을 가지고 있었고 338 nm DAD detector로 분석하였다. 단백질 정량은 Bradford 법으로 bovine serum albumin을 standard로 행하였다. 분석은 3회 시행하였고 평균값을 나타내었다. For the TA activity, a reaction solution containing 50 mM phosphate buffer (pH 7.0), 0.1 mM cofactor pyridoxal phosphate (PLP), 10 mM homoserine, and an appropriate amount of TA enzyme was prepared. The solution was incubated at 37 ° C for about 5 minutes, and 10 mM pyruvate was added to initiate the reaction. After 10 minutes of reaction, 12 mM perchloric acid was added to stop the reaction and centrifuged at 10,000 g for 5 minutes. Then, 5 μL sample was taken and mixed with 12.5 μL of OPA solution (25 mg OPA, 50 μL 2-mercaptoethanol, pH 9.5 0.5 mM saturated sodium borate solution, 4.5 mL methanol), incubated at room temperature and filtered. The alanine modified by OPA was quantitatively analyzed by HPLC on a Zorbax eclipse XBD-C18 column. The OPA-modified Alanine derivative had a retention time of 10.5 minutes and was analyzed with a 338 nm DAD detector. Protein quantification was performed by Bradford method using bovine serum albumin as standard. The analysis was performed 3 times and the mean value was shown.

도 6a에 의하면 TA1, TA2, TA3, 및 TA6 4 종류는 insoluble form으로 생성되었기에, 활성 측정에 이용하지 않았다. 다른 효소 중에서는 5개(TA4, TA5, TA10, TA11, TA15) 만이 호모세린(homoserine)에 대하여 활성을 나타냈다. 이들을 affinity chromatography로 순수분리하여 활성을 측정하였다 (도 7a). TA4가 가장 높은 활성, 즉 3 U/mg protein 의 활성을 보여 주었다.According to Fig. 6A, TA1, TA2, TA3, and TA6 4 were produced in an insoluble form and thus were not used for activity measurement. Of the other enzymes, only 5 (TA4, TA5, TA10, TA11, TA15) showed activity against homoserine. These were purified by affinity chromatography to determine their activity (FIG. 7A). TA4 showed the highest activity, i.e., 3 U / mg protein.

2-2 TA4의 효소 엔지니어링2-2 Enzyme engineering of TA4

TA4 효소가 가장 높은 활성을 나타냈기에, 이 효소를 변이시켜 활성이 증가된 효소를 획득하였다. 먼저 변이 효소 library 를 구축하고 이를 세포성장과 관련된 high throughput screening (HTS) 방법을 이용하여 높은 활성의 효소 변이체를 확보하였다.　 Since TA4 enzyme showed the highest activity, this enzyme was mutated to obtain an enzyme with increased activity. First, a mutant enzyme library was constructed and highly active enzyme mutants were obtained using high throughput screening (HTS) method related to cell growth.

Reporter 균주의 개발 및 조사Development and investigation of Reporter strain

호모세린 트랜스아미네이즈(Homoserine transaminase)는 피루브산(pyruvate)을 알라닌(alanine)으로 바꾸어 주며 alanine의 아미노기는 alanine transaminase 에 의해 다른 아미노산을 생산하는데 이용된다. 즉, 호모세린은 세포성장의 유일한 질소원으로 사용될 수 있으며, 이 경우 세포성장 속도는 본 발명자들이 개발하려는 homoserine transaminase의 활성에 따라 달라질 수 있다. Homoserine transaminase converts pyruvate to alanine, and alanine amino group is used to produce other amino acids by alanine transaminase. That is, homoserine can be used as the only source of nitrogen for cell growth, and the rate of cell growth in this case can vary depending on the activity of the homoserine transaminase that we are developing.

이 전략을 조사하기 위하여 pET-TA4 유전자 재조합 플라스미드를 갖는 E. coli BL21 (DE3) 균주를 IPTG 농도를 달리하여 배양하였다 (도 8). TA4 유전자의 발현은 T7 프로모터에 의해 조절되고 TA4 효소의 활성은 IPTG 농도로 조절할 수 있기 때문이다. 질소원이 배제된 M9 minimal medium을 사용하였고, 질소원으로 10 mM의 호모세린, 그리고 미생물 성장을 유도하기 위해 소량(0.5 mM)의 트레오닌과 메티오닌을 각각 첨가하였다. 도 8 에 나타난 바와 같이, TA4 효소를 갖는 유전자 재조합 균주가 높은 성장 속도를 보여 주었고 그 정도는 IPTG 첨가량이 높을 때 예를 들어, 1.0 mM일 때 가장 분명히 나타났다. 즉 호모세린을 질소원으로 사용하는 균주를 통해 호모세린 TA의 screening이 가능함을 확인하였다. To investigate this strategy, E. coli BL21 (DE3) strains harboring pET-TA4 gene recombinant plasmids were cultured with different IPTG concentrations (FIG. 8). The expression of the TA4 gene is regulated by the T7 promoter and the activity of the TA4 enzyme can be regulated by the IPTG concentration. Nitrogen-free M9 minimal medium was used, 10 mM homoserine was added to the nitrogen source, and small amounts (0.5 mM) of threonine and methionine were added to induce microbial growth. As shown in FIG. 8, the recombinant strain having the TA4 enzyme exhibited a high growth rate, and the degree was most apparent when the IPTG content was high, for example, at 1.0 mM. That is, it was confirmed that homoserine TA can be screened through a strain using homoserine as a nitrogen source.

TA4 효소 라이브러리 제작TA4 enzyme library production

대장균 아스파르트산 아미노산전이효소(PDB ID: 1ASM)의 크리스탈 구조를 주형으로 사용하여, 상동성 모델링을 통해 TA4의 3차원 구조를 구축하였다. 상기 1ASM의 구조와 서열은 https://www.rcsb.org/structure/1ASM을 참고하였다. 1ASM 구조는 조효소로 인산피리독살(pyridoxal-5’-phosphate: PLP)을, 기질의 유사체로 말레산(maleic acid)를 갖고 있다. 이 모델은 MOE(Molecular Operating Environment)를 사용해 제작했으며, PROCHECK 및 ProSA 온라인 구조 분석을 통해 평가하였다.PYMOL뷰어(http://www.pymol.org)를 사용하여 단백질 구조를 확인하였다 (도 10). The 3D structure of TA4 was constructed by homology modeling using the crystal structure of E. coli aspartic acid amino acid transferase (PDB ID: 1 ASM) as a template. The structures and sequences of the above 1ASM are described in https://www.rcsb.org/structure/1ASM. 1ASM structure contains pyridoxal-5'-phosphate (PLP) as a coenzyme and maleic acid as an analogue of the substrate. This model was produced using MOE (Molecular Operating Environment) and evaluated through PROCHECK and ProSA online structure analysis. The protein structure was confirmed using the PYMOL viewer (http://www.pymol.org) (FIG. 10).

1ASM 구조로부터 말레산의 카르복실산(아스파르트산 잔기의 카르복실산)과 상호작용을 하고 있는 4개의 아미노산(Ile17, Gly38, Asn194, Arg386)을 확인하였고, TA4 모델 구조를 통해서 아스파르트산 잔기의 카르복실산과 상호작용을 할 것으로 예상되는 4개의 아미노산(Lys14, Gly40, Asn178, Try364)를 선정하였다 (도 11). The four amino acids (Ile17, Gly38, Asn194, Arg386) interacting with the carboxylic acid (maleic acid residue of aspartic acid residue) of the maleic acid were identified from the 1ASM structure. Through the TA4 model structure, Four amino acids (Lys14, Gly40, Asn178, Try364) expected to interact with the phenyl acid were selected (Fig. 11).

도 10과 도 11의 정보를 이용하여 TA4 효소 mutant library를 구축하였다. 아스파르트산 잔기의 카르복실산과 상호작용을 할 것으로 예상되는　TA4의 4개의 아미노산(Lys14, Gly40, Asn178, Try364)을 에셈블리 PCR 방법으로 무작위로 변형시켜 TA4 라이브러리를 제작하였다. 이 때, pET30b/TA4 플라스미드를 주형으로 사용하였으며, 사용한 프라이머를 표 8에 나타냈다. 제한효소 XbaI 및 XhoI 부위를 이용하여, 프라이머 1 및 2 로부터의 PCR 산물을 pET30b 플라스미드에 클로닝하였다. 생성된 TA4 라이브러리를 대장균 DH10β로 형질전환시켰고, 플라스미드를 분리정제하였다. 아래 표 8은 TA4 변이효소 라이브러리 제작에 사용한 프라이머 서열을 나타낸다. Using the information in FIG. 10 and FIG. 11, a TA4 enzyme mutant library was constructed. TA4 library was constructed by randomly modifying four amino acids of TA4 (Lys14, Gly40, Asn178, and Try364) expected to interact with the carboxylic acid of the aspartic acid residue, by means of the Ebble PCR method. At this time, pET30b / TA4 plasmid was used as a template, and the primers used were shown in Table 8. Using the restriction enzymes XbaI and XhoI sites, the PCR products from primers 1 and 2 were cloned into a pET30b plasmid. The resulting TA4 library was transformed with E. coli DH10?, And the plasmid was isolated and purified. Table 8 below shows the primer sequences used in the construction of the TA4 mutant enzyme library.

아래 프라이머 서열 중 알파벳 m, n, k 의 의미는 다음과 같다.The meanings of the letters m, n, k among the primer sequences shown below are as follows.

m: a (Adenine, 아데닌) 또는 c (Cytosine, 시토신)m: a (Adenine, adenine) or c (Cytosine, cytosine)

n: a(Adenine, 아데닌) 또는 g(Guanine, 구아닌) 또는 c(Cytosine, 시토신) 또는 t(thymine, 티민)n: a (adenine, adenine) or g (guanine, guanine) or c (cytosine, cytosine) or t (thymine, thymine)

k: g(Guanine, 구아닌) 또는 t(thymine, 티민)k: g (guanine, guanine) or t (thymine, thymine)

No.No. PrimerPrimer SequenceSequence 서열번호SEQ ID NO: 1One TA Library FTA Library F ctcactataggggaattgtgagcggataacctcactataggggaattgtgagcggataac 130130 22 TA Library RTA Library R gctagttattgctcagcggtggcagcgctagttattgctcagcggtggcagc 131131 33 TA K14 FTA K14 F gaaggaattgcctNNKcaattcttcgcttcgaaggaattgcctNNKcaattcttcgcttc 132132 44 TA K14 RTA K14 R gaagcgaagaattgMNNaggcaattccttcgaagcgaagaattgMNNaggcaattccttc 133133 55 TA G40 FTA G40 F caatctgggacagNNKaatccagatcagccaatctgggacagNNKaatccagatcagc 134134 66 TA G40 RTA G40 R gctgatctggattMNNctgtcccagattggctgatctggattMNNctgtcccagattg 135135 77 TA N178 FTA N178 F gaattatccgaatNNKccgactggagctggaattatccgaatNNKccgactggagctg 136136 88 TA N178 RTA N178 R cagctccagtcggMNNattcggataattccagctccagtcggMNNattcggataattc 137137 99 TA Y364 FTA Y364 F gaatatggcgagggcNNKgtcagagtcggaatatggcgagggcNNKgtcagagtcg 138138 1010 TA Y364 RTA Y364 R cgactctgacMNNgccctcgccatattccgactctgacMNNgccctcgccatattc 139139

TA4 효소 라이브러리 스크리닝 및 우수 효소 개발TA4 enzyme library screening and excellent enzyme development

제작된 라이브러리를 대장균 BL21(DE3)로 형질전환시킨 후, 50 mL LB 배지에서 밤새 배양하였다. 이후 cell pellet을 원심분리하여 얻고 이를 100 mM 인산 완충용액으로 3번 세척한 후 20 mM homoserine을 질소원으로 갖는 M9 최소배지(50 um/mL kanamycin 포함)에 OD 0.05가 되도록 접종하였다. 이 후. 37℃, 200 rpm에서 3시간 배양하고 0.05 mM IPTG를 첨가하여 TA4 효소를 induction 시켰다. OD 값이 0.5가 되었을 때 배양액을 100 배 희석하여 다시 배양하였다. 이와 같은 배양-희석 cycle을 10회 반복한 후 배양액을 50 μg/mL kanamycin이 포함된 LB 한천배지에 스프레딩하고 잘 자라는 콜로니 50개를 취하여 M9 배지에서 순수 배양하였다. 이 후 이들의 유전자 서열을 조사한 결과 총 5가지 mutants를 얻었으며, 이들을 각각 TA4-1 내지 TA4-5로 명명하였다. The prepared library was transformed with E. coli BL21 (DE3) and then cultured overnight in 50 mL of LB medium. After the cell pellet was centrifuged, it was washed 3 times with 100 mM phosphate buffer and then inoculated to an M9 minimal medium (containing 50 um / mL kanamycin) with 20 mM homoserine as a nitrogen source. after. After incubation at 37 ° C and 200 rpm for 3 hours, TA4 enzyme was induced by adding 0.05 mM IPTG. When the OD value reached 0.5, the culture was diluted 100-fold and then re-cultured. After repeating this culture-dilution cycle 10 times, the culture broth was spread on LB agar medium containing 50 μg / mL kanamycin, and 50 colonies were grown and cultured in M9 medium. The gene sequences of these mutants were examined and a total of five mutants were obtained and designated as TA4-1 to TA4-5, respectively.

다음으로, 얻은 효소를 affinity chromatography로 순수분리하여 측정하였으며, 최종적으로 2개의 TA4 변이체(TA4-1, TA4-2)가 높은 활성을 보였고, 특히 TA4-1는 야생형(wildtype)에 비해 5배 가량 높은 15 U/mg protein 활성을 나타냈다. TA4-1은 Y364Q, TA4-2는 N178D의 아미노산 서열 변이를 갖고 있다 (도 12). 이 결과는 modelling을 통해 얻어진 결과, 즉 Y364 및 N178은 1ASM의 N194, R386과 잘 align　되었다는 사실과 함께, 호모세린과 interaction 하는 site의 중요성을 보여 준다.　Next, the obtained enzyme was purified by affinity chromatography. Finally, two TA4 variants (TA4-1 and TA4-2) showed high activity, and TA4-1 was 5 times more abundant than the wildtype High 15 U / mg protein activity. TA4-1 has Y364Q and TA4-2 has amino acid sequence variation of N178D (Fig. 12) . This result is the result obtained through modeling, ie Y364 and N178 show the importance of sites interacting with homoserine, with the fact that they are well aligned with N194 and R386 of 1ASM.

다음으로 TA4-1과 TA4-2 효소가 갖는 변이를 모두 갖는 TA4-6 효소를 site-directed mutagenesis 방법을 이용하여 제작하였다. TA4-6 효소는 가장 높은 활성, 20 U/mg protein을 나타내었다. Next, the TA4-6 enzyme with both TA4-1 and TA4-2 enzyme mutations was constructed by site-directed mutagenesis. TA4-6 enzyme showed the highest activity, 20 U / mg protein.

이후 실험에는 가장 활성이 좋은 TA4-1과 TA4-6 변이체를 사용하였다.In the subsequent experiments, the most active TA4-1 and TA4-6 variants were used.

실시예 3: 2-Oxo-4-hydroxy butyric acid (OHB)의 (2S)-L-reductase 효소의 스크리닝 및 변이효소 제작Example 3: Screening and mutagenesis of (2S) -L-reductase enzyme of 2-Oxo-4-hydroxy butyric acid (OHB)

3-1. OHB L-reductase 효소의 스크리닝3-1. Screening of OHB L-reductase enzyme

OHB L-reductase 효소의 확보Obtaining of OHB L-reductase enzyme

OHB는 prochiral 화합물로 환원되면 2번 위치에 L (2S) 또는 D (2R) 히드록시기를 갖는 2,4-dihydroxy butyric acid (DHB)로 전환된다. OHB reductase는 2-hydroxy acid dehydrogenase에 속하며 여기에는 lactate dehydrogenase (EC1.1.1.27, EC1.1.1.28), malate dehydrogenase (EC1.1.1.37, EC1.1.1.82, EC1.1.1.299) 그리고 branched chain (D), (L)-2-hydroxyacid dehydrogenase (EC1.1.1. 272, EC1.1.1.345) 등이 잘 알려져 있다. 먼저 L-form isomer를 생산하기 위하여 Alcaligeneseutrophus H16, Cupriavidus basilensis, Achromobacter xylosoxidans, Burkholderiaglumae, Escherichia fergusonii, Escherichia coli, Lactobacillusmali 및 Escherichia coli K-12 등으로부터 OHB에 대해 활성이 높을 것으로 예상되는 (L)-lactate dehydrogenase 효소들을 선정하였다 (표 9).OHB is converted to 2,4-dihydroxy butyric acid (DHB) with a L (2S) or D (2R) hydroxy group at position 2 when reduced to a prochiral compound. OHB reductase belongs to 2-hydroxy acid dehydrogenase, and lactate dehydrogenase (EC1.1.1.27, EC1.1.1.28), malate dehydrogenase (EC1.1.1.37, EC1.1.1.82, EC1.1.1.299) and branched chain (D), and (L) -2-hydroxyacid dehydrogenase (EC1.1.1.272, EC1.1.1.345). (L) -lactate dehydrogenase, which is expected to have high activity against OHB from Alcaligeneseutrophus H16, Cupriavidus basilensis , Achromobacter xylosoxidans , Burkholderiaglumae , Escherichia fergusonii , Escherichia coli , Lactobacillus mali and Escherichia coli K- Enzymes were selected (Table 9).

OHB 2S-reductase 효소 screening을 위한 (L)-lactate dehydrogenase 후보 효소들을 표 9에 나타냈다.(L) -lactate dehydrogenase candidate enzymes for OHB 2S-reductase enzyme screening are shown in Table 9.

GeneGene SourceSource Acc.　　 no.Acc. no. Primer　　 sequence (FP/RP)Primer sequence (FP / RP) 서열번호SEQ ID NO: lactate　　 dehdydrogenases(ldhO)lactate dehydrogenases ( ldHO ) Alcaligenes eutrophus(Ae)Alcaligenes eutrophus (Ae) YP　　 725182.1YP 725182.1 ATAACATATGAAGATCTCCCTCACCAGCGCATAACATATGAAGATCTCCCTCACCAGCGC 140140 TAATAGGATCCTCAGTGATGGTGATGGTGATGGGCCG TGGGGACGGCCACGTTGTAATAGGATCCTCAGTGATGGTGATGGTGATGGGCCG TGGGGACGGCCACGTTG 141141 malate/L-lactate　　 dehydrogenase (ldh-2)malate / L-lactate dehydrogenase ( ldh-2 ) Cupriavidus basilensis(Cb)Cupriavidus basilensis (Cb) WP　　 043344208.1WP 043344208.1 ATAACATATGAAGATAACACTGCAATCATAACATATGAAGATAACACTGCAATC 142142 TAATAGGATCCTCAGTGGTGATGATGGTGATGGCCCGCCGGCAGTGCCACGCCAAGTTCTAATA GGATCC TCAGTGGTGATGATGGTGATGGCCCGCCGGCAGTGCCACGCCAAGTTC 143143 malate/L-lactate　　 dehydrogenase (ldh-2)malate / L-lactate dehydrogenase ( ldh-2 ) Achromobacter xylosoxidans(Ax)Achromobacter xylosoxidans (Ax) WP　　 006389860.1WP 006389860.1 ATAACATATGAAGATCTCCATTACCCAAGATAACATATGAAGATCTCCATTACCCAAG 144144 TAATAGGATCCTCAGTGGTGATGATGGTGATGAGGCAACGCGTCAGCTAATA GGATCC TCAGTGGTGATGATGGTGATGAGGCAACGCGTCAGC 145145 malate/L-lactate　　 dehydrogenase (ldh-2)malate / L-lactate dehydrogenase ( ldh-2 ) Burkholderia glumae(Bg)Burkholderia glumae (Bg) YP　　 002909484.1YP 002909484.1 ATAACATATGCAGATATCCCTCGACGATGATAACATATGCAGATATCCCTCGACGATG 146146 TAATAGGATCCCTAGTGGTGATGATGGTGATGGGCCCGTGCGGCCGGCGGCACCACGCCGAGTTCGTCTAATA GGATCC CTAGTGGTGATGATGGTGATGGGCCCGTGCGGCCGGCGGCACCACGCCGAGTTCGTC 147147 malate/L-lactate　　 dehydrogenase (ldh-2)malate / L-lactate dehydrogenase ( ldh-2 ) Escherichia fergusonii(Ef)Escherichia fergusonii (Ef) WP　　 002431747.1WP 002431747.1 ATAACATATGTATGGGTACAGATACCTTCATAACATATGTATGGGTACAGATACCTTC 148148 TAATAGGATCCTTAGTGGTGATGATGGTGATGATGCTGATTCCTGAGGATGTAACTAATA GGATCC TTAGTGGTGATGATGGTGATGATGCTGATTCCTGAGGATGTAAC 149149 D-2-hydroxyacid　　 dehydrogenase (ldh-2)D-2-hydroxyacid dehydrogenase ( ldh-2 ) Escherichia coli(Ec)Escherichia coli (Ec) WP024240865.1WP024240865.1 ATAACATATGATGTCATTACAAATTGCTGATAACATATGATGTCATTACAAATTGCTG 150150 TAATAGGATCCTCAGTGGTGATGATGGTGATGTCCAGCTAATGCTGATTCCTGTAATA GGATCC TCAGTGGTGATGATGGTGATGTCCAGCTAATGCTGATTCCTG 151151 D-2-hydroxyacid　　 dehydrogenase (ldhA)D-2-hydroxyacid dehydrogenase ( ldhA ) Lactobacillus mali(Lm)Lactobacillus mali (Lm) WP　　 003689565.1WP 003689565.1 ATAACATATGACAAGAATAATTGCTTATCATGATAACATATGACAAGAATAATTGCTTATCATG 152152 TAATAGGATCCTTAGTGGTGATGATGGTGATGTTCTCCCTTGAAACTTATTTCATGTGTAATA GGATCC TTAGTGGTGATGATGGTGATGTTCTCCCTTGAAACTTATTTCATGTG 153153 D-lactate　　 dehydrogenase(ldhA)D-lactate dehydrogenase ( ldhA ) Escherichia coli K-12(Eck)Escherichia coli K-12 (Eck) NP　　 415898.1NP 415898.1 ATAACATATGAAACTCGCCGTTTATAGATAACATATGAAACTCGCCGTTTATAG 154154 TAATAGGATCCTTAGTGGTGATGATGGTGATGAACCAGTTCGTTCGGGCAGTAATA GGATCC TTAGTGGTGATGATGGTGATGAACCAGTTCGTTCGGGCAG 155155

선정된 효소들의 아미노산 서열을 비교한 결과, 대개 44-66%의 높은 유사성을 보였다. 그러나 A. eutrophus, L.mali 그리고 E. coli 유래 효소는 낮은 유사성을 보였다. The amino acid sequences of the selected enzymes were compared, showing a high similarity of 44-66%. However, enzymes derived from A. eutrophus , L. mali and E. coli showed low affinity.

OHB L-reductase 효소의 생산 및 활성 측정Production and activity measurement of OHB L-reductase enzyme

효소발현을 위해 E. coli BL21(DE3) star 균주를 숙주균주로 이용하였으며, 클로닝과 플라스미드 보존을 위해서는 E. coli DH5α 균주를 숙주로 이용하였다. kanamycin 50 μg/mL이 포함된 LB medium을 일반적인 배양과 클로닝 후 재조합 균주 배양에 사용하였다. 유전자 발현 및 효소 생산을 위해 pET 플라스미드와 T7 프로모터를 사용하였다. Ldh 유전자는 PCR 증폭하였고 이 때 C 말단에 His-tag을 붙여 주었다. PCR fragment는 pET vector (표 10)에 넣은 후 E. coli DH5α 균주에 클로닝하고 유전자 서열을 확인한 후 E. coli BL21(DE3) star에 도입하였다. Ldh 유전자의 soluble expression을 촉진하기 위해 Chaperon 을 발현하는 pG-Tf2 플라스미드를 추가로 도입해 주었다.　 E. coli BL21 (DE3) star strain was used as a host strain for the expression of the enzyme, and E. coli DH5α strain was used as a host for cloning and plasmid preservation. LB medium containing kanamycin 50 μg / mL was used for culture of recombinant strains after general culture and cloning. PET plasmid and T7 promoter were used for gene expression and enzyme production. The Ldh gene was amplified by PCR and the His-tag was attached to the C-terminus. The PCR fragment was inserted into a pET vector (Table 10), cloned into the E. coli strain DH5α, the gene sequence was confirmed, and introduced into E. coli BL21 (DE3) star. In order to promote the soluble expression of the Ldh gene, the pG-Tf2 plasmid expressing Chaperon was further introduced.

LDH 효소 생산을 위해 유전자 재조합 BL21(pET-Xx-LDH, Xx는 표 9에 나오는 LDH 유전자를 가진 플라스미드) 균주를 50 μg/mL kanamycin을 포함하는 LB 배지에서 호기적으로 배양하였다. 배양온도는 30℃, 교반속도는 100 rpm 이었고 1 L 플라스크에 250 mL 배지를 넣어 주었다. 세포농도가 0.5 OD에 도달했을 때, 0.1 mM의 IPTG를 첨가하고 추가적으로 10시간 배양하였다. 이 후 세포를 원심분리하고 25 mM 인산 완충용액 (pH 7.0)으로 3번 세척한 후 binding buffer (20 mM pH 7.0 인산 완충용액, 0.5 M NaCl, 10 mM imidazole)에 현탁한 후 sonicator를 이용하여 파쇄하였다. 세포 파쇄액은 4℃, 25,000 g 에서 30분 원심분리하여 비용해성 부분을 제거하고 용해성 부분을 모아 효소 활성 분석이나 추가적인 효소분리에 이용하였다. For the production of LDH enzyme, recombinant BL21 (pET-Xx-LDH, Xx is a plasmid with the LDH gene shown in Table 9) was aerobically cultured in LB medium containing 50 μg / mL kanamycin. The incubation temperature was 30 ° C, stirring speed was 100 rpm, and 250 ml medium was added to a 1 L flask. When the cell concentration reached 0.5 OD, 0.1 mM IPTG was added and incubated for an additional 10 hours. The cells were then centrifuged, washed three times with 25 mM phosphate buffer (pH 7.0), suspended in binding buffer (20 mM pH 7.0 phosphate buffer, 0.5 M NaCl, 10 mM imidazole) and sonicated Respectively. The cell lysate was centrifuged at 25,000 g for 30 min at 4 ° C to remove the insoluble portion, and the soluble portion was collected and used for enzyme activity analysis or additional enzyme separation.

LDH 효소활성은 pyruvate 와 OHB를 기질로 사용하여 분석하였다. OHB는 상업적으로 구입할 수 없으므로 homoserine으로부터 합성하였다. 즉, 125 mM homoserine 용액을 pH 7.8, 100 mL Tris 완충용액으로 제조하고　1.25 U/mL의 snake venom (L)-amino acid oxidase 효소와 4400 U/mL의 catalase 효소와 혼합한 후 37℃에서 90분간 반응시켰다. 이 후 반응액을 Ultracentrifugal filter (10 kDa, Amicon)필터로 여과하여 OHB를 얻었다. 효소 활성 측정을 위해 효소 반응액은 60 mM Hepes buffer (pH 7.0), 50 mM NaCl, 5 mM MgCl₂, 5 mM fructose-1,6-bisphosphate, 적당량의 효소 등을 혼합하여 제조하고 37℃에서 2분간 incubation 하였다. 이 후 0.1 mM의 NAD(P)H 와 적당량의 OHB 혹은 pyruvate를 첨가하여 반응을 개시하였다. 효소 활성은 NAD(P)H의 감소 속도와 extinction coefficient를 이용하여 계산하였다.LDH enzyme activity was analyzed using pyruvate and OHB as substrates. OHB was synthesized from homoserine since it was not commercially available. The 125 mM homoserine solution was mixed with 1.25 U / mL of snake venom (L) -amino acid oxidase enzyme and 4400 U / mL of catalase enzyme, and the mixture was incubated at 37 ° C for 90 minutes Lt; / RTI > After that, the reaction solution was filtered with an ultracentrifugal filter (10 kDa, Amicon) to obtain OHB. For enzyme activity, the enzyme reaction mixture was prepared by mixing 60 mM Hepes buffer (pH 7.0), 50 mM NaCl, 5 mM MgCl ₂ , 5 mM fructose-1,6-bisphosphate and an appropriate amount of enzyme. Minute incubation. Then, 0.1 mM NAD (P) H and an appropriate amount of OHB or pyruvate were added to initiate the reaction. Enzyme activity was calculated using the rate of decrease of NAD (P) H and the extinction coefficient.

아래 표 10에는 OHB L-reductase 스크리닝을 위한 플라스미드를 기재하였다.Table 10 below describes plasmids for OHB L-reductase screening.

PlasmidsPlasmids DescriptionDescription SourceSource pACYC-DeutpACYC-Deut lacIq; expression vector; p15A-ori; His6-N; CmRLaCIq; expression vector; p15A-ORi; His6-N; CmR 　 pACYC-tacpACYC-tac Replacment of T7 promoter of pACYC-Deut by tac promoterReplacment of T7 promoter of pACYC-Deut by tac promoter 　 pET-DeutpET-Deut lacIq; expression vector; f1-ori; KanRLaCIq; expression vector; f1-ORi; KanR 　 pET-Ae-LDHpET-Ae-LDH pET containing LDH from Alcaligenes　　 eutrophus pET containing LDH from Alcaligenes eutrophus This studyThis study pET-Cb-LDHpET-Cb-LDH pET containing LDH from Cupriavidus　　 basilensis pET containing LDH from Cupriavidus basilensis This studyThis study pET-Ax-LDHpET-Ax-LDH pET containing LDH from Achromobacter　　 xylosoxidans pET containing LDH from Achromobacter xylosoxidans This studyThis study pET-Bg-LDHpET-Bg-LDH pET containing LDH from Burkholderia　　 glumae pET containing LDH from Burkholderia glumae This studyThis study pET-Ef-LDHpET-Ef-LDH pET containing LDH from Escherichia　　 fergusonii pET containing LDH from Escherichia fergusonii This studyThis study pET-Ec-LDHpET-Ec-LDH pET containing LDH from Escherichia　　 coli pET containing LDH from Escherichia coli This studyThis study pET-Lm-LDHpET-Lm-LDH pET containing LDH from Lactobacillus　　 mali pET containing LDH from Lactobacillus mali This studyThis study pET-LDHA1pET-LDHA1 pET containing mutant enzyme LDH I48S from Alcaligenes eutrophus pET containing mutant enzyme LDH I48S from Alcaligenes eutrophus This studyThis study pET-LDHA2pET-LDHA2 pET containing mutant enzyme LDH I48T from Alcaligenes eutrophus pET containing mutant enzyme LDH I48T from Alcaligenes eutrophus This studyThis study pET-LDHA3pET-LDHA3 pET containing mutant enzyme LDH I48N from Alcaligenes eutrophus pET containing mutant enzyme LDH I48N from Alcaligenes eutrophus This studyThis study pET-LDHA4pET-LDHA4 pET containing mutant enzyme LDH I48D from Alcaligenes eutrophus pET containing mutant enzyme LDH I48D from Alcaligenes eutrophus This studyThis study pET-LDHA5pET-LDHA5 pET containing mutant enzyme LDH I48K from Alcaligenes eutrophus pET containing mutant enzyme LDH I48K from Alcaligenes eutrophus This studyThis study

대부분의 효소들은 pyruvate에 높은 활성을 가지고 있었고 그에 비해 OHB에는 낮은 활성을 보였다 (도 13). pyruvate를 기질로 할 경우 Ec/Eck-LDH 가 6 μmol/mg protein/min 로 가장 높은 활성을 나타내었다. OHB에 대해서는 Ae-LDH가 1.8 μmol/mg protein/min의　가장 높은 활성을 보였다. 따라서 Ae-LDH를 선택하여 OHB에 높은 활성을 갖는 변이 효소를 제작하였다. Most enzymes had high activity on pyruvate and low activity on OHB (Fig. 13). When pyruvate was used as a substrate, Ec / Eck-LDH showed the highest activity at 6 μmol / mg protein / min. OHB, Ae-LDH showed the highest activity of 1.8 μmol / mg protein / min. Therefore, Ae-LDH was selected to produce mutant enzymes with high activity in OHB.

OHB L-reductase 효소의 생성물의 광학활성 측정Measurement of optical activity of OHB L-reductase enzyme product

Ae-LDH가 생성하는 DHB 2번 위치 hydroxy 기가 (R) 혹은 (S) 중 어느 것인지 알아보기 위해 광학활성을 갖는 HPLC와 colorimetric 분석 kit을 사용하여 분석하였다. 먼저 생성된 lactate에 대하여 HPLC 분석을 실시하였다. Agilent 사의 chiral column (EC 250/4 NUCLEOSIL Chiral-1, Germany)을 사용하였고 분석온도은 35 ℃, 이동상으로는 0.2 mM CuSO₄그리고 flow rate는 0.5 ml min¹ 이었다. 이 조건에서 L-lactate는 약 7.3 분에, 그리고 D-Lactate는 약 9.0분에 peak가 나타났다. Ae-LDH에 의해 생성된 lactate는 모두 L-form으로 나타났다. 또한 상업적으로 판매되는 lactate 분석 kit (BioVision, CA, USA)으로 확인하였으며 역시 생성되는 lactate는 L-form으로 확인되었다.To investigate whether the hydroxy group at position 2 of DHB produced by Ae-LDH is (R) or (S), we analyzed by HPLC with optical activity and colorimetric analysis kit. First, HPLC analysis was performed on the lactate produced. The chiral column (EC 250/4 NUCLEOSIL Chiral-1, Germany) from Agilent was used. The analytical temperature was 35 ° C, the mobile phase was 0.2 mM CuSO ₄ and the flow rate was 0.5 ml min ^-1 . At this condition, the peak appeared at about 7.3 minutes for L-lactate and about 9.0 minutes for D-lactate. The lactate produced by Ae-LDH was all L-form. Also, a commercially available lactate assay kit (BioVision, CA, USA) was also confirmed, and the lactate produced was also identified as L-form.

OHB의 환원반응에 의해 생성되는 DHB에 대해서도 2번 위치 OH기의 chirality를 조사하였다.　 그러나 DHB의 경우 상업제품이 없고 더 나아가 광학적으로 순수한 화합물은 구할 수 없었다. 그리하여 2번 위치 OH 기가 R-form 및 S-form으로 섞여있는 racemic 2-hydroxy gamma butyrolactone (HGBL)를 구입(Sigma-Aldrich, MO, USA)하여 화학적으로 분해시켜 racemic DHB를 합성하였다. 합성 방법은 다음과 같았다.The chirality of the OH group at position 2 was also investigated for the DHB produced by the reduction reaction of OHB. However, for DHB, there are no commercial products and further optically pure compounds were not available. Racemic DHB was synthesized by chemically degrading racemic 2-hydroxy gamma butyrolactone (HGBL) (Sigma-Aldrich, MO, USA) in which the 2-position OH group was mixed with R-form and S-form. The synthesis method was as follows.

Racemic HGBL을 metahnol에 녹인 후 동일 당량의 NaOH를 첨가하고 상온에서 18시간 반응시켰다. 그리고 진공여과 후 건조시켰다. NMR로 확인한 결과 100% 전환이 확인되었다.　　 Racemic HGBL was dissolved in metahnol, and the same amount of NaOH was added and reacted at room temperature for 18 hours. After vacuum filtration, it was dried. NMR analysis confirmed 100% conversion.

lactate의 chirality 분석에 사용된 동일한 chiral column을 사용하였을 때 HGBL로부터 합성된 DHB는 2개의 HPLC peak를 9.9분 및 11.3분에 보여주었다. lactate 의 경우 L-form이 D-form보다 먼저 나타났으므로 DHB의 경우도 L-form이 먼저 나타날 것으로 예상되었다. HGBL로부터 합성된 racemic DHB를 colorimetric 방법으로 분석한 결과 L-form과 D-form이 약 1:1의 비율로 얻어졌다. lactate assay kit은 원래 lactate를 분석하도록 고안되어 있으나 DHB와 lactate 간의 화학적 구조 유사성으로 인해 DHB의 chirality도 분석되는 것으로 추정했다. Ae-LDH와 반응시킨 OHB 생성물에 대해 DHB 분석을 chiral column과 colorimetric 분석 kit을 이용하여 동시에 실시하였다. HPLC 분석의 경우 하나의 peak만 얻어졌고 retention time은 L-form으로 추정되는 9.9분 시간대에 나타났다. 또한 colorimetric 분석에서도 L-form 만 얻어졌다. 따라서 Ae-LDH와의 반응으로 얻어진 DHB는 광학적으로 순수한 L-form 혹은 (S)-form isomer로 확인했다.DHB synthesized from HGBL showed two HPLC peaks at 9.9 and 11.3 min when the same chiral column used for the chirality analysis of lactate was used. lactate, L-form was found earlier than D-form, and DHB was also expected to show L-form first. The racemic DHB synthesized from HGBL was analyzed by colorimetric method, and L-form and D-form were obtained at a ratio of about 1: 1. The lactate assay kit was originally designed to analyze lactate, but the chirality of DHB was also analyzed because of the chemical similarity between DHB and lactate. DHB analysis of OHB products reacted with Ae-LDH was carried out simultaneously using chiral column and colorimetric assay kit. For HPLC analysis, only one peak was obtained and the retention time appeared at 9.9 min, which is estimated to be L-form. L-form was obtained in colorimetric analysis. Therefore, DHB obtained by reaction with Ae-LDH was confirmed as optically pure L-form or (S) -form isomer.

3-2. site-directed mutagenesis를 이용한 OHB L-reductase 효소의 변이체 개발3-2. Mutants of OHB L-reductase enzyme by site-directed mutagenesis

먼저 대장균 락트산탈수소효소의 크리스탈 구조(PDB ID: 2G8Y)를 주형으로 사용하여, 상동성 모델링을 통해 Ae_ldhA의 3차원 구조를 구축하였다. Ae_ldhA 모델은 MOE(Molecular Operating Environment)를 사용하여 제작했으며, PROCHECK 및 ProSA 온라인 구조 분석을 통해 평가하였다.PYMOL뷰어(http://www.pymol.org)를 사용하여 단백질 구조를 확인하였다 (도 14). First, the three - dimensional structure of Ae_ldhA was constructed by homology modeling using the crystal structure of E. coli lactate dehydrogenase (PDB ID: 2G8Y) as a template. The Ae_ldhA model was produced using MOE (Molecular Operating Environment) and evaluated through PROCHECK and ProSA online structure analysis. The protein structure was confirmed using the PYMOL viewer (http://www.pymol.org) (FIG. 14).

Ae_ldhA 변이체 디자인을 위해 피부르산과 HOB 구조(Pubchem Database)의 구조를 Ae_ldhA1의 효소 활성 위치에 Triangular Matching 방법을 사용하여 도킹 시뮬레이션(docking simulation)을 하였고, 이를 이용하여 효소의 아미노산 잔기와 피부르산 및 HOB와의 상호작용을 검토하였다(도 15).　 For the Ae_ldhA mutant design, docking simulation was performed by using the triangular matching method on the enzyme active site of Ae_ldhA1 and the structure of the leucine and HOB structure (Pubchem Database), and the amino acid residues of the enzyme, (Fig. 15).

도킹 시뮬레이션 결과를 바탕으로 Ile48 위치가 피부르산의 메틸 그룹 또는 HOB의 히드록시 에틸 그룹과의 상호작용에 중요한 역할을 할 수 있다고 가정하였고, 이를 친수성(Ser, Thr, Asn) 또는 전하를 띄는 아미노산(Asp, Lys)으로 치환하였다. HOB가 피부르산보다 크기 때문에 치환되는 아미노산을 동일 종류 중에 작은 잔기를 갖는 아미노산으로 한정하였다.Based on the results of the docking simulation, it was assumed that Ile48 position could play an important role in the interaction with the methyl group of hyaluronic acid or with the hydroxyethyl group of HOB. It was assumed that hydrophilic (Ser, Thr, Asn) Asp, Lys). Since the HOB is larger than the skin acidic acid, the substituted amino acid is limited to an amino acid having a small residue in the same kind.

Ile48 위치의 mutation은 site directed mutagenesis kit을 이용하여 수행하였다. 아래 표 11은 Ae-LdhA의 site directed mutagenesis 에 사용된 primer 서열을 보여준다. 변이된 Ae_ldhA을 발현하는 플라스미드(pET24ma-Ae-ldhO, 참고문헌: Zhang et al. “NADH-dependent lactate dehydrogenase from Alcaligenes eutrophus H16 reduces 2-oxoadipate to 2-hydroxyadipate” Biotechnology and Bioprocess Engineering, 19: 1048-1057 (2014))를 pET plasmid에 클로닝 한 후 서열을 확인(Macrogen, Seoul, Korea)하고 E. coli BL21(DE3)에 형질전환하여 발현하였다. 세포 파쇄액에 존재하는 효소는 Ni-NTA 레진을 사용하여 정제하고 10 kDa molecular cutoff membrane을 이용하여 염을 제거하였다. 이후 Ultra-15 30K centrifugal filter (Amicon, Merck Millipore Co., Darmstadt, Germany)로 다시 한 번 여과한 후 -80℃에 보관하였다.　 Mutations at Ile48 sites were performed using a site directed mutagenesis kit. Table 11 below shows the primer sequences used for site directed mutagenesis of Ae-LdhA. The plasmid expressing mutated Ae_ldhA (pET24ma-Ae-ldhO, refer to Zhang et al., "NADH-dependent lactate dehydrogenase from Alcaligenes eutrophus H16 reduces 2-oxoadipate to 2-hydroxyadipate" Biotechnology and Bioprocess Engineering, 19: 1048-1057 (2014)) was cloned into pET plasmid and sequenced (Macrogen, Seoul, Korea) and transformed into E. coli BL21 (DE3). The enzyme present in the cell lysate was purified using Ni-NTA resin and the salt was removed using a 10 kDa molecular cutoff membrane. After further filtration with an Ultra-15 30K centrifugal filter (Amicon, Merck Millipore Co., Darmstadt, Germany), the solution was stored at -80 ° C.

Primers　　　　　　　　　　 Primers 염기서열Base sequence 서열번호SEQ ID NO: LDH1_FPLDH1_FP ctatatcagccacggcctgtcgagtctgcccaactaccgcaccgccctcgctatatcagccacggcctgtcgagtctgcccaactaccgcaccgccctcg 156156 LDH1_RPLDH1_RP cgagggcggtgcggtagttgggcagactcgacaggccgtggctgatatagcgagggcggtgcggtagttgggcagactcgacaggccgtggctgatatag 157157 LDH2_FPLDH2_FP ctatatcagccacggcctgtcgactctgcccaactaccgcaccgccctcgctatatcagccacggcctgtcgactctgcccaactaccgcaccgccctcg 158158 LDH2_RPLDH2_RP cgagggcggtgcggtagttgggcagagtcgacaggccgtggctgatatagcgagggcggtgcggtagttgggcagagtcgacaggccgtggctgatatag 159159 LDH3_FPLDH3_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcgctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 160160 LDH3_RPLDH3_RP cgagggcggtgcggtagttgggcagattcgacaggccgtggctgatatagcgagggcggtgcggtagttgggcagattcgacaggccgtggctgatatag 161161 LDH4_FPLDH4_FP ctatatcagccacggcctgtcggacctgcccaactaccgcaccgccctcgctatatcagccacggcctgtcggacctgcccaactaccgcaccgccctcg 162162 LDH4_RPLDH4_RP cgagggcggtgcggtagttgggcaggtccgacaggccgtggctgatatagcgagggcggtgcggtagttgggcaggtccgacaggccgtggctgatatag 163163 LDH5_FPLDH5_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcgctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 164164 LDH5_RPLDH5_RP cgagggcggtgcggtagttgggcagattcgacaggccgtggctgatatagcgagggcggtgcggtagttgggcagattcgacaggccgtggctgatatag 165165

효소의 활성은 NADH의 산화 정도를 340nm의 흡광도를 관찰함으로 측정하였고, 효소의 활성(specific activity; 1U)은 1분 동안 1 μmol의 NADH를 NAD로 산화시키는데 필요한 효소의 양으로 정의하였다. 변이체 효소는 모두 pyruvate 와 OHB에 대하여 감소된 활성을 보여 주었다. I48K 효소는 pyruvate 에 대하여 5배 이상 감소된 활성을 보여 주었고 다른 효소들은 0.8-3배 감소한 활성을 보여 주었다 (도 16). OHB에 대한 활성도 대부분 감소하였다. 그러나 I48T (LDH2)의 경우 pyruvate에 대해서는 약 50% 정도의 활성이 감소하였으나 OHB에 대하여는 미미하지만 약간 증가된 활성을 보였다. 즉 pyruvate 대비 OHB에 대한 선택성이 2배 이상 증가한 것으로 나타났다. 향후 실험에서는 LDH2를 선택하였다.The activity of the enzyme was measured by measuring the absorbance of NADH at 340 nm and the specific activity (1 U) of the enzyme was defined as the amount of enzyme required to oxidize 1 μmol of NADH to NAD for 1 minute. All of the mutant enzymes showed reduced activity against pyruvate and OHB. The I48K enzyme showed a 5-fold reduction in activity against pyruvate and the other enzymes showed a 0.8-3-fold reduced activity (Fig. 16). Most of OHB activity decreased. However, in the case of I48T (LDH2), about 50% of the activity of pyruvate was decreased, but the activity of OHB was slight but slightly increased. That is, the selectivity to OHB compared to pyruvate was more than doubled. LDH2 was selected in future experiments.

실시예 4: 2-Oxo-4-hydroxy butyric acid (OHB)의 (2R)-D-reductase 효소의 스크리닝 및 변이효소 제작Example 4: Screening of 2R-D-reductase enzyme of 2-Oxo-4-hydroxy butyric acid (OHB) and preparation of mutant enzyme

4-1. OHB D-reductase 효소의 스크리닝4-1. Screening of OHB D-reductase enzyme

OHB D-reductase 효소의 확보Obtaining of OHB D-reductase enzyme

L-reductase의 경우를 참고하여 D-lactate dehydrogenase 를 대상으로 OHB 2R-reductase 효소를 스크리닝 하였다. E. coli, Pediococcus acidilactici, Pseudomonas aeruginosa, Leuconostoc mesenteroides cremoris, Lactobacillus bulgaricus, L. jensenii, Oenococcus oenii, L. plantarum, L. reuteri and L. casei 등으로부터 OHB에 대해 활성이 높을 것으로 예상되는 (D)-lactate dehydrogenase 효소들을 선정하였다 (표 12).In the case of L-reductase, OHB 2R-reductase enzyme was screened for D-lactate dehydrogenase. (D) - expected to be highly active against OHB from E. coli , Pediococcus acidilactici , Pseudomonas aeruginosa , Leuconostoc mesenteroides cremoris , Lactobacillus bulgaricus , L. jensenii , Oenococcus oenii , L. plantarum , L. reuteri and L. lactate dehydrogenase enzymes were selected (Table 12).

표 12에는 OHB D-reductase 효소 screening을 위한 (D)-lactate dehydrogenase 후보 효소들을 나타냈다.Table 12 shows (D) -lactate dehydrogenase candidate enzymes for OHB D-reductase enzyme screening.

GeneGene SourceSource Acc. no.Acc. no. Primer sequence (FP /RP)Primer sequence (FP / RP) 서열번호SEQ ID NO: D-lactate dehydrogenases (ldh)D-lactate dehydrogenases ( ldH ) Escherichia　　 coli K-12Escherichia coli K-12 NP 415898.1NP 415898.1 ATAACATATGAAACTCGCCGTTTATAGATAA CATATG AAACTCGCCGTTTATAG 166166 TAATAGGATCCTTAGTGGTGATGATGGTGATGAACCAGTTCGTTCGGGCAGTAATA GGATCC TTAGTGGTGATGATGGTGATGAACCAGTTCGTTCGGGCAG 167167 D-lactate dehydrogenases D-lactate dehydrogenases Lactobacillus　　 helveticusLactobacillus helveticus WP 003628108.1 1WP 003628108.1 1 ATAACATATGACAAAGGTTTTTGCATAA CATATG ACAAAGGTTTTTGC 168168 TAATAGGATCCTCAGTGGTGATGATGGTGATGAAACTTGTTCTTGTTCAAAGCAACTAATA GGATCC TCAGTGGTGATGATGGTGATGAAACTTGTTCTTGTTCAAAGCAAC 169169 D-lactate/D-glycerate dehydrogenase (ldhD)D-lactate / D-glycerate dehydrogenase ( ldhD ) Pediococcus　　 acidilacticiPediococcus acidilactici X70925.1X70925.1 ATAACATATGAAGATTATTGCTTATGGATAA CATATG AAGATTATTGCTTATGG 170170 TAATAGGATCCTCAGTGGTGATGATGGTGATGCTCAAACTTAACTTCATTCTTTGTAATA GGATCC TCAGTGGTGATGATGGTGATGCTCAAACTTAACTTCATTCTTTG 171171 D-lactate dehydrogenases (ldhD)D-lactate dehydrogenases ( ldhD ) Pseudomonas　　 aeruginosaPseudomonas aeruginosa NP_249618.1NP_249618.1 ATAACATATGCGCATCCTGTTCTTCAGATAACATATGCGCATCCTGTTCTTCAG 172172 TAATAGGATCCCTAGTGGTGATGATGGTGATGGGCCCGGACCCGATTGCGTAATA GGATCC CTAGTGGTGATGATGGTGATGGGCCCGGACCCGATTGCG 173173 D-lactate dehydrogenases (ldhD)D-lactate dehydrogenases ( ldhD ) Clostridium　　 perfringens 13Clostridium perfringens 13 NP_561446.1NP_561446.1 ATAACATATGATGATAAAATTAGTATGTTATATAA CATATG ATGATAAAATTAGTATGTTAT 174174 TAATAGGATCCTTAGTGGTGATGATGGTGATGATGATGCTATTGCATTTTTACAAGTATTTCTAATA GGATCC TTAGTGGTGATGATGGTGATGATGATGCTATTGCATTTTTACAAGTATTTC 175175 D-lactate dehydrogenases (ldhD)D-lactate dehydrogenases ( ldhD ) Lactobacillus　　 reuteriLactobacillus reuteri ZP_03974385.1ZP_03974385.1 ATAACATATGAAAGGAATGGGAAAACATAA CATATG AAAGGAATGGGAAAAC 176176 TAATAGGATCCTCAGTGGTGATGATGGTGATGCATTCTTATTTCATTTCGTGTAATA GGATCC TCAGTGGTGATGATGGTGATGCATTCTTATTTCATTTCGTG 177177 D-lactate dehydrogenases (ldhD)D-lactate dehydrogenases ( ldhD ) Leuconostoc　　 mesenteroides cremorisLeuconostoc mesenteroides cremoris ZP_03913173.1ZP_03913173.1 ATAACATATGAAGATTTTTGCTTACGATAA CATATG AAGATTTTTGCTTACG 178178 TAATAGGATCCTTAGTGGTGATGATGGTGATGATATTCAACAGCAATAGCTGGTAATA GGATCC TTAGTGGTGATGATGGTGATGATATTCAACAGCAATAGCTGG 179179 D-lactate dehydrogenases (ldhD)D-lactate dehydrogenases ( ldhD ) Oenococcus　　 oeniOenococcus oeni ZP_01544962.1ZP_01544962.1 ATAACATATGAAAATTTATGCTTATGATAA CATATG AAAATTTATGCTTATG 180180 TAATAGGATCCTTAGTGGTGATGATGGTGATGGAATTTAACGAGATTCTTGTCTCTAATA GGATCC TTAGTGGTGATGATGGTGATGGAATTTAACGAGATTCTTGTCTC 181181 D-lactate dehydrogenases (ldhD)D-lactate dehydrogenases ( ldhD ) Lactobacillus　　 delbrueckii subsp. bulgaricusLactobacillus delbrueckii subsp. bulgaricus CAI96942.1CAI96942.1 ATAACATATGACTAAAATTTTTGCTTAC ATAA CATATGACTAAAATTTTTGCTTAC 182182 TAATAGGATCCTTAGTGGTGATGATGGTGATGGAAACTCCAGTTAAGGTTGGCTAATA GGATCC TTAGTGGTGATGATGGTGATGGAAACTCCAGTTAAGGTTGGC 183183

OHB D-reductase 효소의 생산 및 활성 측정Production and activity measurement of OHB D-reductase enzyme

　L-LDH의 경우와 마찬가지로 효소발현을 위해 E. coli BL21(DE3) star 균주를 숙주균주로 이용하였고 클로닝과 플라스미드 보존을 위해서는 E. coli DH5α 균주를 숙주로 이용하였다. kanamycin 50 ug/mL이 포함된 LB medium을 일반적인 배양과 클로닝 후 재조합 균주 배양에 사용하였다. 유전자 발현 및 효소 생산을 위해 pET 플라스미드와 T7 프로모터를 사용하였다. Ldh 유전자는 아래 표 10에 있는 primer를 사용하여 PCR 증폭하였고 이 때 C 말단에 His-tag을 붙여 주었다. PCR fragment는 pET vector에 넣은 후 E. coli DH5a 균주에 클로닝하고 유전자 서열을 확인한 후 E. coli BL21(DE3) star에 도입하였다. Ldh 유전자의 soluble expression을 촉진하기 위해 Chaperon 을 발현하는 pG-Tf2 플라스미드를 추가로 도입해 주었다.　 As in the case of L-LDH, E. coli BL21 (DE3) star strain was used as a host strain for enzyme expression and E. coli DH5α strain was used as a host for cloning and plasmid preservation. LB medium containing kanamycin 50 ug / mL was used for culture of recombinant strains after normal culture and cloning. PET plasmid and T7 promoter were used for gene expression and enzyme production. The Ldh gene was amplified by PCR using primers shown in Table 10 below, and a His-tag was attached to the C-terminus. The PCR fragment was inserted into a pET vector, cloned into E. coli strain DH5a, and sequenced into E. coli BL21 (DE3) star. In order to promote the soluble expression of the Ldh gene, the pG-Tf2 plasmid expressing Chaperon was further introduced.

D-LDH 효소 생산을 위해 유전자 재조합 BL21(pET-Xx-LDH) 균주를 50 μg/mL kanamycin을 포함하는 LB 배지에서 호기적으로 배양하였다. 배양온도는 30ºC, 교반속도는 100 rpm 이었고 1 L 플라스크에 250 mL 배지를 넣어 주었다. 세포농도가 0.5 OD에 도달했을 때, 0.1 mM의 IPTG를 첨가하고 추가적으로 10시간 배양하였다. 이 후 세포를 원심분리하고 25 mM 인산 완충용액 (pH 7.0)으로 3번 세척한 후 binding buffer (20 mM pH 7.0 인산 완충용액, 0.5 M NaCl, 10 mM imidazole)에 현탁한 후 sonicator를 이용하여 파쇄하였다. 세포 파쇄액은 4℃, 25,000 g 에서 30분 원심분리하여 비용해성 부분을 제거하고 용해성 부분을 모아 효소 활성 분석이나 추가적인 효소분리에 이용하였다. For the production of D-LDH enzyme, recombinant BL21 (pET-Xx-LDH) strain was aerobically cultured in LB medium containing 50 μg / mL kanamycin. The incubation temperature was 30 º C, stirring speed was 100 rpm, and 250 mL medium was added to a 1 L flask. When the cell concentration reached 0.5 OD, 0.1 mM IPTG was added and incubated for an additional 10 hours. The cells were then centrifuged, washed three times with 25 mM phosphate buffer (pH 7.0), suspended in binding buffer (20 mM pH 7.0 phosphate buffer, 0.5 M NaCl, 10 mM imidazole) and sonicated Respectively. The cell lysate was centrifuged at 25,000 g for 30 min at 4 ° C to remove the insoluble portion, and the soluble portion was collected and used for enzyme activity analysis or additional enzyme separation.

D-LDH 효소활성은 L-LDH의 경우와 동일한 방법으로 측정하였다. pyruvate 와 OHB를 기질로 사용하여 분석하였다. L-LDH와 마찬가지로 대부분의 효소들은 pyruvate에 3-10배 높은 활성을 가지고 있었고 그에 비해 OHB에는 낮은 활성을 보였다 (도 17). L-LDH와 비교할 때 D-LDH의 활성은 전반적으로 3-5배 낮았다. Lb-LDH 및 Lp-LDH 가 가장 높은 2.2 μmol/mg protein/min 의 활성을 보였다. OHB에 대해서는 Lb-LDH가 0.8 μmol/mg protein/min의 가장 높은 활성을 보였다. pyruvate 와 OHB에 대한 선택도는 약 1:0.2 수준이었다. Lb-LDH를 선택하여 OHB에 높은 활성을 갖는 D-reductase 변이 효소를 제작하였다. D-LDH enzyme activity was measured in the same manner as in the case of L-LDH. pyruvate and OHB as substrates. Like L-LDH, most of the enzymes had 3-10-fold higher activity on pyruvate than on OHB (Fig. 17). The activity of D-LDH was generally 3-5 times lower than that of L-LDH. Lb-LDH and Lp-LDH showed the highest activities of 2.2 μmol / mg protein / min. OHB, Lb-LDH showed the highest activity of 0.8 μmol / mg protein / min. The selectivity for pyruvate and OHB was about 1: 0.2. L-LDH was selected to produce D-reductase mutant enzyme with high activity in OHB.

OHB D-reductase 효소의 생성물의 광학활성 측정Measurement of optical activity of OHB D-reductase enzyme product

Lb-LDH가 생성하는 DHB 2번 위치 hydroxy 기가 (R) 혹은 (S) 중 어느 것인지 알아보기 위해 광학활성을 갖는 HPLC와 colorimetric 분석 kit을 사용하여 분석하였다. Lb-LDH와 반응시킨 OHB 생성물의 HPLC 분석의 경우 하나의 peak만 얻어졌고 retention time은 D-form으로 추정되는 11.3분 시간대에 나타났다. 또한 colorimetric 분석에서도 D-form 만 얻어졌다. 따라서 Lb-LDH와의 반응으로 얻어진 DHB는 광학적으로 순수한 D-form 혹은 (R)-form isomer로 확인하였다.To investigate whether the hydroxy group at position 2 of DHB produced by Lb-LDH is either (R) or (S), it was analyzed using optically active HPLC and colorimetric analysis kit. HPLC analysis of the OHB product reacted with Lb-LDH showed only one peak, and the retention time was 11.3 minutes at the estimated D-form. D-form was obtained in colorimetric analysis. Therefore, the DHB obtained by the reaction with Lb-LDH was confirmed to be optically pure D-form or (R) -form isomer.

4-2. site-directed mutagenesis를 이용한 OHB D-reductase 효소의 변이체 개발4-2. Site-directed mutagenesis to develop OHB D-reductase enzyme variants

PDB databank에 나와있는 Lb-LDH 효소의 결정구조를 이용하여 engineering 해야 할 아미노산 잔기를 확인하였다 (도 18). 즉 피루브산과의 docking을 통해 His296, Arg235, Glu264 등 3개의 잔기가 피루브산과의 반응 활성부위에 존재하고 추가적으로 두 개의 hydrophobic 아미노산 잔기인 Val78과 Tyr101이 활성부위 주위에 존재하는 것을 확인하였다. 이러한 hydrophobic 잔기들은 OHB와의 반응에 방해요인으로 작용할 가능성이 크므로 이들을 (크기가 작은) hydrophilic 아미노 잔기 (Ser, Thr, Asn, Asp, Lys)로 바꾸어 주었다.　　 The amino acid residues to be engineered were identified using the crystal structure of the Lb-LDH enzyme in the PDB databank (FIG. 18). That is, through docking with pyruvic acid, three residues such as His296, Arg235 and Glu264 exist in the reactive site with pyruvic acid, and additionally two hydrophobic amino acid residues Val78 and Tyr101 exist around the active site. These hydrophobic residues are likely to interfere with the reaction with OHB, so they have been replaced with hydrophilic amino residues (Ser, Thr, Asn, Asp, Lys).

Mutation은 site directed mutagenesis kit을 이용하여 수행하였다. 표 13는 Lb-LDH의 site-directed mutagenesis에 사용한 primer 염기서열을 나타낸다. 변이된 Lb-LDH를 발현하는 플라스미드를 pET plasmid에 클로닝한 후 서열을 확인(Macrogen, Seoul, Korea)하고 E. coli BL21(DE3)에 형질전환하여 발현하였다. 세포 파쇄액에 존재하는 효소는 Ni-NTA 레진을 사용하여 정제하고 10 kDa molecular cutoff membrane을 이용하여 염을 제거하였다. 이후 Ultra-15 30K centrifugal filter (Amicon, Merck Millipore Co., Darmstadt, Germany)로 다시 한 번 여과한 후 -80℃에 보관하였다.　 Mutation was performed using a site directed mutagenesis kit. Table 13 shows the primer sequences used for the site-directed mutagenesis of Lb-LDH. The plasmid expressing the mutated Lb-LDH was cloned into the pET plasmid and sequenced (Macrogen, Seoul, Korea) and transformed into E. coli BL21 (DE3). The enzyme present in the cell lysate was purified using Ni-NTA resin and the salt was removed using a 10 kDa molecular cutoff membrane. After further filtration with an Ultra-15 30K centrifugal filter (Amicon, Merck Millipore Co., Darmstadt, Germany), the solution was stored at -80 ° C.

아래 표 13에는 Lb-LDH의 site-directed mutagenesis에 사용한 primer 염기서열을 나타냈다.Table 13 below shows the primer sequences used for the site-directed mutagenesis of Lb-LDH.

Primers　Primers 염기서열Base sequence 서열번호SEQ ID NO: LDH6_FPLDH6_FP catcactaagatgagcctgcgtaactccggtgttgacaacatcgacatggcta catcactaagatgagcctgcgtaactccggtgttgacaacatcgacatggcta 184184 LDH6_RPLDH6_RP tagccatgtcgatgttgtcaacaccggagttacgcaggctcatcttagtgatgtagccatgtcgatgttgtcaacaccggagttacgcaggctcatcttagtgatg 185185 LDH7_FPLDH7_FP catcactaagatgagcctgcgtaacaacggtgttgacaacatcgacatggctacatcactaagatgagcctgcgtaacaacggtgttgacaacatcgacatggcta 186186 LDH7_RPLDH7_RP tagccatgtcgatgttgtcaacaccgttgttacgcaggctcatcttagtgatgtagccatgtcgatgttgtcaacaccgttgttacgcaggctcatcttagtgatg 187187 LDH8_FPLDH8_FP catcactaagatgagcctgcgtaacaccggtgttgacaacatcgacatggctacatcactaagatgagcctgcgtaacaccggtgttgacaacatcgacatggcta 188188 LDH8_RPLDH8_RP tagccatgtcgatgttgtcaacaccggtgttacgcaggctcatcttagtgatgtagccatgtcgatgttgtcaacaccggtgttacgcaggctcatcttagtgatg 189189 LDH9_FPLDH9_FP catcactaagatgagcctgcgtaacgacggtgttgacaacatcgacatggcta　　 catcactaagatgagcctgcgtaacgacggtgttgacaacatcgacatggcta 190190 LDH9_RPLDH9_RP tagccatgtcgatgttgtcaacaccgtcgttacgcaggctcatcttagtgatgtagccatgtcgatgttgtcaacaccgtcgttacgcaggctcatcttagtgatg 191191 LDH10_FPLDH10_FP catcactaagatgagcctgcgtaacaagggtgttgacaacatcgacatggcta　catcactaagatgagcctgcgtaacaagggtgttgacaacatcgacatggcta 192192 LDH10_RPLDH10_RP tagccatgtcgatgttgtcaacacccttgttacgcaggctcatcttagtgatg　tagccatgtcgatgttgtcaacacccttgttacgcaggctcatcttagtgatg 193193

효소의 활성은 NADH의 산화 정도를 340nm의 흡광도를 관찰해 측정하였고, 효소의 활성(specific activity; 1U)은 1분 동안 1 μmol의 NADH를 NAD+로 산화시키는데 필요한 효소의 양으로 정의하였다. 변이체 효소는 모두 pyruvate 와 OHB에 대하여 감소된 활성을 나타냈다. 이들 중 V78D (LDH-9)는 pyruvate에 대하여 거의 4배 가량 감소한 활성을 보여주었다. 가장 우수한 효소는 V78T (LDH-8) 로 나타났으며, pyruvate에 대해서는 약 45% 활성이 감소한 반면 HOB에 대해서는 약 10% 활성이 증가한 것으로 나타났다 (도 19). 향후 실험에서는 OHB D-reductase 효소로 V78T (LDH-8)를 선택하였다.　The enzyme activity was determined by measuring the degree of oxidation of NADH by observing the absorbance at 340 nm and the specific activity (1 U) of the enzyme was defined as the amount of enzyme required to oxidize 1 μmol of NADH to NAD + for 1 minute. All of the mutant enzymes showed reduced activity against pyruvate and OHB. Of these, V78D (LDH-9) showed almost four-fold reduced activity against pyruvate. The most excellent enzyme was V78T (LDH-8), with about 45% activity reduced for pyruvate while about 10% increased for HOB (FIG. 19). In future experiments, V78T (LDH-8) was selected as the OHB D-reductase enzyme.

실시예 5: DHB (2,4-dihydroxybutyric acid) 생산용 균주의 제작Example 5: Preparation of strains for producing DHB (2,4-dihydroxybutyric acid)

DHB의 최적 생산을 위해서는 트랜스아미네이즈 및 OHB reductase가 최적발현되어야 한다. 또한 TA활성의 증가에는 TA 효소 자체의 발현은 물론 TA 반응의 cofactor인 pyridoxal-5-phosphate (PLP), 즉 비타민 B6 생합성이 증대 되어야 한다. 더 나아가 DHB의 세포외 이송속도가 증가되어야 한다. 이를 위해 앞서 실시예 1에서 homoserine 생산을 위해 제작된 균주를 추가적으로 변형시켜 DHB 생산 균주를 제작하였다.For optimal production of DHB, transaminase and OHB reductase should be optimally expressed. In addition, an increase in TA activity requires an increase in the biosynthesis of pyridoxal-5-phosphate (PLP), the cofactor of the TA response, as well as the expression of the TA enzyme itself. Furthermore, the extracellular delivery rate of DHB should be increased. For this, DHB production strains were prepared by further modifying strains prepared for homoserine production in Example 1 above.

5-1. Vitamin B6 생산 증가5-1. Increased Vitamin B6 production

대장균의 경우 PLP는 DXP 의존 경로를 통해 생합성되며, 이 경우 PLP생합성 속도는 epd, dxs, pdxJ 유전자 등이 코딩하는 단백질에 의해 조절된다고 알려져 있다(도 20). PLP 생합성 속도를 향상시키기 위하여 이 세 유전자의 프로모터를 합성 프로모터 5 (표 5)로 치환하였으며, Pop-in pop-out 방법을 사용하였다. 사용한 primer를 아래 표 14에 나타냈다. 표 14은 비타민 B6 생합성을 증가시키기 위하여 epd, dxs, pdxJ 유전자의 발현을 증대시키기 위해 사용된 프라이머 염기서열을 나타낸다.In the case of Escherichia coli, PLP is biosynthesized through a DXP-dependent pathway. In this case, it is known that the rate of PLP biosynthesis is regulated by proteins encoded by epd, dxs, pdxJ gene, etc. (Fig. In order to improve the rate of PLP biosynthesis, the promoters of these three genes were replaced with synthetic promoter 5 (Table 5) and pop-in pop-out method was used. The primers used are shown in Table 14 below. Table 14 shows the primer sequences used to increase the expression of epd, dxs, and pdxJ genes to increase vitamin B6 biosynthesis.

결과적으로 PLP 생합성이 강화된 3개의 균주를 얻을 수 있었다(표 15).As a result, three strains with enhanced PLP biosynthesis were obtained (Table 15).

PrimersPrimers 염기서열Base sequence 서열번호SEQ ID NO: US-Psp5-epd-FPUS-Psp5-EPd-FP atctaagcggccgcggaattatgcaattcgtggtacatctaagcggccgcggaattatgcaattcgtggtac 194194 US-Psp5-epd-RPUS-Psp5-EPd-RP aaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatgaccgtacgcgtagcgataaatgaaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatgaccgtacgcgtagcgataaatg 195195 Psp5-epd-FPPsp5-epd-FP gccagtttagtatcgaccgccagtttagtatcgacc 196196 Psp5-epd-RPPsp5-epd-RP acaaaagcatgatcctgttgaagatgcgacaaaagcatgatcctgttgaagatgcg 197197 DS- Psp5-epd-FPDS- Psp5-epd-FP catttatcgctacgcgtacggtcattccacacttataggtacaaaaagatgcgaaagcaaataaatttttttccatttatcgctacgcgtacggtcattccacacttataggtacaaaaagatgcgaaagcaaataaatttttttc 198198 DS- Psp5-epd-RPDS- Psp5-epd-RP tagtaatctagaggagttggcatctttctgcgatttctagtaatctagaggagttggcatctctttctgcgatttc 199199 US-Psp5-dxs-FPUS-Psp5-dxs-FP atctaagcggccgctcgacatttcattgtcgttgagatctaagcggccgctcgacatttcattgtcgttgag 200200 US-Psp5-dxs-RPUS-Psp5-dxs-RP caaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatgagttttgatattgccaaataccaaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatgagttttgatattgccaaatac 201201 Psp5-dsx-FPPsp5-dsx-FP gtaaagcttaccggaaagcagctgt　gtaaagcttaccggaaagcagctgt 202202 Psp5-dsx-RPPsp5-dsx-RP agcaactcgaagcctgcgttaagagcaactcgaagcctgcgttaag 203203 DS- Psp5-dxs-FPDS- Psp5-dxs-FP tccacacttataggtacaaaaagatgcgaaagcaaataaattttttcggcaccctggcgttttcccaacgttgcagtccacacttataggtacaaaaagatgcgaaagcaaataaattttttcggcaccctggcgttttcccaacgttgcag 204204 DS- Psp5-dsx-RPDS- Psp5-dsx-RP tagtaatctagaggtcatatgttcggcgttagcacaaactagtaatctagaggtcatatgttcggcgttagcacaaac 205205 US-Psp5-pdxj-FPUS-Psp5-PDxJ-FP atctaagcggccgcggcttgcggcaaaggtcgatctaagcggccgcggcttgcggcaaaggtcg 206206 US-Psp5-pdxj-RPUS-Psp5-PDxJ-RP aaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatggctgaattactgttaggcgtcaaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatggctgaattactgttaggcgtc 207207 Psp5- pdxj-FPPsp5- pdxj-FP ctttcacgttgtgataggtcagctttcacgttgtgataggtcag 208208 Psp5- pdxj-RPPsp5-pdxj-RP aaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatggctgaattactgttaggcgtcaaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatggctgaattactgttaggcgtc 209209 DS-Psp5-pdxj-FPDS-Psp5-PDXj-FP gacgcctaacagtaattcagccattccacacttataggtacaaaaagatgcgaaagcgacgcctaacagtaattcagccattccacacttataggtacaaaaagatgcgaaagc 210210 DS-Psp5-pdxj-RPDS-Psp5-PDxj-RP cgatctttaagagtaactccgatgcgatctttaagagtaactccgatg 211211

균주명　Strain name 특성characteristic 기타Other EcW13EcW13 pts eda lacI thrB metA lysA adhE pflB ldhA iclR; replacing acs native promoter by Ptrc promoter; replacing ppc native promoter by synthetic promoter 8; pUCPK-Ptrc-metL If pts is the thrB metadata, then the adhE pflB ldhA iclR; replacing acs native promoter by P trc promoter; replacing ppc native promoter by synthetic promoter 8; pUCPK-P trc - metL This studyThis study EcW14EcW14 Ec13, ΔP_epd::P_SP5 Ec13, [Delta] P _epd :: P _SP5 This studyThis study EcW15EcW15 Ec14, ΔP_dxs::P_SP5 Ec14,? P _dxs :: P _SP5 This studyThis study EcW16EcW16 Ec15, ΔP_pdxJ::P_SP5 Ec15, [Delta] _PpdxJ :: P _SP5 This studyThis study

상기 EcW13 균주는 2018년 06월 22일자로 대한민국 서울시 서대문구에 소재하는 한국미생물보존센터에 기탁하여 기탁번호 KCCM12281P를 수여받았으며, 상기 EcW20 균주는 2018년 06월 22일자로 한국미생물보존센터에 기탁하여 기탁번호 KCCM12282P를 수여받았습니다.The EcW13 strain was deposited on June 22, 2018 with the deposit number KCCM12281P deposited at the Korean Microorganism Conservation Center in Seodaemun-gu, Seoul, Korea. The EcW20 strain was deposited with the Korean Center for Microorganism Preservation on June 22, 2018, I received the number KCCM12282P.

5-2. DHB 세포막 이송 단백질의 발현 조절5-2. Regulation of Expression of DHB Membrane Transfer Protein

DHB의 세포외 이송속도를 증대시키기 위하여 이송단백질 후보를 먼저 스크리닝(screening) 하였다. 알려진 유기산 막 이송 단백질, 특히 lactic acid 와 저분자량의 carboxylic acid 이송 단백질을 query로 사용하여 importer 및 exporter를 선정하였다. 표 16에는 2,4-DHB exporter 후보로 선정된 단백질 목록을 나타냈으며, 표 17에는 2,4-DHB importer 후보로 선정된 단백질 목록을 나타냈다.To increase the extracellular delivery rate of DHB, the transferred protein candidates were first screened. The known organic acid membrane transfer proteins, especially lactic acid and low molecular weight carboxylic acid transfer protein, were used as query to select importer and exporter. Table 16 lists selected proteins as candidates for the 2,4-DHB exporter, and Table 17 lists the proteins selected as candidates for the 2,4-DHB importer.

Protein　　 NamesProtein Names GeneGene MFS type transporterMFS type transporter YhjXYhjX Probable formate transporter　Probable formate transporter focAfocA Lactate premease　Lactate premease lldplldp Inner membrane metabolite transporterInner membrane metabolite transporter yhjEyhjE C4-dicarboxylate transporterC4-dicarboxylate transporter dcuAdcuA C4-dicarboxylate transporterC4-dicarboxylate transporter dcuBdCuB C4-dicarboxylate transporterC4-dicarboxylate transporter dcuCdcuC

Protein　　 NamesProtein Names GeneGene Alpha-ketoglutarate　　 permeaseAlpha-ketoglutarate permease kgtPkgtP Glycolate　　 permeaseGlycolate permease glcAglcA Acetate　　 symporter/ inner membrane proteinAcetate symporter / inner membrane protein actPactP Proline　　 symporterProline symporter putPputP Pantothenate　　 synmportePantothenate synmporte panFpanF Succinate　　 and acetate symporterSuccinate and acetate symporter satPsatP Short　　 chain fatty acid transporterShort chain fatty acid transporter atoEatoE DSdX　　 permeaseDSdX permease dsdXdsdX UncharacterizedUncharacterized yhbMyhbM

이들 중 가장 가능성이 높은 2개의 exporter, 즉 ldp, dcuA의 발현양을 합성 promoter를 이용하여 높여 주었다. 막 단백질의 과량 발현은 세포성장에 방해가 되므로 중간 세기의 합성 프로모터인 SP5를 사용하였다. 또한 importer 로 가장 가능성이 높을 것으로 예상되는 3개의 막 단백질, kgtP, dsdx, actP을 제거했다. Transporter engineering 에 사용한 primer 서열을 표 18에 나타내었다.The expression levels of the two most promising exporters, ldp and dcuA, were elevated using synthetic promoters. Since the overexpression of membrane protein interferes with cell growth, we used SP5, a medium - intensity synthetic promoter. We also removed the three most promising membrane proteins, kgtP, dsdx, and actP as the importer . The primer sequences used in the transporter engineering are shown in Table 18.

Primer (과발현)Primer (overexpression) 　염기서열Base sequence 서열번호SEQ ID NO: US-Psp4-lldp-FPUS-Psp4-lldp-FP atctaagcggccgcgattggaatgcccatcgcacatctaagcggccgcgattggaatgcccatcgcac 212212 US-Psp4-lldp-RPUS-Psp4-lLDP-RP ttgacaattaatcatccggctcgtaatttatgtggaatgaatctctggcaacaaaactacgttgacaattaatcatccggctcgtaatttatgtggaatgaatctctggcaacaaaactacg 213213 Psp5-lldp-FPPsp5-lldp-FP ccatctgaccaatctcaaagctgtccatctgaccaatctcaaagctgt 214214 Psp5-lldp-RPPsp5-lLDP-RP ttgtgaacaaagcacctggtcgcgttgtgaacaaagcacctggtcgcg 215215 DS- Psp4-lldp-FPDS- Psp4-lldp-FP tccacataaattacgagccggatgattaattgtcaattggtagggccaattcttgtgtccacataaattacgagccggatgattaattgtcaattggtagggccaattcttgtg 216216 DS- Psp4-lldp-RPDS-Psp4-lldp-RP tagtaatctagagaaagaagtcgaaaaaaacgaaatctagtaatctagagaaagaagtcgaaaaaaacgaaatc 217217 US-Psp4-ducA-FPUS-Psp4-ducA-FP atctaagcggccgcgttgatgattgcatagataaccatctaagcggccgcgttgatgattgcatagataacc 218218 US- Psp4-ducA -RPUS-Psp4-ducA-RP ttgacaattaatcatccggctcgtaatttatgtggaatgcaggttgctggcggtctggactatctggttcttgacaattaatcatccggctcgtaatttatgtggaatgcaggttgctggcggtctggactatctggttc 219219 Psp4-ducA-FPPsp4-ducA-FP ccatctgaccaatctcaaagctgtccatctgaccaatctcaaagctgt 220220 Psp4-ducA-RPPsp4-ducA-RP ttgtgaacaaagcacctggtcgcgttgtgaacaaagcacctggtcgcg 221221 DS-Psp4-ducA -FPDS-Psp4-ducA-FP tccacataaattacgagccggatgattaattgtcaatgttttttaacaagttgatattagattgtccacataaattacgagccggatgattaattgtcaatgttttttaacaagttgatattagattg 222222 DS-Psp4-ducA -RPDS-Psp4-ducA -RP tagtaatctagagaaagaagtcgaaaaaaacgaaatctagtaatctagagaaagaagtcgaaaaaaacgaaatc 223223 　 Primers (제거)Primers (removal) 염기서열Base sequence US-dsdx-FPUS-dsdx-FP atctaagcggccgcggtatccagcgacgattttagcgatctaagcggccgcggtatccagcgacgattttagcg 224224 US-dsdx-RPUS-DSDx-RP gtacgtctctttgcgcttacatatctcaccttcccctg
　gtacgtctctttgcgcttacatatctcaccttcccctg
225225 G-dsdx -FPG-dsdx -FP gcgtgtaaagtcacctaatgcgcgtgtaaagtcacctaatgc 226226 G-dsdx -RPG-dsdx -RP gggcatcagcagacatatggggcatcagcagacatatg 227227 DS-dsdx-FPDS-dsdx-FP caggggaaggtgagatatgtaagcgcaaagagacgtaccaggggaaggtgagatatgtaagcgcaaagagacgtac 228228 DS- dsdx-RPDS-dsdx-RP tagtaatctagacgctcataatgccgattgataactagtaatctagacgctcataatgccgattgataac 229229 US-kgtP-FPUS-kgtP-FP atctaagcggccgcggagctgatccgcgagcatacatctaagcggccgcggagctgatccgcgagcatac 230230 US- kgtP -RPUS- kgtP -RP acggcaggagacataatggcatgtagtgacgggtcagttgccagacacggcaggagacataatggcatgtagtgacgggtcagttgccagac 231231 G- kgtP -FPG-kgtP-FP tggcgtctggatctggaaaacgtggcgtctggatctggaaaacg 232232 G- kgtP -RPG-kgtP-RP tgtgtaagcgcagcgatgctgtgtaagcgcagcgatgc 233233 DS- kgtP -FPDS-kgtP-FP gtctggcaactgacccgtcactacatgccattatgtctcctgccgtgtctggcaactgacccgtcactacatgccattatgtctcctgccgt 234234 DS- kgtP-RPDS-kgtP-RP ta tagtaatctagagaagctgttttggcggatgata tagtaatctagagaagctgttttggcggatga 235235 US-actP-FPUS-actP-FP atctaagcggccgccttatctttattaaggtaaacatctaagcggccgccttatctttattaaggtaaac 236236 US- actP -RPUS-actP-RP agtcctgcatgaggtacaagcatcatgtaatctctccccttccccggtcgtctgagtcctgcatgaggtacaagcatcatgtaatctctccccttccccggtcgtctg 237237 G- actP -FPG-actP -FP ctgtgggatttcgatagtatctgtgggatttcgatagtat 238238 G- actP -RPG-actP-RP agcaacctgggcgatacctcgacagcaacctgggcgatacctcgac 239239 DS- actP -FPDS-actP -FP cagacgaccggggaaggggagagattacatgatgcttgtacctcatgcaggactcagacgaccggggaaggggagagattacatgatgcttgtacctcatgcaggact 240240 DS- actP-RPDS-actP-RP tagtaatctagaaagctgcttgaagagaagcagtagtaatctagaaagctgcttgaagagaagcag 241241

최종적으로 transporter와 PLP 생합성 경로가 변화된 DHB 생산 숙주 균주 대장균이 아래 표 19과 같이 얻어졌다.Finally, E. coli DHB-producing host strains with altered transporter and PLP biosynthetic pathway were obtained as shown in Table 19 below.

균주명Strain name 특성characteristic 기타Other EcW16EcW16 pts eda lacI thrB metA lysA adhE pflB ldhA iclR; replacing acs native promoter by Ptrc promoter;replacing ppc native promoter by synthetic promoter 8; pUCPK-Ptrc-metL;ΔP_epd::P_SP9ΔP_dxs::P_SPΔP_pdxJ::P_SP9 If pts is the thrB metadata, then the adhE pflB ldhA iclR; replacing acs native promoter by P trc promoter; replacing ppc native promoter by synthetic promoter 8; pUCPK-P trc - metL; ΔP _epd :: P _SP9 ΔP _dxs :: P _SP ΔP _pdxJ :: P _SP9 homoserine 경로, PLP 생합성 경로homoserine pathway, PLP biosynthetic pathway EcW17EcW17 EcW16, ΔP_ldp::P_SP5 EcW16, [Delta] P _ldp :: P _SP5 exporter 발현expression of exporter EcW18EcW18 EcW16, ΔP_dcuA::P_SP5 EcW16,? P _dcuA :: P _SP5 exporter 발현expression of exporter EcW19EcW19 EcW16, ΔP_ldp::P_SP5;ΔP_dcuA::P_SP5 _{_{EcW16, ΔP ldp :: P SP5;}} ΔP dcuA :: P SP5 exporter 발현expression of exporter EcW20EcW20 EcW19, ΔkgtP, Δdsdx and ΔactP EcW19,? KgtP ,? Dsdx and? ActP Importer 제거Remove Importer

실시예Example 6: 광학적으로 순수한 S- 6: Optically pure S- form 또는form or L-form 2,4-디히드록시 부티르산 (2,4- L-form 2,4-dihydroxybutyric acid (2,4- dihydroxybutyricdihydroxybutyric acid, S- 혹은 R- acid, S- or R- DHBDHB ) ) 생산균주의Production strain 제작 및 발효 Production and fermentation

6-1. DHB의 생산을 위한 유전자 재조합 플라스미드 제작6-1. Genetically recombinant plasmids for production of DHB

pET-TA4-1과 LDH를 발현하는 플라스미드 (pET-LDHA2 또는 pET-LDHD3)를　이용하여 TA4-1 효소와 OHB reductase 효소를 동시에 발현하는 유전자 재조합 플라스미드를 제작하였다. 각각의 유전자 절편을 PCR 증폭한 후 overlap 하고 pBAD plasmid의 NdeI 및 EcoRI site에 ligation 해 주었다. 두 개의 새로운 플라스미드, 즉 L-form DHB를 위한 pBAD_TA4-1_LDHA2 (이후 pDHB-L로 명명) 플라스미드와 D-form DHB를 위한 pBAD_TA4-1_LDHD3 (이후 pDHB-D로 명명) 플라스미드를 얻었다. 이 두 플라스미드에서는 TA4-1과 OHB reductase 유전자가 동시에 polycistronic 하게 전사되도록 아라비노우즈에 의해 유도되는 프로모터 아래 클로닝하였다(도 21).A plasmid (pET-LDHA2 or pET-LDHD3) expressing pET-TA4-1 and LDH was used to construct a recombinant plasmid expressing TA4-1 enzyme and OHB reductase enzyme simultaneously. Each gene fragment was amplified by PCR and ligated to NdeI and EcoRI site of pBAD plasmid. Two new plasmids were obtained: pBAD_TA4-1_LDHA2 (hereinafter referred to as pDHB-L) plasmid for L-form DHB and pBAD_TA4-1_LDHD3 (hereinafter referred to as pDHB-D) plasmid for D-form DHB. In these two plasmids, TA4-1 and OHB reductase genes were simultaneously cloned under the promoter induced by Arabinose to be transcribed polycistronic (Fig. 21).

6-2. DHB 생산 6-2. DHB production

DHB 생산을 위하여 실시예 1에서 개발된 homoserine 생산 균주에 DHB생산 경로 플라스미드 pDHB-L 및 pDHB-D를 각각 도입하였다. 사용한 homoserine 생산균주는 EcW20(표 19 참고)로 10개의 유전자 결실 (pts, eda, lacI, thrB, metA, lysA, adhE, pflB, ldhA, iclR)과 2개의 프로모터 치환 (Pacs::Ptrc), Pppc::PSP8), 그리고 metL 유전자를 과발현하기 위한 플라스미드 pUCPK-Ptrc-metL 를 가지고 있었다. 더 나아가, 이 균주는 PLP 과생산을 위한 생산 경로 핵심 효소 발현 강화, importer 막단백질의 제거, exporter 막단백의 발현 강화 등이 추가적으로 도입되었다. EcW20 균주에 DHB생산 경로 플라스미드 pDHB-L 혹은 pDHB-D가 도입된 균주를 각각 EcW20(pDHB-L) 및 EcW20(pDHB-D)으로 명명하였다.DHB production pathway plasmids pDHB-L and pDHB-D were respectively introduced into the homoserine producing strains developed in Example 1 for DHB production. (Pts , eda, lacI , thrB , metA, lysA, adhE, pflB, ldhA and iclR ) and two promoter substitutions (P acs :: P trc ) with EcW20 , P ppc :: P SP8 ), and the plasmid pUCPK-P trc - metL to overexpress the metL gene. Furthermore, this strain has been further supplemented with PLP and enhanced production pathway key enzyme expression for production, elimination of importer membrane protein, and enhancement of expression membrane protein expression. The strains into which the DHB production pathway plasmid pDHB-L or pDHB-D was introduced into the EcW20 strain were named EcW20 (pDHB-L) and EcW20 (pDHB-D), respectively.

먼저 pDHBL이 도입된 EcW20(pDHB-L) 균주를 통해 L-DHB생산을 조사하였다. EcW20(pDHB-L) 균주를 50 mL 배지 부피로 250 mL 플라스크에서 200 rpm으로 교반하면서 호기조건에서 배양하였다. 포도당을 탄소원으로 하는 TPM2 배지(yeast extract, 2 g; MgSO₄7-H₂O, 2 g; KH₂PO₄, 2 g; (NH₄)₂SO₄, 10 g; L-methionine, 0.2 g; L-lysine, 0.2g; L-Threonine, 0.2 g ;L-isoleucine, 0.05 g; trace metal solution, 10 ml)를 사용하였다. 포도당농도는 10 g/L, Kanamycin과 ampicillin은 각각 50 mg/L로 첨가하였다. Arabinose는 배양 3시간 후 0 - 1 g/L 범위에서 농도를 달리하면서 첨가해 주었다. First, the production of L-DHB was investigated through the EcW20 (pDHB-L) strain in which pDHBL was introduced. The EcW20 (pDHB-L) strain was cultivated under aerobic conditions with stirring at 200 rpm in a 250 mL flask with a volume of 50 mL of medium. TPM2 medium glucose as a carbon source (yeast extract, 2 g; MgSO 4 7-H 2 O, 2 g; KH 2 PO 4, 2 g; (NH 4) 2 SO 4, 10 g; L-methionine, 0.2 g L-isoleucine, 0.05 g; trace metal solution, 10 ml) was used. Glucose concentration was 10 g / L, Kanamycin and ampicillin were added at 50 mg / L, respectively. Arabinose was added at different concentrations in the range of 0 - 1 g / L after 3 hours of incubation.

도 22에 나타낸 바와 같이, DHB 생산 플라스미드를 갖지 않는 EcW20 균주는 DHB를 전혀 생산하지 않았다. DHB 생산 플라스미드를 갖는 EcW20 균주의 경우 arabinose 첨가량에 따라 DHB의 생산량이 변하였다. 즉, arabinose를 첨가하지 않은 경우 0.005 g/L의 소량이 얻어진데 비해 0.5 g/L의 농도로 첨가한 경우 0.33 g/L로 가장 많은 양이 얻어졌다. 더 높은 1 g/L의 arabinose가 첨가된 경우 세포성장, 포도당 소모 등이 저하되었고 DHB 생산도 0.19 g/L로 낮아졌다. 이는 DHB 생산 경로 효소의 과발현에 따른 부작용으로 판단된다. 생산된 DHB는 모두 L-form으로 나타났다. As shown in Fig. 22, the EcW20 strain having no DHB production plasmid produced no DHB at all. In the case of the EcW20 strain having a DHB production plasmid, the amount of DHB produced varied depending on the amount of arabinose added. That is, when arabinose was not added, a small amount of 0.005 g / L was obtained, whereas when it was added at a concentration of 0.5 g / L, the highest amount of 0.33 g / L was obtained. When 1 g / L of arabinose was added, cell growth, glucose consumption, and DHB production were reduced to 0.19 g / L. This is considered to be a side effect of overexpression of DHB production pathway enzyme. All DHB produced were in L-form.

동일한 방법으로 pDHBD 가 도입된 EcW20(pDHB-D) 균주를 통해 D-DHB생산을 조사하였다. L-DHB 의 경우와 마찬가지로 DHB 생산 플라스미드를 갖지 않는 EcW20 균주는 DHB를 전혀 생산하지 않았다. EcW20(pDHB-D) 균주의 경우 arabinose 첨가량에 따라 DHB의 생산량이 변하였다. 즉, arabinose를 첨가하지 않은 경우 0.005 g/L의 소량이 얻어진데 비해 0.5 g/L의 농도로 첨가한 경우 0.20 g/L로 가장 많은 양이 얻어졌다. 더 높은 1 g/L의 arabinose가 첨가된 경우 세포성장, 포도당 소모 등이 저하되었고 DHB 생산도 0.17 g/L로 낮아졌다. 이는 DHB 생산 경로 효소의 과발현에 따른 부작용으로 판단되었다. 생산된 DHB는 모두 D-form으로 나타났다. 전반적으로 L-DHB 보다 낮은 양의 D-DHB가 생성되었다. 그 이유는 재조합 균주 제작에 사용된 LdhD 효소의 활성이 LdhA 활성보다 낮기 때문으로 확인했다. (도22)D-DHB production was investigated in the same manner through the EcW20 (pDHB-D) strain in which pDHBD was introduced. As in the case of L-DHB, the EcW20 strain without the DHB production plasmid produced no DHB at all. In the case of the EcW20 (pDHB-D) strain, the amount of DHB produced varied depending on the amount of arabinose added. That is, a small amount of 0.005 g / L was obtained when arabinose was not added, whereas 0.20 g / L was obtained when 0.5 g / L was added. When 1 g / L of arabinose was added, cell growth, glucose consumption, and DHB production were reduced to 0.17 g / L. This was considered to be a side effect due to overexpression of DHB production pathway enzyme. The DHB produced was all D-form. Overall, lower amounts of D-DHB were produced than L-DHB. This is because the activity of the LdhD enzyme used in the production of the recombinant strain is lower than that of the LdhA activity. (Fig. 22)

EcW20(pDHB-L) 및 EcW20(pDHB-D) 균주를 이용하여 생물반응기 실험을 실시하였다 (도 24a). 반응기 부피는 3 L, 그리고 초기 배지 부피는 1 L 였다. 플라스크실험과 동일한 TPM2 배지를 사용하였고 발효 중 포도당을 적당한 수준으로 첨가시켜 주었다. 호기 조건을 유지하기 위하여 1 vvm의 속도로 공기를 주입하였고 교반속도는 500 - 900 rpm 사이에서 적절히 조절하였다. Arabinose는 6시간 배양 후 0.5 g/되도록 첨가하였다. 세포는 초기부터 18시간까지 성장하였다. DHB 생산은 9 시간부터 시작되어 배양이 끝난 48시간까지 지속시켰다. Bioreactor experiments were performed using EcW20 (pDHB-L) and EcW20 (pDHB-D) strains (Fig. 24A). The reactor volume was 3 L, and the initial medium volume was 1 L. The same TPM2 medium as in the flask experiment was used and glucose was added at an appropriate level during fermentation. Air was injected at a rate of 1 vvm to maintain aerobic conditions, and the stirring speed was suitably adjusted between 500 - 900 rpm. Arabinose was added at a concentration of 0.5 g / ml after 6 hours of incubation. Cells grew up to 18 hours from the beginning. DHB production started from 9 hours and lasted up to 48 hours after cultivation.

도 24a 및 24b에서 검은 동그라미 기호의 실선은 세포농도를 의미하며, 흰색 동그라미 기호의 점선은 생성물인 DHB를 의미하고, 역삼각형 기호의 점선은 포도당 농도를 의미한다. In FIGS. 24A and 24B, the solid line of the black circles indicates the cell concentration, the white circles indicate the product DHB, and the inverted triangles indicate the glucose concentration.

그 결과, D-DHB와 L-DHB의 경우 세포성장, 생성물 생산, 최종농도 등에서 차이가 났다. 즉 L-DHB의 경우 최종 세포 농도는 10 g/L 이었고 최종 L-DHB 농도는 20 g/L이었다. 이에 비해 D-DHB의 경우 최종 세포 농도는 8 g/L 이었고 최종 L-DHB 농도는 14 g/L이었다. As a result, D-DHB and L-DHB differed in cell growth, product production, and final concentration. In the case of L-DHB, the final cell concentration was 10 g / L and the final L-DHB concentration was 20 g / L. In contrast, the final cell concentration of D-DHB was 8 g / L and the final L-DHB concentration was 14 g / L.

실시예 7: 2-hydroxy gamma butyrolactone (HGBL) 생산균주의 제작 및 발효Example 7: Production and fermentation of 2-hydroxy gamma butyrolactone (HGBL) producing strain

7-1 HGBL의 생산을 위한 유전자 재조합 플라스미드 및 재조합 균주의 제작7-1 Production of recombinant plasmids and recombinant strains for the production of HGBL

DHB를 HGBL로 전환하기 위해서는 lactonase 효소가 필요하다. 락토네이즈 효소로 paraoxonase가 사용되었으며, 이 효소는 칼슘을 필요로 하고 여러 기질에 대하여 활성을 가지고 있다. 이 효소반응은 산성 범위에서 최적이므로 세포막이나 periplasmic space에서 발현하는 것이 필요하다. 또 이 효소가 동물유래이므로 미생물 발현을 위해서는 적절한 변이가 필요하다. 따라서 본 연구에서는 pon1 유전자 변이체 (G3C9)를 사용하고 또한 N-말단에 signal sequence를 부착하여 periplasmic space에 발현되도록 하였다. 먼저 pon1(G3C9) 유전자를 합성한 후 PCR 증폭(FP, tagacacatatggctaaactgacagcg; RP, tacatactcgagttacagctcacagtaaagagctttg)하고 낮은 copy 수를 갖는 pACYC-Duet 플라스미드에 클로닝하였다. pon1(G3C9) 의 발현에는 tac 프로모터를 사용하였다 (도 18). 이 플라스미드를 pACYC_Pon1으로 명명하였다.　　 A lactonase enzyme is required to convert DHB to HGBL. Paraoxonase was used as a lactonease enzyme, which requires calcium and is active against several substrates. Since this enzyme reaction is optimal in the acidic range, it is necessary to express it in cell membrane or periplasmic space. Since this enzyme is derived from an animal, appropriate mutation is necessary for microbial expression. Therefore, in this study, the pon1 gene mutant (G3C9) was used and the signal sequence was attached to the N-terminus to be expressed in the periplasmic space. First, pon1 (G3C9) gene was synthesized and then PCR amplified (FP, tagacacatatggctaaactgacagcg; RP, tacatactcgagttacagctcacagtaaagagctttg) and cloned into pACYC-Duet plasmid with low copy number. The tac promoter was used for the expression of pon1 (G3C9) (Fig. 18). This plasmid was named pACYC_Pon1.

얻어진 Pon1 plasmid를 앞서 얻은 2개의 균주 즉 L-DHB와 D-DHB를 생산하기 위한 균주에 각각 도입하였다. 이 경우 DHB의 세포외 이송 속도를 높이는 것이 바람직하지 않으므로 숙주세포로 EcW16이 사용하였고 (표 19), homoserine에서 DHB를 생산하기 위한 플라스미드는 앞서 DHB생산의 경우와 동일하게 pDHB-L 및 pDHB-D를 사용하였다 (도 21).　 그리고 이들 균주를 각각 EcW16(pHGBL-L), EcW16(pHGBL-D)라고 명명하였다The obtained Pon1 plasmids were introduced into the strains for producing the two strains, L-DHB and D-DHB, respectively. In this case, it is not preferable to increase the extracellular transport rate of DHB. Therefore, EcW16 was used as a host cell (Table 19). Plasmids for producing DHB in homoserine were pDHB-L and pDHB-D (Fig. 21). These strains were named EcW16 (pHGBL-L) and EcW16 (pHGBL-D), respectively

7-2 HGBL의 생산을 생물반응기 운전7-2 Production of HGBL by bioreactor operation

HGBL 생산을 위하여 2단 배양을 실시하였다. 먼저 1단 배양에서 DHB를 생산한 후 2단에서 pH를 낮추어 HGBL을 생산하였다. 한천배지에서 단일 균주를 얻은 후, LB 배지를 이용하여 starter culture를 시행하였다. 그리고 앞서 기술한 바와 같이 TPM2 배지를 이용하여 seed culture를 실시하였다. 본 배양은 지수성장기에 있는 seed culture 세포를 접종하여 TPM2 배지에서 실시하였다.　 Two - stage cultivation was performed for HGBL production. First, DHB was produced in one culture and then HGBL was produced by lowering pH in the second stage. After obtaining a single strain from agar medium, starter culture was performed using LB medium. The seed culture was performed using TPM2 medium as described above. This culture was carried out in TPM2 medium inoculated with seed culture cells in the exponential growth phase.

L-DHB의 경우 48시간 1단 배양 동안 약 16 g/L의 DHB가 생산되었다. 세포 농도나 포도당의 소모 속도도 L-DHB의 생산을 위한 발효에 비해 감소하였다. transporter의 engineering 부재, pon1 발현을 위한 플라스미드 도입과 추가적인 단백질, 특히 pon1 막 단백질의 생산이 그 원인으로 추정되었다. In the case of L-DHB, about 16 g / L of DHB was produced during one 48-hour incubation. The cell concentration and glucose consumption rate were also decreased compared to fermentation for L-DHB production. the lack of engineering of the transporter, the introduction of plasmids for the expression of pon1, and the production of additional proteins, particularly the pon1 membrane protein.

D-DHB 의 경우 48시간 1단 배양 동안 약 9 g/L의 DHB가 생산되었다. 세포 농도나 포도당의 소모 속도도　D-DHB의 생산을 위한 발효에 비해 감소하였다. pon1 발현을 위한 플라스미드 도입과 추가적인 단백질, 특히 pon1 막 단백질의 생산이 그 원인으로 추정되었다. 또한 L-DHB가 D-DHB보다 높은 농도로 얻어졌는데 이는 앞서 설명한 바와 같이 2-oxo-reductase의 활성이 L-form이 D-form에 비해 현저히 높았기 때문으로 추정된다.In the case of D-DHB, about 9 g / L of DHB was produced during the first 48-hour incubation. Cell concentration and glucose uptake rate were also decreased compared to fermentation for D-DHB production. The introduction of plasmids for the expression of pon1 and the production of additional proteins, particularly the pon1 membrane protein, were presumed to be the cause. In addition, L-DHB was obtained at a higher concentration than D-DHB because the activity of 2-oxo-reductase was remarkably higher than that of D-form as described above.

이후 생산된 DHB를 HGBL로 전환하기 위하여 염산용액을 이용하여 배양액의 pH를 6.2로 낮추고 배양을 계속 하였다. 그 결과 L-HGBL의 경우 약 24시간의 추가 배양을 통해 약 5 g/L의 락톤을 얻을 수 있었다. 대부분의 DHB는 HGBL로 전환되지 않고 남아 있었다 (도 26a). The pH of the medium was lowered to 6.2 using a hydrochloric acid solution to convert the produced DHB to HGBL, and the culture was continued. As a result, about 5 g / L of lactone was obtained by further culturing for about 24 hours in case of L-HGBL. Most of the DHB remained unconverted to HGBL (Fig. 26A).

한편 D-HGBL을 얻기 위해 동일한 실험을 실시하였다 (도 26b) 즉 48시간 1단 배양 후 24시간 2단 배양을 pH 6.2에서 실시하였다. L-HGBL의 경우와는 달리 0.3 g/L의 아주 낮은 농도만 얻어졌다. 그 이유는 사용한 pon1 효소가 L-form 의 DHB에만 활성이 있게 때문으로 추정되었다. Meanwhile, the same experiment was carried out to obtain D-HGBL (Fig. 26B). That is, a 48-hour single-stage culture and a 24-hour two-stage culture were carried out at pH 6.2. Unlike the case of L-HGBL, only a very low concentration of 0.3 g / L was obtained. The reason is that the used pon1 enzyme is active only in DHB of L-form.

화학적 반응을 통해 2,4-dihydroxy butyric acid를 Lactone으로 전환시켜 보았다. 이 경우 도 23 및 도 24에서 보여준 1단 배양이 끝난 배지를 원심분리하여 세포를 제거하고 배양액에 염산용액을 가하여 pH를 1.0까지 떨어뜨렸다. 그리고 10시간 정치반응 시켰다. 이 경우 L-DHB와 D-DHB 모두 약 50%가 lactone으로 전환되었다. 그 결과 L-HGBL은 약 9 g/L 그리고 D-HGBL은 약 6 g/L가 얻어졌다. 이로 미루어 DHB의 에스테르화 반응은 D-form, L-form 모두 산촉매 화학 반응을 이용하는 것이 pon1 효소를 이용하는 것보다 효율적임을 알 수 있었다. Through chemical reaction, 2,4-dihydroxy butyric acid was converted to lactone. In this case, the culture medium having the single-stage culture shown in FIGS. 23 and 24 was centrifuged to remove cells, and a hydrochloric acid solution was added to the culture to lower the pH to 1.0. Then, the reaction was allowed to proceed for 10 hours. In this case, about 50% of L-DHB and D-DHB were converted to lactone. As a result, about 9 g / L of L-HGBL and about 6 g / L of D-HGBL were obtained. Thus, DHB esterification was found to be more efficient in the D-form and L-form reactions than the pon1 enzyme.

기탁기관명 : 한국미생물보존센터(국외)Name of depository: Korea Microorganism Conservation Center (overseas)

수탁번호 : KCCM12281PAccession number: KCCM12281P

수탁일자 : 20180622Checked on: 20180622

수탁번호 : KCCM12282PAccession number: KCCM12282P

수탁일자 : 20180622Checked on: 20180622

<110> UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> Method of producing 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutanoic acid <130> DPP20173744KR <150> 10-2017-0088465 <151> 2017-07-12 <160> 241 <170> KopatentIn 3.0 <210> 1 <211> 1197 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase[Bacillus subtilis subsp. subtilis str. 168]: AQR85543.1) <400> 1 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaatattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa taatccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggct acgtcagagt cggactttta acaagtgaag aaagacttaa ggaagccgct 1140 tatcgaattg gcaaactgaa cctgtttacc caaaaaagca ttgacaagac cttataa 1197 <210> 2 <211> 1197 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase: AQR85543.1 (Y364Q)) <400> 2 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaatattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa taatccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggcc aggtcagagt cggactttta acaagtgaag aaagacttaa ggaagccgct 1140 tatcgaattg gcaaactgaa cctgtttacc caaaaaagca ttgacaagac cttataa 1197 <210> 3 <211> 1197 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D)) <400> 3 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaatattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa tgacccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggcc aggtcagagt cggactttta acaagtgaag aaagacttaa ggaagccgct 1140 tatcgaattg gcaaactgaa cctgtttacc caaaaaagca ttgacaagac cttataa 1197 <210> 4 <211> 1196 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D Y364Q)) <400> 4 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaatattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa tgacccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggcc aggtcgagtc ggacttttaa caagtgaaga aagacttaag gaagccgctt 1140 atcgaattgg caaactgaac ctgtttaccc aaaaaagcat tgacaagacc ttataa 1196 <210> 5 <211> 1050 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (L-lactate dehydrogenase [Alcaligenes eutrophus]: YP_725182.1) <400> 5 atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60 gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120 tatatcagcc acggcctgtc gatcctgccc aactaccgca ccgccctcga cggccacagc 180 gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240 ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300 gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360 ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420 atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480 ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540 accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600 gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660 gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720 gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780 ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840 ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900 gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960 gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020 tcgctcaacg tggccgtccc cacggcctga 1050 <210> 6 <211> 1050 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (L-lactate dehydrogenase: YP_725182.1 (I48N)) <400> 6 atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60 gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120 tatatcagcc acggcctgtc gaatctgccc aactaccgca ccgccctcga cggccacagc 180 gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240 ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300 gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360 ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420 atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480 ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540 accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600 gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660 gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720 gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780 ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840 ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900 gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960 gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020 tcgctcaacg tggccgtccc cacggcctga 1050 <210> 7 <211> 1050 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (L-lactate dehydrogenase: YP_725182.1(I48T)) <400> 7 atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60 gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120 tatatcagcc acggcctgtc gactctgccc aactaccgca ccgccctcga cggccacagc 180 gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240 ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300 gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360 ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420 atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480 ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540 accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600 gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660 gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720 gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780 ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840 ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900 gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960 gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020 tcgctcaacg tggccgtccc cacggcctga 1050 <210> 8 <211> 1002 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase [Lactobacillus delbrueckii subsp. bulgaricus]: CAI96942.1) <400> 8 atgactaaaa tttttgctta cgcaattcgt gaagatgaaa agccattctt gaaggaatgg 60 gaagacgctc acaaggacgt cgaagttgaa tacactgaca agcttttgac cccagaaact 120 gttgctttgg caaagggtgc tgacggtgtt gttgtttacc aacaacttga ctacaccgct 180 gaaactctgc aagctttggc agacaacggc atcactaaga tgagcctgcg taacgttggt 240 gttgacaaca tcgacatggc taaggctaag gaacttggct tccaaatcac caacgttcca 300 gtttactcac caaacgccat cgcagaacac gctgctatcc aagctgcccg catcctgcgt 360 caagacaagg ctatggacga aaaggttgcc cgtcacgact tgcgttgggc accaactatc 420 ggccgtgaag ttcgcgacca agttgttggt gttataggta ctggccacat cggtcaagtc 480 ttcatgcaaa tcatggaagg cttcggcgct aaggttatcg cttacgacat cttccgcaac 540 ccagaattgg aaaagaaggg ctactacgta gactcacttg acgacctgta caagcaagct 600 gacgttattt ccctgcacgt tcctgacgtt ccagctaacg ttcacatgat caacgacgag 660 tcaatcgcta aaatgaagca agacgtagtt atcgttaacg tatcacgtgg tccattggtt 720 gacactgacg cggttatccg tggtttggac tcaggcaaga tcttcggtta cgcaatggac 780 gtttacgaag gtgaagttgg catcttcaac gaagactggg aaggcaagga attcccagac 840 gcacgtttag ctgacttaat cgctcgtcca aacgttctgg taactccaca cactgctttc 900 tacactactc acgctgttcg caacatggta gttaaggcct tcgacaacaa ccttgaattg 960 gttgaaggca aggaagctga aactccagtt aaggttggct aa 1002 <210> 9 <211> 1002 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase: CAI96942.1 (V79N)) <400> 9 atgactaaaa tttttgctta cgcaattcgt gaagatgaaa agccattctt gaaggaatgg 60 gaagacgctc acaaggacgt cgaagttgaa tacactgaca agcttttgac cccagaaact 120 gttgctttgg caaagggtgc tgacggtgtt gttgtttacc aacaacttga ctacaccgct 180 gaaactctgc aagctttggc agacaacggc atcactaaga tgagcctgcg taacaacggt 240 gttgacaaca tcgacatggc taaggctaag gaacttggct tccaaatcac caacgttcca 300 gtttactcac caaacgccat cgcagaacac gctgctatcc aagctgcccg catcctgcgt 360 caagacaagg ctatggacga aaaggttgcc cgtcacgact tgcgttgggc accaactatc 420 ggccgtgaag ttcgcgacca agttgttggt gttataggta ctggccacat cggtcaagtc 480 ttcatgcaaa tcatggaagg cttcggcgct aaggttatcg cttacgacat cttccgcaac 540 ccagaattgg aaaagaaggg ctactacgta gactcacttg acgacctgta caagcaagct 600 gacgttattt ccctgcacgt tcctgacgtt ccagctaacg ttcacatgat caacgacgag 660 tcaatcgcta aaatgaagca agacgtagtt atcgttaacg tatcacgtgg tccattggtt 720 gacactgacg cggttatccg tggtttggac tcaggcaaga tcttcggtta cgcaatggac 780 gtttacgaag gtgaagttgg catcttcaac gaagactggg aaggcaagga attcccagac 840 gcacgtttag ctgacttaat cgctcgtcca aacgttctgg taactccaca cactgctttc 900 tacactactc acgctgttcg caacatggta gttaaggcct tcgacaacaa ccttgaattg 960 gttgaaggca aggaagctga aactccagtt aaggttggct aa 1002 <210> 10 <211> 1002 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase: CAI96942.1 (V79T)) <400> 10 atgactaaaa tttttgctta cgcaattcgt gaagatgaaa agccattctt gaaggaatgg 60 gaagacgctc acaaggacgt cgaagttgaa tacactgaca agcttttgac cccagaaact 120 gttgctttgg caaagggtgc tgacggtgtt gttgtttacc aacaacttga ctacaccgct 180 gaaactctgc aagctttggc agacaacggc atcactaaga tgagcctgcg taacaccggt 240 gttgacaaca tcgacatggc taaggctaag gaacttggct tccaaatcac caacgttcca 300 gtttactcac caaacgccat cgcagaacac gctgctatcc aagctgcccg catcctgcgt 360 caagacaagg ctatggacga aaaggttgcc cgtcacgact tgcgttgggc accaactatc 420 ggccgtgaag ttcgcgacca agttgttggt gttataggta ctggccacat cggtcaagtc 480 ttcatgcaaa tcatggaagg cttcggcgct aaggttatcg cttacgacat cttccgcaac 540 ccagaattgg aaaagaaggg ctactacgta gactcacttg acgacctgta caagcaagct 600 gacgttattt ccctgcacgt tcctgacgtt ccagctaacg ttcacatgat caacgacgag 660 tcaatcgcta aaatgaagca agacgtagtt atcgttaacg tatcacgtgg tccattggtt 720 gacactgacg cggttatccg tggtttggac tcaggcaaga tcttcggtta cgcaatggac 780 gtttacgaag gtgaagttgg catcttcaac gaagactggg aaggcaagga attcccagac 840 gcacgtttag ctgacttaat cgctcgtcca aacgttctgg taactccaca cactgctttc 900 tacactactc acgctgttcg caacatggta gttaaggcct tcgacaacaa ccttgaattg 960 gttgaaggca aggaagctga aactccagtt aaggttggct aa 1002 <210> 11 <211> 1065 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Paraoxonase PON1 variant: AAR95986.1 G3C9 ) <400> 11 atggctaaac tgacagcgct cacactcttg gggctgggat tggcactctt cgatggacag 60 aagtcttctt tccaaacacg atttaatgtt caccgtgaag taactccagt ggaacttcct 120 aactgtaatt tagttaaagg ggttgacaat ggttctgaag acttggaaat actgcccaat 180 ggactggctt tcatcagctc cggattaaag tatcctggaa taatgagctt tgaccctgat 240 aagtctggaa agatacttct aatggacctg aatgaggaag acccagtagt gttggaactg 300 ggcattactg gaaatacatt ggatatatct tcatttaacc ctcatgggat tagcacattc 360 acagatgaag ataacactgt gtacctactg gtggtaaacc atccagactc ctcgtccacc 420 gtggaggtgt ttaaatttca agaagaagaa aaatcacttt tgcatctgaa aaccatcaga 480 cacaagcttc tgcctagtgt gaatgacatt gtcgctgtgg gacctgaaca cttttatgcc 540 acaaatgatc actattttgc tgacccttac ttaaaatcct gggaaatgca tttgggatta 600 gcgtggtcat ttgttactta ttatagtccc aatgatgttc gagtagtggc agaaggattt 660 gattttgcta acggaatcaa catctcacca gacggcaagt atgtctatat agctgagttg 720 ctggctcata agatccatgt gtatgaaaag cacgctaatt ggactttaac tccattgaag 780 tccctcgact ttgacaccct tgtggataac atctctgtgg atcctgtgac aggggacctc 840 tgggtgggat gccatcccaa cggaatgcga atcttctact atgacccaaa gaatcctccc 900 ggctcagagg tgcttcgaat ccaggacatt ttatccgaag agcccaaagt gacagtggtt 960 tatgcagaaa atggcactgt gttacagggc agcacggtgg ccgctgtgta caaagggaaa 1020 ctgctgattg gcacagtgtt tcacaaagct ctttactgtg agctg 1065 <210> 12 <211> 398 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase [Bacillus subtilis subsp. subtilis str. 168]: AQR85543.1 (TA4)) <400> 12 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asn Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Tyr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Tyr Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 13 <211> 398 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase: AQR85543.1 (Y364Q)_TA4-1) <400> 13 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asn Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Tyr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Gln Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 14 <211> 398 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D)_TA4-2) <400> 14 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asp Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Tyr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Gln Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 15 <211> 398 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D Y364Q)_TA4-6) <400> 15 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asp Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Tyr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Gln Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 16 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (L-lactate dehydrogenase [Alcaligenes eutrophus]: YP_725182.1_) <400> 16 Met Lys Ile Ser Leu Thr Ser Ala Arg Gln Leu Ala Arg Asp Ile Leu 1 5 10 15 Ala Ala Gln Gln Val Pro Ala Asp Ile Ala Asp Asp Val Ala Glu His 20 25 30 Leu Val Glu Ser Asp Arg Cys Gly Tyr Ile Ser His Gly Leu Ser Ile 35 40 45 Leu Pro Asn Tyr Arg Thr Ala Leu Asp Gly His Ser Val Asn Pro Gln 50 55 60 Gly Arg Ala Lys Cys Val Leu Asp Gln Gly Thr Leu Met Val Phe Asp 65 70 75 80 Gly Asp Gly Gly Phe Gly Gln His Val Gly Lys Ser Val Met Gln Ala 85 90 95 Ala Ile Glu Arg Val Arg Gln His Gly His Cys Ile Val Thr Leu Arg 100 105 110 Arg Ser His His Leu Gly Arg Met Gly His Tyr Gly Glu Met Ala Ala 115 120 125 Ala Ala Gly Phe Val Leu Leu Ser Phe Thr Asn Val Ile Asn Arg Ala 130 135 140 Pro Val Val Ala Pro Phe Gly Gly Arg Val Ala Arg Leu Thr Thr Asn 145 150 155 160 Pro Leu Cys Phe Ala Gly Pro Met Pro Asn Gly Arg Pro Pro Leu Val 165 170 175 Val Asp Ile Ala Thr Ser Ala Ile Ala Ile Asn Lys Ala Arg Val Leu 180 185 190 Ala Glu Lys Gly Glu Pro Ala Pro Glu Gly Ser Ile Ile Gly Ala Asp 195 200 205 Gly Asn Pro Thr Thr Asp Ala Ser Thr Met Phe Gly Glu His Pro Gly 210 215 220 Ala Leu Leu Pro Phe Gly Gly His Lys Gly Tyr Ala Leu Gly Val Val 225 230 235 240 Ala Glu Leu Leu Ala Gly Val Leu Ser Gly Gly Gly Thr Ile Gln Pro 245 250 255 Asp Asn Pro Arg Gly Gly Val Ala Thr Asn Asn Leu Phe Ala Val Leu 260 265 270 Leu Asn Pro Ala Leu Asp Leu Gly Leu Asp Trp Gln Ser Ala Glu Val 275 280 285 Glu Ala Phe Val Arg Tyr Leu His Asp Thr Pro Pro Ala Pro Gly Val 290 295 300 Asp Arg Val Gln Tyr Pro Gly Glu Tyr Glu Ala Ala Asn Arg Ala Gln 305 310 315 320 Ala Ser Asp Thr Leu Asn Ile Asn Pro Ala Ile Trp Arg Asn Leu Glu 325 330 335 Arg Leu Ala Gln Ser Leu Asn Val Ala Val Pro Thr Ala 340 345 <210> 17 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (L-lactate dehydrogenase: YP_725182.1 (I48T)) <400> 17 Met Lys Ile Ser Leu Thr Ser Ala Arg Gln Leu Ala Arg Asp Ile Leu 1 5 10 15 Ala Ala Gln Gln Val Pro Ala Asp Ile Ala Asp Asp Val Ala Glu His 20 25 30 Leu Val Glu Ser Asp Arg Cys Gly Tyr Ile Ser His Gly Leu Ser Thr 35 40 45 Leu Pro Asn Tyr Arg Thr Ala Leu Asp Gly His Ser Val Asn Pro Gln 50 55 60 Gly Arg Ala Lys Cys Val Leu Asp Gln Gly Thr Leu Met Val Phe Asp 65 70 75 80 Gly Asp Gly Gly Phe Gly Gln His Val Gly Lys Ser Val Met Gln Ala 85 90 95 Ala Ile Glu Arg Val Arg Gln His Gly His Cys Ile Val Thr Leu Arg 100 105 110 Arg Ser His His Leu Gly Arg Met Gly His Tyr Gly Glu Met Ala Ala 115 120 125 Ala Ala Gly Phe Val Leu Leu Ser Phe Thr Asn Val Ile Asn Arg Ala 130 135 140 Pro Val Val Ala Pro Phe Gly Gly Arg Val Ala Arg Leu Thr Thr Asn 145 150 155 160 Pro Leu Cys Phe Ala Gly Pro Met Pro Asn Gly Arg Pro Pro Leu Val 165 170 175 Val Asp Ile Ala Thr Ser Ala Ile Ala Ile Asn Lys Ala Arg Val Leu 180 185 190 Ala Glu Lys Gly Glu Pro Ala Pro Glu Gly Ser Ile Ile Gly Ala Asp 195 200 205 Gly Asn Pro Thr Thr Asp Ala Ser Thr Met Phe Gly Glu His Pro Gly 210 215 220 Ala Leu Leu Pro Phe Gly Gly His Lys Gly Tyr Ala Leu Gly Val Val 225 230 235 240 Ala Glu Leu Leu Ala Gly Val Leu Ser Gly Gly Gly Thr Ile Gln Pro 245 250 255 Asp Asn Pro Arg Gly Gly Val Ala Thr Asn Asn Leu Phe Ala Val Leu 260 265 270 Leu Asn Pro Ala Leu Asp Leu Gly Leu Asp Trp Gln Ser Ala Glu Val 275 280 285 Glu Ala Phe Val Arg Tyr Leu His Asp Thr Pro Pro Ala Pro Gly Val 290 295 300 Asp Arg Val Gln Tyr Pro Gly Glu Tyr Glu Ala Ala Asn Arg Ala Gln 305 310 315 320 Ala Ser Asp Thr Leu Asn Ile Asn Pro Ala Ile Trp Arg Asn Leu Glu 325 330 335 Arg Leu Ala Gln Ser Leu Asn Val Ala Val Pro Thr Ala 340 345 <210> 18 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (L-lactate dehydrogenase: YP_725182.1(I48N)) <400> 18 Met Lys Ile Ser Leu Thr Ser Ala Arg Gln Leu Ala Arg Asp Ile Leu 1 5 10 15 Ala Ala Gln Gln Val Pro Ala Asp Ile Ala Asp Asp Val Ala Glu His 20 25 30 Leu Val Glu Ser Asp Arg Cys Gly Tyr Ile Ser His Gly Leu Ser Asn 35 40 45 Leu Pro Asn Tyr Arg Thr Ala Leu Asp Gly His Ser Val Asn Pro Gln 50 55 60 Gly Arg Ala Lys Cys Val Leu Asp Gln Gly Thr Leu Met Val Phe Asp 65 70 75 80 Gly Asp Gly Gly Phe Gly Gln His Val Gly Lys Ser Val Met Gln Ala 85 90 95 Ala Ile Glu Arg Val Arg Gln His Gly His Cys Ile Val Thr Leu Arg 100 105 110 Arg Ser His His Leu Gly Arg Met Gly His Tyr Gly Glu Met Ala Ala 115 120 125 Ala Ala Gly Phe Val Leu Leu Ser Phe Thr Asn Val Ile Asn Arg Ala 130 135 140 Pro Val Val Ala Pro Phe Gly Gly Arg Val Ala Arg Leu Thr Thr Asn 145 150 155 160 Pro Leu Cys Phe Ala Gly Pro Met Pro Asn Gly Arg Pro Pro Leu Val 165 170 175 Val Asp Ile Ala Thr Ser Ala Ile Ala Ile Asn Lys Ala Arg Val Leu 180 185 190 Ala Glu Lys Gly Glu Pro Ala Pro Glu Gly Ser Ile Ile Gly Ala Asp 195 200 205 Gly Asn Pro Thr Thr Asp Ala Ser Thr Met Phe Gly Glu His Pro Gly 210 215 220 Ala Leu Leu Pro Phe Gly Gly His Lys Gly Tyr Ala Leu Gly Val Val 225 230 235 240 Ala Glu Leu Leu Ala Gly Val Leu Ser Gly Gly Gly Thr Ile Gln Pro 245 250 255 Asp Asn Pro Arg Gly Gly Val Ala Thr Asn Asn Leu Phe Ala Val Leu 260 265 270 Leu Asn Pro Ala Leu Asp Leu Gly Leu Asp Trp Gln Ser Ala Glu Val 275 280 285 Glu Ala Phe Val Arg Tyr Leu His Asp Thr Pro Pro Ala Pro Gly Val 290 295 300 Asp Arg Val Gln Tyr Pro Gly Glu Tyr Glu Ala Ala Asn Arg Ala Gln 305 310 315 320 Ala Ser Asp Thr Leu Asn Ile Asn Pro Ala Ile Trp Arg Asn Leu Glu 325 330 335 Arg Leu Ala Gln Ser Leu Asn Val Ala Val Pro Thr Ala 340 345 <210> 19 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase [Lactobacillus delbrueckii subsp. bulgaricus]: CAI96942.1) <400> 19 Met Thr Lys Ile Phe Ala Tyr Ala Ile Arg Glu Asp Glu Lys Pro Phe 1 5 10 15 Leu Lys Glu Trp Glu Asp Ala His Lys Asp Val Glu Val Glu Tyr Thr 20 25 30 Asp Lys Leu Leu Thr Pro Glu Thr Val Ala Leu Ala Lys Gly Ala Asp 35 40 45 Gly Val Val Val Tyr Gln Gln Leu Asp Tyr Thr Ala Glu Thr Leu Gln 50 55 60 Ala Leu Ala Asp Asn Gly Ile Thr Lys Met Ser Leu Arg Asn Val Gly 65 70 75 80 Val Asp Asn Ile Asp Met Ala Lys Ala Lys Glu Leu Gly Phe Gln Ile 85 90 95 Thr Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ala Ala 100 105 110 Ile Gln Ala Ala Arg Ile Leu Arg Gln Asp Lys Ala Met Asp Glu Lys 115 120 125 Val Ala Arg His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val 130 135 140 Arg Asp Gln Val Val Gly Val Ile Gly Thr Gly His Ile Gly Gln Val 145 150 155 160 Phe Met Gln Ile Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp 165 170 175 Ile Phe Arg Asn Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser 180 185 190 Leu Asp Asp Leu Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro 195 200 205 Asp Val Pro Ala Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys 210 215 220 Met Lys Gln Asp Val Val Ile Val Asn Val Ser Arg Gly Pro Leu Val 225 230 235 240 Asp Thr Asp Ala Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly 245 250 255 Tyr Ala Met Asp Val Tyr Glu Gly Glu Val Gly Ile Phe Asn Glu Asp 260 265 270 Trp Glu Gly Lys Glu Phe Pro Asp Ala Arg Leu Ala Asp Leu Ile Ala 275 280 285 Arg Pro Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His 290 295 300 Ala Val Arg Asn Met Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu 305 310 315 320 Val Glu Gly Lys Glu Ala Glu Thr Pro Val Lys Val Gly 325 330 <210> 20 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase: CAI96942.1 (V79T)) <400> 20 Met Thr Lys Ile Phe Ala Tyr Ala Ile Arg Glu Asp Glu Lys Pro Phe 1 5 10 15 Leu Lys Glu Trp Glu Asp Ala His Lys Asp Val Glu Val Glu Tyr Thr 20 25 30 Asp Lys Leu Leu Thr Pro Glu Thr Val Ala Leu Ala Lys Gly Ala Asp 35 40 45 Gly Val Val Val Tyr Gln Gln Leu Asp Tyr Thr Ala Glu Thr Leu Gln 50 55 60 Ala Leu Ala Asp Asn Gly Ile Thr Lys Met Ser Leu Arg Asn Thr Gly 65 70 75 80 Val Asp Asn Ile Asp Met Ala Lys Ala Lys Glu Leu Gly Phe Gln Ile 85 90 95 Thr Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ala Ala 100 105 110 Ile Gln Ala Ala Arg Ile Leu Arg Gln Asp Lys Ala Met Asp Glu Lys 115 120 125 Val Ala Arg His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val 130 135 140 Arg Asp Gln Val Val Gly Val Ile Gly Thr Gly His Ile Gly Gln Val 145 150 155 160 Phe Met Gln Ile Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp 165 170 175 Ile Phe Arg Asn Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser 180 185 190 Leu Asp Asp Leu Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro 195 200 205 Asp Val Pro Ala Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys 210 215 220 Met Lys Gln Asp Val Val Ile Val Asn Val Ser Arg Gly Pro Leu Val 225 230 235 240 Asp Thr Asp Ala Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly 245 250 255 Tyr Ala Met Asp Val Tyr Glu Gly Glu Val Gly Ile Phe Asn Glu Asp 260 265 270 Trp Glu Gly Lys Glu Phe Pro Asp Ala Arg Leu Ala Asp Leu Ile Ala 275 280 285 Arg Pro Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His 290 295 300 Ala Val Arg Asn Met Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu 305 310 315 320 Val Glu Gly Lys Glu Ala Glu Thr Pro Val Lys Val Gly 325 330 <210> 21 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase: CAI96942.1 (V79N)) <400> 21 Met Thr Lys Ile Phe Ala Tyr Ala Ile Arg Glu Asp Glu Lys Pro Phe 1 5 10 15 Leu Lys Glu Trp Glu Asp Ala His Lys Asp Val Glu Val Glu Tyr Thr 20 25 30 Asp Lys Leu Leu Thr Pro Glu Thr Val Ala Leu Ala Lys Gly Ala Asp 35 40 45 Gly Val Val Val Tyr Gln Gln Leu Asp Tyr Thr Ala Glu Thr Leu Gln 50 55 60 Ala Leu Ala Asp Asn Gly Ile Thr Lys Met Ser Leu Arg Asn Asn Gly 65 70 75 80 Val Asp Asn Ile Asp Met Ala Lys Ala Lys Glu Leu Gly Phe Gln Ile 85 90 95 Thr Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ala Ala 100 105 110 Ile Gln Ala Ala Arg Ile Leu Arg Gln Asp Lys Ala Met Asp Glu Lys 115 120 125 Val Ala Arg His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val 130 135 140 Arg Asp Gln Val Val Gly Val Ile Gly Thr Gly His Ile Gly Gln Val 145 150 155 160 Phe Met Gln Ile Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp 165 170 175 Ile Phe Arg Asn Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser 180 185 190 Leu Asp Asp Leu Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro 195 200 205 Asp Val Pro Ala Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys 210 215 220 Met Lys Gln Asp Val Val Ile Val Asn Val Ser Arg Gly Pro Leu Val 225 230 235 240 Asp Thr Asp Ala Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly 245 250 255 Tyr Ala Met Asp Val Tyr Glu Gly Glu Val Gly Ile Phe Asn Glu Asp 260 265 270 Trp Glu Gly Lys Glu Phe Pro Asp Ala Arg Leu Ala Asp Leu Ile Ala 275 280 285 Arg Pro Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His 290 295 300 Ala Val Arg Asn Met Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu 305 310 315 320 Val Glu Gly Lys Glu Ala Glu Thr Pro Val Lys Val Gly 325 330 <210> 22 <211> 355 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (Paraoxonase PON1 variant: AAR95986.1_G3C9) <400> 22 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu 1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg 20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val 35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe 50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val 85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe 100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe 130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys 180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr 195 200 205 Ser Pro Asn Asp Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser 260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr 340 345 350 Cys Glu Leu 355 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (synthetic promoter 8) <400> 23 tttcaattta atcatccggc tcgtataatg tgtgga 36 <210> 24 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pts_RP primer) <400> 24 cggaaccgcc tgcttctgcc ataacatgcg atacaacggc ggttacagca gaatacctgc 60 gataggcagt acggataccg gcagcatc 88 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pts_seq_FP primer) <400> 25 ggcgtcggtt ccgcgaattt c 21 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pts_seq_RP primer) <400> 26 ggtgcagacc aaacggtacc 20 <210> 27 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta eda_FP primer) <400> 27 caactgcaca ctcggtacgc agagtcactt ccagaacgcg cattacaccg cgtgttccag 60 tttttttacc acgataaccg gtacaacc 88 <210> 28 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta eda_RP primer) <400> 28 ggttgtaccg gttatcgtgg taaaaaaact ggaacacgcg gtgtaatgcg cgttctggaa 60 gtgactctgc gtaccgagtg tgcagttg 88 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta eda_seq_FP primer) <400> 29 ccgatggcaa aagcgttgg 19 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta eda_seq_RP primer) <400> 30 cgcgccagct tagtaatgc 19 <210> 31 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta poxB_RP primer) <400> 31 ggcaggggtg aaacgcatct ggggagtcac aggcgactct ctgaactaac catcgagtgg 60 atgtccaccc gccacgaaga agtggcggcc 90 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta poxB_seq_FP primer) <400> 32 tggaataacg cagcagttg 19 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta poxB_seq_RPprimer) <400> 33 ggtcttagtg acagtcttaa tc 22 <210> 34 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pta_FP primer) <400> 34 gaccagcgtc agccttggcg tgatccgtgc aatggaacgc aaataactca gccgcgtacc 60 ggtggcgatg cgcccgatca gactacg 87 <210> 35 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pta_RP primer) <400> 35 cgtagtctga tcgggcgcat cgccaccggt acgcggctga gttatttgcg ttccattgca 60 cggatcacgc caaggctgac gctggtc 87 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pta_seq_FP) <400> 36 ggcgttcgtc tgagcgtttt c 21 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pta_seq_RP) <400> 37 ggattcttgc agcttcgcc 19 <210> 38 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ackA_FP) <400> 38 gaaatttgcc atcatcgatg cagtaaatgg tgaagagtac ctttgaaagc acgtatcaaa 60 tggaaaatgg acggcaataa acaggaagc 89 <210> 39 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ackA_RP) <400> 39 gcttcctgtt tattgccgtc cattttccat ttgatacgtg ctttcaaagg tactcttcac 60 catttactgc atcgatgatg gcaaatttc 89 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ackA_seq_FP) <400> 40 tctggtttag ccgaatgttt cc 22 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ackA_seq_RP) <400> 41 atgttcagtt cttctaccgg 20 <210> 42 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ldhA_RP) <400> 42 gcaacaggtg aacgagtcct ttggctttga gctggaattt ttttaactgc caatggctgc 60 gaagcggtat gtattttcgt aaacgatg 88 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ldhA_seq_FP) <400> 43 ttttccggtg tcagcggg 18 <210> 44 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta ldhA_seq_RP) <400> 44 gactttctgc tgacgg 16 <210> 45 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta adhE_RP) <400> 45 gtaaaaaaag cccagcgtga atatgccagt ttcactcaag agcaataact gctgcagatg 60 ctcgaatccc actcgcgaaa atggccgttg 90 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta adhE_seq_FP) <400> 46 ggagctgtat gcggctttaa c 21 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta adhE_seq_RP) <400> 47 gtagacaaaa tcttccgcgc cg 22 <210> 48 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pflB_RP) <400> 48 ccaaaggtga ctggcagaat gaagtaaacg tccgtgactt catttaaagt ccttcctggc 60 tggcgctact gaagcgacca ccaccctg 88 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pflB_seq_FP) <400> 49 tgcagagaag tgaactgtgc c 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta pflB_seq_RP) <400> 50 cagaaaaact acactccgta cg 22 <210> 51 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta iclR_FP) <400> 51 cccggcttgt tgcgccagtt ccgtgagtgc cacactgcca ttcagccacg cgttaaagac 60 tgaacctgtc cagtcgctgg tgcggtggc 89 <210> 52 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta iclR_RP) <400> 52 gccaccgcac cagcgactgg acaggttcag tctttaacgc gtggctgaat ggcagtgtgg 60 cactcacgga actggcgcaa caagccggg 89 <210> 53 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta iclR_seq_FP) <400> 53 gcgcgtttgg gcgagatc 18 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta iclR_seq_RP) <400> 54 ctgaaattac tggagtgg 18 <210> 55 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_FP) <400> 55 ggctttctgt gcaaagcgca ccacatcaaa ctgtttcagc gctgttagac ccacaccggg 60 cagccaaatt cagcgggcaa acgcagcag 89 <210> 56 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_RP) <400> 56 ctgctgcgtt tgcccgctga atttggctgc ccggtgtggg tctaacagcg ctgaaacagt 60 ttgatgtggt gcgctttgca cagaaagcc 89 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_seq_FP) <400> 57 cctgttccag atgggcataa tc 22 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_seq_RP) <400> 58 gggtctacga tgcgcaaatt attcg 25 <210> 59 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta thrB_RP) <400> 59 cttatcggca aagcgtccga ggttgttgag actgaatgtc tctgttatgc accatcaaca 60 ggtgtcaccg ccgccccgag cacatcaaac 90 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta thrB_seq_FP) <400> 60 ttgctcggag atgtagtcac gg 22 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta thrB_seq_RP) <400> 61 gcgatactgc gccggtaaaa tag 23 <210> 62 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta metA_FP) <400> 62 gaagaaaacg tctttgtgat gacaacttct cgtgcgtctg gttaattaac ctgatgccga 60 agaagattga aactgaaaat cagtttctg 89 <210> 63 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta metA_RP) <400> 63 cagaaactga ttttcagttt caatcttctt cggcatcagg ttaattaacc agacgcacga 60 gaagttgtca tcacaaagac gttttcttc 89 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta metA_seq_FP) <400> 64 ctggtcagga aattcgtcca c 21 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta metA_seq_RP) <400> 65 cgaatcaacg ctgccggaaa g 21 <210> 66 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lacI_RP) <400> 66 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttgacga tggcggagct 60 gaattacatt cccaaccgcg tggcacaac 89 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lacI_seq_FP) <400> 67 gatatttatg ccagccagcc 20 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lacI_seq_RP) <400> 68 ccacgtttct gcgaaaacgc ggg 23 <210> 69 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Ptrc-acs-FP) <400> 69 gtttaatcgg tacccgggga tcgcggccgc cgcgcccttc ctgccagtca attttc 56 <210> 70 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Ptrc-acs-RP) <400> 70 cttttaagaa ggagatatac atatgagcca aattcacaaa cacacc 46 <210> 71 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Ptrc-acs-FP) <400> 71 ggtgtgtttg tgaatttggc tcatatgtat atctccttct taaaag 46 <210> 72 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Ptrc-acs-RP) <400> 72 cggcgtgcgt ttatttttat ccttgtcatc gactgcacgg tgcaccaatg c 51 <210> 73 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-Ptrc-acs-FP) <400> 73 gcattggtgc accgtgcagt cgatgacaag gataaaaata aacgcacgcc g 51 <210> 74 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-Ptrc-acs-RP) <400> 74 gggcgcgcgc cattctccgg tcgactctag atcatgccgt catcccac 48 <210> 75 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-ldhA-FP) <400> 75 atcgcggccg ctgtctgttt tgcggtc 27 <210> 76 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-ldhA-RP) <400> 76 ctggagaaag tcttatgtaa tcttgccgct cccc 34 <210> 77 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-ldhA-FP) <400> 77 ggggagcggc aagattacat aagactttct ccag 34 <210> 78 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-ldhA-RP) <400> 78 ctagaggatc ccaagcagaa tcaagttcta ccg 33 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Gn-ldhA-FP) <400> 79 gatggtacgg cgattgggat g 21 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Gn-ldhA-RP) <400> 80 gccagggaga aaaaatcag 19 <210> 81 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-iclR-FP) <400> 81 ctgcgcggac gctgaggatc ccggtgatcc cgtcctctca cg 42 <210> 82 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-iclR-RP) <400> 82 ttcgaacccc agagtcccgc ctgccgctcg taggtcctg 39 <210> 83 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-iclR-FP) <400> 83 gcaggaccta cgagcggcag gcgggactct ggggttcgaa atg 43 <210> 84 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-iclR-RP) <400> 84 tgcctgcagg tcgactctag attatttgtt aactgttaat tgtccttgtt c 51 <210> 85 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Gn-iclR-FP) <400> 85 ctgaacagca ggtcgtcc 18 <210> 86 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Gn-iclR-RP) <400> 86 gcgtcgaaac cttcgatg 18 <210> 87 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP1 promoter) <400> 87 aaaaagagta ttgacttcgc atctttttgt acctataatg tgtgga 46 <210> 88 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP2 promoter) <400> 88 aaaaaattta tttgcttatt aattcatccg gctcgtatat gtgtgga 47 <210> 89 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP3 promoter) <400> 89 ttgacatcag gaaaattttt ctgtataatg tgtgga 36 <210> 90 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP4 promoter) <400> 90 ttgacaatta atcatccggc tcgtaattta tgtgga 36 <210> 91 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP5 promoter) <400> 91 aaaaaattta tttgctttcg catctttttg tacctataag tgtgga 46 <210> 92 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP6 promoter) <400> 92 aaaaaattta tttgctttcg catctttttg tacctgtaat gtgtgga 47 <210> 93 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP7 promoter) <400> 93 ttcactttta atcatccggc tcgtataatg tgtgga 36 <210> 94 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP8 promoter) <400> 94 tttcaattta atcatccggc tcgtataatg tgtgga 36 <210> 95 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (SP9 promoter) <400> 95 ttccctatta atcatccggc tcgtataatg tgtgga 36 <210> 96 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT_Aspatate transaminase_M.loti_ANN59010.1_F primer) <400> 96 gaccatggaa ttcatggaag agtttcacaa ggtc 34 <210> 97 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT_Aspatate transaminase_M.loti_ANN59010.1_R primer) <400> 97 acagccaagc ttttaccggt gggcggacag c 31 <210> 98 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase M. loti ANN57047.1_F primer) <400> 98 gaccatggaa ttcatgctcc acacgatttc g 31 <210> 99 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase M. loti ANN57047.1_R primer) <400> 99 agtttggatc ctcacttggc gaggaacg 28 <210> 100 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase A. tumefaciens ACM26415.1_F primer) <400> 100 gaccatggaa ttcatggaag agtttcataa ag 32 <210> 101 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase A. tumefaciens ACM26415.1_R primer) <400> 101 agtttggatc cttaagaacg atgagcg 27 <210> 102 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase B. subtilis AQR85543.1_F primer) <400> 102 ttcagaattc aaaagatctt ttaagaagga gatatacata tgcagagcaa aggcg 55 <210> 103 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase B. subtilis AQR85543.1_R primer) <400> 103 agtttggatc cttaatggtg atggtgatgg tg 32 <210> 104 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase E. coli AVI55449.1_F primer) <400> 104 gaccatggaa ttcatgactg aaccccgttc ag 32 <210> 105 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase E. coli AVI55449.1_R primer) <400> 105 acagccaagc ttttagtccg ccttcgccgg c 31 <210> 106 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (bcAT branched chain transaminase D. mccartyi AQY72500.1_F primer) <400> 106 gaaggagata tacatatggc tagcatgac 29 <210> 107 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (bcAT branched chain transaminase D. mccartyi AQY72500.1_R primer) <400> 107 ctttgttagc agccggatct cagtggtggt ggtgg 35 <210> 108 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (bcAT branched chain transaminase E. coli AVI53929.1_F primer) <400> 108 aagaaggaga tatacatatg gctagcatga ctgg 34 <210> 109 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (bcAT branched chain transaminase E. coli AVI53929.1_R primer) <400> 109 ccggatctca gtggtggtgg tggtggtgct cg 32 <210> 110 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (bcAT branched chain transaminase E. spp AOL13801.1_F primer) <400> 110 taagaaggag atatacatat ggctagcatg actgg 35 <210> 111 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (bcAT branched chain transaminase E. spp AOL13801.1_R primer) <400> 111 gtgctcgagt tgattaactt gatctaacca gccgtat 37 <210> 112 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase E. coli AUV21492.1_F primer) <400> 112 taagaaggag atatacatat gtttcaaaaa gttg 34 <210> 113 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase E. coli AUV21492.1_R primer) <400> 113 gtgctcgagt tgattaagca gataatcg 28 <210> 114 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase E. coli AVI54196.1_F primer) <400> 114 gaccatggaa ttcatggctg acactcgccc tg 32 <210> 115 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase E. coli AVI54196.1_R primer) <400> 115 aacagccaag cttttattcc gcgttttcgt g 31 <210> 116 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. denitrificans AGI22743.1_F primer) <400> 116 cagaattcaa aagatctttt aagaaggaga tatacatatg actgaacccc g 51 <210> 117 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. denitrificans AGI22743.1_R primer) <400> 117 agatccttac tcgagtttgg atccttagtc cgccttcgcc 40 <210> 118 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. putida AE015451.2_F primer) <400> 118 cagaattcaa aagatctttt aagaaggaga tatacatatg gccaacccag gttcg 55 <210> 119 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. putida AE015451.2_R primer) <400> 119 ctcgagtttg gatccttact tacgagtcag gcc 33 <210> 120 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1_F primer) <400> 120 ttcagaattc aaaagatctt ttaagaagga gatatacata tggctgacac tcgc 54 <210> 121 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1_R primer) <400> 121 cgagtttgga tccttattcc gcgttttcgt g 31 <210> 122 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1 A140P-Y275D_F primer) <400> 122 ttcagaattc aaaagatctt ttaagaagga gatatacata tggctgacac tcgc 54 <210> 123 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1 A140P-Y275D_R primer) <400> 123 cgagtttgga tccttattcc gcgttttcgt g 31 <210> 124 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT aspatate transaminase P. denitrificans AGI22891.1_F primer) <400> 124 ttcagaattc aaaagatctt ttaagaagga gatatacata tgctcggacc cggcg 55 <210> 125 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT aspatate transaminase P. denitrificans AGI22891.1_R primer) <400> 125 tcgagtttgg atcctcagcg attctggtgc gcg 33 <210> 126 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase P. denitrificans AGI22971.1_F primer) <400> 126 gtggtggtgg tggtggtgct cgagcagttt caggcgcagc gc 42 <210> 127 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase P. denitrificans AGI22971.1_R primer) <400> 127 aactttaaga aggagatata catatgatga gcaagttctg gagtccc 47 <210> 128 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase P. fluorescens AUM68313.1_F primer) <400> 128 gtggtggtgg tggtggtgct cgaggagttc gccgagggc 39 <210> 129 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase P. fluorescens AUM68313.1_R primer) <400> 129 actttaagaa ggagatatac atatgatgag taaattctgg agcccg 46 <210> 130 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA Library F) <400> 130 ctcactatag gggaattgtg agcggataac 30 <210> 131 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA Library R) <400> 131 gctagttatt gctcagcggt ggcagc 26 <210> 132 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA K14 F) <400> 132 gaaggaattg cctnnkcaat tcttcgcttc 30 <210> 133 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA K14 R) <400> 133 gaagcgaaga attgmnnagg caattccttc 30 <210> 134 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA G40 F) <400> 134 caatctggga cagnnkaatc cagatcagc 29 <210> 135 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA G40 R) <400> 135 gctgatctgg attmnnctgt cccagattg 29 <210> 136 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA N178 F) <400> 136 gaattatccg aatnnkccga ctggagctg 29 <210> 137 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA N178 R) <400> 137 cagctccagt cggmnnattc ggataattc 29 <210> 138 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA Y364 F) <400> 138 gaatatggcg agggcnnkgt cagagtcg 28 <210> 139 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA Y364 R) <400> 139 cgactctgac mnngccctcg ccatattc 28 <210> 140 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (lactate dehdydrogenases(ldhO) Alcaligenes eutrophus(Ae) YP725182.1_F primer) <400> 140 ataacatatg aagatctccc tcaccagcgc 30 <210> 141 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (lactate dehdydrogenases(ldhO) Alcaligenes eutrophus(Ae) YP725182.1_R primer) <400> 141 taataggatc ctcagtgatg gtgatggtga tgggccgtgg ggacggccac gttg 54 <210> 142 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Cupriavidus basilensis(Cb) WP 043344208.1_F primer) <400> 142 ataacatatg aagataacac tgcaatc 27 <210> 143 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Cupriavidus basilensis(Cb) WP 043344208.1_R primer) <400> 143 taataggatc ctcagtggtg atgatggtga tggcccgccg gcagtgccac gccaagttc 59 <210> 144 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Achromobacter xylosoxidans(Ax) WP 006389860.1_F primer) <400> 144 ataacatatg aagatctcca ttacccaag 29 <210> 145 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Achromobacter xylosoxidans(Ax) WP 006389860.1_R primer) <400> 145 taataggatc ctcagtggtg atgatggtga tgaggcaacg cgtcagc 47 <210> 146 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Burkholderia glumae(Bg) YP 002909484.1_F primer) <400> 146 ataacatatg cagatatccc tcgacgatg 29 <210> 147 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Burkholderia glumae(Bg) YP 002909484.1_R primer) <400> 147 taataggatc cctagtggtg atgatggtga tgggcccgtg cggccggcgg caccacgccg 60 agttcgtc 68 <210> 148 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Escherichia fergusonii(Ef) WP 002431747.1_F primer) <400> 148 ataacatatg tatgggtaca gataccttc 29 <210> 149 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate/L-lactate dehydrogenase (ldh-2) Escherichia fergusonii(Ef) WP 002431747.1_R primer) <400> 149 taataggatc cttagtggtg atgatggtga tgatgctgat tcctgaggat gtaac 55 <210> 150 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-2-hydroxyacid dehydrogenase (ldh-2) Escherichia coli(Ec) WP024240865.1_F primer) <400> 150 ataacatatg atgtcattac aaattgctg 29 <210> 151 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-2-hydroxyacid dehydrogenase (ldh-2) Escherichia coli(Ec) WP024240865.1_R primer) <400> 151 taataggatc ctcagtggtg atgatggtga tgtccagcta atgctgattc ctg 53 <210> 152 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-2-hydroxyacid dehydrogenase (ldhA) Lactobacillus mali(Lm) WP 003689565.1_F primer) <400> 152 ataacatatg acaagaataa ttgcttatca tg 32 <210> 153 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-2-hydroxyacid dehydrogenase (ldhA) Lactobacillus mali(Lm) WP 003689565.1_R primer) <400> 153 taataggatc cttagtggtg atgatggtga tgttctccct tgaaacttat ttcatgtg 58 <210> 154 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase(ldhA) Escherichia coli K-12(Eck) NP 415898.1_F primer) <400> 154 ataacatatg aaactcgccg tttatag 27 <210> 155 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenase(ldhA) Escherichia coli K-12(Eck) NP 415898.1_R primer) <400> 155 taataggatc cttagtggtg atgatggtga tgaaccagtt cgttcgggca g 51 <210> 156 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH1_FP) <400> 156 ctatatcagc cacggcctgt cgagtctgcc caactaccgc accgccctcg 50 <210> 157 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH1_RP) <400> 157 cgagggcggt gcggtagttg ggcagactcg acaggccgtg gctgatatag 50 <210> 158 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH2_FP) <400> 158 ctatatcagc cacggcctgt cgactctgcc caactaccgc accgccctcg 50 <210> 159 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH2_RP) <400> 159 cgagggcggt gcggtagttg ggcagagtcg acaggccgtg gctgatatag 50 <210> 160 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH3_FP) <400> 160 ctatatcagc cacggcctgt cgaatctgcc caactaccgc accgccctcg 50 <210> 161 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH3_RP) <400> 161 cgagggcggt gcggtagttg ggcagattcg acaggccgtg gctgatatag 50 <210> 162 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH4_FP) <400> 162 ctatatcagc cacggcctgt cggacctgcc caactaccgc accgccctcg 50 <210> 163 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH4_RP) <400> 163 cgagggcggt gcggtagttg ggcaggtccg acaggccgtg gctgatatag 50 <210> 164 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH5_FP) <400> 164 ctatatcagc cacggcctgt cgaatctgcc caactaccgc accgccctcg 50 <210> 165 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH5_RP) <400> 165 cgagggcggt gcggtagttg ggcagattcg acaggccgtg gctgatatag 50 <210> 166 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldh) Escherichia coli K-12 NP 415898.1_F primer) <400> 166 ataacatatg aaactcgccg tttatag 27 <210> 167 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldh) Escherichia coli K-12 NP 415898.1_R primer) <400> 167 taataggatc cttagtggtg atgatggtga tgaaccagtt cgttcgggca g 51 <210> 168 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases Lactobacillus helveticus WP 003628108.1 1_F primer) <400> 168 ataacatatg acaaaggttt ttgc 24 <210> 169 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases Lactobacillus helveticus WP 003628108.1 1_R primer) <400> 169 taataggatc ctcagtggtg atgatggtga tgaaacttgt tcttgttcaa agcaac 56 <210> 170 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate/D-glycerate dehydrogenase (ldhD) Pediococcus acidilactici X70925.1_F primer) <400> 170 ataacatatg aagattattg cttatgg 27 <210> 171 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate/D-glycerate dehydrogenase (ldhD) Pediococcus acidilactici X70925.1_R primer) <400> 171 taataggatc ctcagtggtg atgatggtga tgctcaaact taacttcatt ctttg 55 <210> 172 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Pseudomonas aeruginosa NP_249618.1_F primer) <400> 172 ataacatatg cgcatcctgt tcttcag 27 <210> 173 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Pseudomonas aeruginosa NP_249618.1_R primer) <400> 173 taataggatc cctagtggtg atgatggtga tgggcccgga cccgattgcg 50 <210> 174 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Clostridium perfringens 13 NP_561446.1_F primer) <400> 174 ataacatatg atgataaaat tagtatgtta t 31 <210> 175 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Clostridium perfringens 13 NP_561446.1_R primer) <400> 175 taataggatc cttagtggtg atgatggtga tgatgatgct attgcatttt tacaagtatt 60 tc 62 <210> 176 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus reuteri ZP_03974385.1_F primer) <400> 176 ataacatatg aaaggaatgg gaaaac 26 <210> 177 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus reuteri ZP_03974385.1_R primer) <400> 177 taataggatc ctcagtggtg atgatggtga tgcattctta tttcatttcg tg 52 <210> 178 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Leuconostoc mesenteroides cremoris ZP_03913173.1_F primer) <400> 178 ataacatatg aagatttttg cttacg 26 <210> 179 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Leuconostoc mesenteroides cremoris ZP_03913173.1_R primer) <400> 179 taataggatc cttagtggtg atgatggtga tgatattcaa cagcaatagc tgg 53 <210> 180 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Oenococcus oeni ZP_01544962.1_F primer) <400> 180 ataacatatg aaaatttatg cttatg 26 <210> 181 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Oenococcus oeni ZP_01544962.1_R primer) <400> 181 taataggatc cttagtggtg atgatggtga tggaatttaa cgagattctt gtctc 55 <210> 182 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus delbrueckii subsp. bulgaricus CAI96942.1_F primer) <400> 182 ataacatatg actaaaattt ttgcttac 28 <210> 183 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus delbrueckii subsp. bulgaricus CAI96942.1_R primer) <400> 183 taataggatc cttagtggtg atgatggtga tggaaactcc agttaaggtt ggc 53 <210> 184 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH6_FP) <400> 184 catcactaag atgagcctgc gtaactccgg tgttgacaac atcgacatgg cta 53 <210> 185 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH6_RP) <400> 185 tagccatgtc gatgttgtca acaccggagt tacgcaggct catcttagtg atg 53 <210> 186 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH7_FP) <400> 186 catcactaag atgagcctgc gtaacaacgg tgttgacaac atcgacatgg cta 53 <210> 187 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH7_RP) <400> 187 tagccatgtc gatgttgtca acaccgttgt tacgcaggct catcttagtg atg 53 <210> 188 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH8_FP) <400> 188 catcactaag atgagcctgc gtaacaccgg tgttgacaac atcgacatgg cta 53 <210> 189 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH8_RP) <400> 189 tagccatgtc gatgttgtca acaccggtgt tacgcaggct catcttagtg atg 53 <210> 190 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH9_FP) <400> 190 catcactaag atgagcctgc gtaacgacgg tgttgacaac atcgacatgg cta 53 <210> 191 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH9_RP) <400> 191 tagccatgtc gatgttgtca acaccgtcgt tacgcaggct catcttagtg atg 53 <210> 192 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH10_FP) <400> 192 catcactaag atgagcctgc gtaacaaggg tgttgacaac atcgacatgg cta 53 <210> 193 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH10_RP) <400> 193 tagccatgtc gatgttgtca acacccttgt tacgcaggct catcttagtg atg 53 <210> 194 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp5-epd-FP) <400> 194 atctaagcgg ccgcggaatt atgcaattcg tggtac 36 <210> 195 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp5-epd-RP) <400> 195 aaaaaattta tttgctttcg catctttttg tacctataag tgtggaatga ccgtacgcgt 60 agcgataaat g 71 <210> 196 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5-epd-FP) <400> 196 gccagtttag tatcgacc 18 <210> 197 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5-epd-RP) <400> 197 acaaaagcat gatcctgttg aagatgcg 28 <210> 198 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- Psp5-epd-FP) <400> 198 catttatcgc tacgcgtacg gtcattccac acttataggt acaaaaagat gcgaaagcaa 60 ataaattttt ttc 73 <210> 199 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- Psp5-epd-RP) <400> 199 tagtaatcta gaggagttgg catctttctg cgatttc 37 <210> 200 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp5-dxs-FP) <400> 200 atctaagcgg ccgctcgaca tttcattgtc gttgag 36 <210> 201 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp5-dxs-RP) <400> 201 caaaaaattt atttgctttc gcatcttttt gtacctataa gtgtggaatg agttttgata 60 ttgccaaata c 71 <210> 202 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5-dsx-FP) <400> 202 gtaaagctta ccggaaagca gctgt 25 <210> 203 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5-dsx-RP) <400> 203 agcaactcga agcctgcgtt aag 23 <210> 204 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- Psp5-dxs-FP) <400> 204 tccacactta taggtacaaa aagatgcgaa agcaaataaa ttttttcggc accctggcgt 60 tttcccaacg ttgcag 76 <210> 205 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- Psp5-dsx-RP) <400> 205 tagtaatcta gaggtcatat gttcggcgtt agcacaaac 39 <210> 206 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp5-pdxj-FP) <400> 206 atctaagcgg ccgcggcttg cggcaaaggt cg 32 <210> 207 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp5-pdxj-RP) <400> 207 aaaaaattta tttgctttcg catctttttg tacctataag tgtggaatgg ctgaattact 60 gttaggcgtc 70 <210> 208 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5- pdxj-FP) <400> 208 ctttcacgtt gtgataggtc ag 22 <210> 209 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5- pdxj-RP) <400> 209 aaaaaattta tttgctttcg catctttttg tacctataag tgtggaatgg ctgaattact 60 gttaggcgtc 70 <210> 210 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-Psp5-pdxj-FP) <400> 210 gacgcctaac agtaattcag ccattccaca cttataggta caaaaagatg cgaaagc 57 <210> 211 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-Psp5-pdxj-RP) <400> 211 cgatctttaa gagtaactcc gatg 24 <210> 212 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp4-lldp-FP) <400> 212 atctaagcgg ccgcgattgg aatgcccatc gcac 34 <210> 213 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp4-lldp-RP) <400> 213 ttgacaatta atcatccggc tcgtaattta tgtggaatga atctctggca acaaaactac 60 g 61 <210> 214 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5-lldp-FP) <400> 214 ccatctgacc aatctcaaag ctgt 24 <210> 215 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp5-lldp-RP) <400> 215 ttgtgaacaa agcacctggt cgcg 24 <210> 216 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- Psp4-lldp-FP) <400> 216 tccacataaa ttacgagccg gatgattaat tgtcaattgg tagggccaat tcttgtg 57 <210> 217 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- Psp4-lldp-RP) <400> 217 tagtaatcta gagaaagaag tcgaaaaaaa cgaaatc 37 <210> 218 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-Psp4-ducA-FP) <400> 218 atctaagcgg ccgcgttgat gattgcatag ataacc 36 <210> 219 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US- Psp4-ducA -RP) <400> 219 ttgacaatta atcatccggc tcgtaattta tgtggaatgc aggttgctgg cggtctggac 60 tatctggttc 70 <210> 220 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp4-ducA-FP) <400> 220 ccatctgacc aatctcaaag ctgt 24 <210> 221 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (Psp4-ducA-RP) <400> 221 ttgtgaacaa agcacctggt cgcg 24 <210> 222 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-Psp4-ducA -FP) <400> 222 tccacataaa ttacgagccg gatgattaat tgtcaatgtt ttttaacaag ttgatattag 60 attg 64 <210> 223 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-Psp4-ducA -RP) <400> 223 tagtaatcta gagaaagaag tcgaaaaaaa cgaaatc 37 <210> 224 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-dsdx-FP) <400> 224 atctaagcgg ccgcggtatc cagcgacgat tttagcg 37 <210> 225 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-dsdx-RP) <400> 225 gtacgtctct ttgcgcttac atatctcacc ttcccctg 38 <210> 226 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (G-dsdx -FP) <400> 226 gcgtgtaaag tcacctaatg c 21 <210> 227 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (G-dsdx -RP) <400> 227 gggcatcagc agacatatg 19 <210> 228 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS-dsdx-FP) <400> 228 caggggaagg tgagatatgt aagcgcaaag agacgtac 38 <210> 229 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- dsdx-RP) <400> 229 tagtaatcta gacgctcata atgccgattg ataac 35 <210> 230 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-kgtP-FP) <400> 230 atctaagcgg ccgcggagct gatccgcgag catac 35 <210> 231 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US- kgtP -RP) <400> 231 acggcaggag acataatggc atgtagtgac gggtcagttg ccagac 46 <210> 232 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (G- kgtP -FP) <400> 232 tggcgtctgg atctggaaaa cg 22 <210> 233 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (G- kgtP -RP) <400> 233 tgtgtaagcg cagcgatgc 19 <210> 234 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- kgtP -FP) <400> 234 gtctggcaac tgacccgtca ctacatgcca ttatgtctcc tgccgt 46 <210> 235 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- kgtP-RP) <400> 235 tatagtaatc tagagaagct gttttggcgg atga 34 <210> 236 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US-actP-FP) <400> 236 atctaagcgg ccgccttatc tttattaagg taaac 35 <210> 237 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (US- actP -RP) <400> 237 agtcctgcat gaggtacaag catcatgtaa tctctcccct tccccggtcg tctg 54 <210> 238 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (G- actP -FP) <400> 238 ctgtgggatt tcgatagtat 20 <210> 239 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (G- actP -RP) <400> 239 agcaacctgg gcgatacctc gac 23 <210> 240 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- actP -FP) <400> 240 cagacgaccg gggaagggga gagattacat gatgcttgta cctcatgcag gact 54 <210> 241 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DS- actP-RP) <400> 241 tagtaatcta gaaagctgct tgaagagaag cag 33 <110> UNIST (ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> Method of producing 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutanoic acid <130> DPP20173744 <150> 10-2017-0088465 <151> 2017-07-12 <160> 241 <170> Kopatentin 3.0 <210> 1 <211> 1197 <212> DNA <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase [Bacillus subtilis subsp. subtilis str. 168]: AQR85543.1) <400> 1 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaattattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa taatccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggct acgtcagagt cggactttta acaagtgaag aaagacttaa ggaagccgct 1140 tatcgaattg gcaaactgaa cctgtttacc caaaaaagca ttgacaagac cttataa 1197 <210> 2 <211> 1197 <212> DNA <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase: AQR85543.1 (Y364Q)) <400> 2 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaattattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa taatccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggcc aggtcagagt cggactttta acaagtgaag aaagacttaa ggaagccgct 1140 tatcgaattg gcaaactgaa cctgtttacc caaaaaagca ttgacaagac cttataa 1197 <210> 3 <211> 1197 <212> DNA <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D)) <400> 3 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaattattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa tgacccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggcc aggtcagagt cggactttta acaagtgaag aaagacttaa ggaagccgct 1140 tatcgaattg gcaaactgaa cctgtttacc caaaaaagca ttgacaagac cttataa 1197 <210> 4 <211> 1196 <212> DNA <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D Y364Q)) <400> 4 atgaaatttg aacagtctca tgtattgaag gaattgccta aacaattctt cgcttctctg 60 gtgcagaaag tgaaccgaaa gcttgcagaa ggacatgacg ttatcaatct gggacaggga 120 aatccagatc agccgactcc tgagcatatt gttgaggaga tgaaacgagc tgtcgctgat 180 cctgagaatc ataaattattc gtcttttcgc ggctcatacc gtctgaagtc agcggctgct 240 gcattttaca aaagagaata cggcattgat ctggatccgg aaaccgaagt cgctgtatta 300 ttcggcggaa aagccggtct agtcgagctc ccgcaatgct tgttgaatcc cggtgatacg 360 attttagttc ccgatccggg ttatcctgat tactggtcgg gtgtcacact tgcaaaagca 420 aagatggaaa tgatgccgct cgtaaaggac agagcgtttc tccctgatta cagcagcata 480 accgctgaaa taagggaaca ggcgaaattg atgtatttga attatccgaa tgacccgact 540 ggagctgttg ctacttccga gttctttgaa gataccgtgc gttttgcggc tgaaaacgga 600 atctgcgtcg ttcacgattt tgcttacggc gctgtaggat ttgacggctg caagccttta 660 agctttttgc agactgaagg tgcgaaggat atcggaattg aaatttacac gctgtcaaaa 720 acgtataata tggcaggatg gcgggttggc tttgccgtcg gaaacgcttc ggtcattgaa 780 gcgatcaatc tttatcaaga ccatatgttt gtcagtcttt tcagagcgac tcaggaggct 840 gcagcagagg cactgttagc cgatcagacg tgtgtggcag agcaaaatgc caggtatgag 900 agcaggagaa acgcttggat cacggcatgc cgggaaatcg gctgggatgt aacagctccc 960 gcaggttctt tttttgcatg gctgcctgtg cctgaaggct atacttctga gcagttctca 1020 gatcttttgc tggaaaaagc gaatgtggca gttgcggctg gaaacggctt cggtgaatat 1080 ggcgagggcc aggtcgagtc ggacttttaa caagtgaaga aagacttaag gaagccgctt 1140 atcgaattgg caaactgaac ctgtttaccc aaaaaagcat tgacaagacc ttataa 1196 <210> 5 <211> 1050 <212> DNA <213> Artificial Sequence <220> Synthetic (L-lactate dehydrogenase [Alcaligenes eutrophus]: YP_725182.1) <400> 5 atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60 gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120 tatatcagcc acggcctgtc gatcctgccc aactaccgca ccgccctcga cggccacagc 180 gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240 ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300 gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360 ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420 atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480 ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540 accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600 gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660 gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720 gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780 ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840 ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900 gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960 gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020 tcgctcaacg tggccgtccc cacggcctga 1050 <210> 6 <211> 1050 <212> DNA <213> Artificial Sequence <220> Synthetic (L-lactate dehydrogenase: YP_725182.1 (I48N)) <400> 6 atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60 gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120 tatatcagcc acggcctgtc gaatctgccc aactaccgca ccgccctcga cggccacagc 180 gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240 ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300 gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360 ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420 atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480 ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540 accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600 gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660 gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720 gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780 ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840 ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900 gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960 gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020 tcgctcaacg tggccgtccc cacggcctga 1050 <210> 7 <211> 1050 <212> DNA <213> Artificial Sequence <220> Synthetic (L-lactate dehydrogenase: YP_725182.1 (I48T)) <400> 7 atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60 gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120 tatatcagcc acggcctgtc gactctgccc aactaccgca ccgccctcga cggccacagc 180 gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240 ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300 gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360 ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420 atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480 ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540 accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600 gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660 gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720 gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780 ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840 ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900 gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960 gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020 tcgctcaacg tggccgtccc cacggcctga 1050 <210> 8 <211> 1002 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase [Lactobacillus delbrueckii subsp. bulgaricus]: CAI96942.1) <400> 8 atgactaaaa tttttgctta cgcaattcgt gaagatgaaa agccattctt gaaggaatgg 60 gaagacgctc acaaggacgt cgaagttgaa tacactgaca agcttttgac cccagaaact 120 gttgctttgg caaagggtgc tgacggtgtt gttgtttacc aacaacttga ctacaccgct 180 gaaactctgc aagctttggc agacaacggc atcactaaga tgagcctgcg taacgttggt 240 gttgacaaca tcgacatggc taaggctaag gaacttggct tccaaatcac caacgttcca 300 gtttactcac caaacgccat cgcagaacac gctgctatcc aagctgcccg catcctgcgt 360 caagacaagg ctatggacga aaaggttgcc cgtcacgact tgcgttgggc accaactatc 420 ggccgtgaag ttcgcgacca agttgttggt gttataggta ctggccacat cggtcaagtc 480 ttcatgcaaa tcatggaagg cttcggcgct aaggttatcg cttacgacat cttccgcaac 540 ccagaattgg aaaagaaggg ctactacgta gactcacttg acgacctgta caagcaagct 600 gacgttattt ccctgcacgt tcctgacgtt ccagctaacg ttcacatgat caacgacgag 660 tcaatcgcta aaatgaagca agacgtagtt atcgttaacg tatcacgtgg tccattggtt 720 gacactgacg cggttatccg tggtttggac tcaggcaaga tcttcggtta cgcaatggac 780 gtttacgaag gtgaagttgg catcttcaac gaagactggg aaggcaagga attcccagac 840 gcacgtttag ctgacttaat cgctcgtcca aacgttctgg taactccaca cactgctttc 900 tacactactc acgctgttcg caacatggta gttaaggcct tcgacaacaa ccttgaattg 960 gttgaaggca aggaagctga aactccagtt aaggttggct aa 1002 <210> 9 <211> 1002 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase: CAI 96942.1 (V79N)) <400> 9 atgactaaaa tttttgctta cgcaattcgt gaagatgaaa agccattctt gaaggaatgg 60 gaagacgctc acaaggacgt cgaagttgaa tacactgaca agcttttgac cccagaaact 120 gttgctttgg caaagggtgc tgacggtgtt gttgtttacc aacaacttga ctacaccgct 180 gaaactctgc aagctttggc agacaacggc atcactaaga tgagcctgcg taacaacggt 240 gttgacaaca tcgacatggc taaggctaag gaacttggct tccaaatcac caacgttcca 300 gtttactcac caaacgccat cgcagaacac gctgctatcc aagctgcccg catcctgcgt 360 caagacaagg ctatggacga aaaggttgcc cgtcacgact tgcgttgggc accaactatc 420 ggccgtgaag ttcgcgacca agttgttggt gttataggta ctggccacat cggtcaagtc 480 ttcatgcaaa tcatggaagg cttcggcgct aaggttatcg cttacgacat cttccgcaac 540 ccagaattgg aaaagaaggg ctactacgta gactcacttg acgacctgta caagcaagct 600 gacgttattt ccctgcacgt tcctgacgtt ccagctaacg ttcacatgat caacgacgag 660 tcaatcgcta aaatgaagca agacgtagtt atcgttaacg tatcacgtgg tccattggtt 720 gacactgacg cggttatccg tggtttggac tcaggcaaga tcttcggtta cgcaatggac 780 gtttacgaag gtgaagttgg catcttcaac gaagactggg aaggcaagga attcccagac 840 gcacgtttag ctgacttaat cgctcgtcca aacgttctgg taactccaca cactgctttc 900 tacactactc acgctgttcg caacatggta gttaaggcct tcgacaacaa ccttgaattg 960 gttgaaggca aggaagctga aactccagtt aaggttggct aa 1002 <210> 10 <211> 1002 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase: CAI 96942.1 (V79T)) <400> 10 atgactaaaa tttttgctta cgcaattcgt gaagatgaaa agccattctt gaaggaatgg 60 gaagacgctc acaaggacgt cgaagttgaa tacactgaca agcttttgac cccagaaact 120 gttgctttgg caaagggtgc tgacggtgtt gttgtttacc aacaacttga ctacaccgct 180 gaaactctgc aagctttggc agacaacggc atcactaaga tgagcctgcg taacaccggt 240 gttgacaaca tcgacatggc taaggctaag gaacttggct tccaaatcac caacgttcca 300 gtttactcac caaacgccat cgcagaacac gctgctatcc aagctgcccg catcctgcgt 360 caagacaagg ctatggacga aaaggttgcc cgtcacgact tgcgttgggc accaactatc 420 ggccgtgaag ttcgcgacca agttgttggt gttataggta ctggccacat cggtcaagtc 480 ttcatgcaaa tcatggaagg cttcggcgct aaggttatcg cttacgacat cttccgcaac 540 ccagaattgg aaaagaaggg ctactacgta gactcacttg acgacctgta caagcaagct 600 gacgttattt ccctgcacgt tcctgacgtt ccagctaacg ttcacatgat caacgacgag 660 tcaatcgcta aaatgaagca agacgtagtt atcgttaacg tatcacgtgg tccattggtt 720 gacactgacg cggttatccg tggtttggac tcaggcaaga tcttcggtta cgcaatggac 780 gtttacgaag gtgaagttgg catcttcaac gaagactggg aaggcaagga attcccagac 840 gcacgtttag ctgacttaat cgctcgtcca aacgttctgg taactccaca cactgctttc 900 tacactactc acgctgttcg caacatggta gttaaggcct tcgacaacaa ccttgaattg 960 gttgaaggca aggaagctga aactccagtt aaggttggct aa 1002 <210> 11 <211> 1065 <212> DNA <213> Artificial Sequence <220> Synthetic (Paraoxonase PON1 variant: AAR95986.1 G3C9) <400> 11 atggctaaac tgacagcgct cacactcttg gggctgggat tggcactctt cgatggacag 60 aagtcttctt tccaaacacg atttaatgtt caccgtgaag taactccagt ggaacttcct 120 aactgtaatt tagttaaagg ggttgacaat ggttctgaag acttggaaat actgcccaat 180 ggactggctt tcatcagctc cggattaaag tatcctggaa taatgagctt tgaccctgat 240 aagtctggaa agatacttct aatggacctg aatgaggaag acccagtagt gttggaactg 300 ggcattactg gaaatacatt ggatatatct tcatttaacc ctcatgggat tagcacattc 360 acagatgaag ataacactgt gtacctactg gtggtaaacc atccagactc ctcgtccacc 420 gtggaggtgt ttaaatttca agaagaagaa aaatcacttt tgcatctgaa aaccatcaga 480 cacaagcttc tgcctagtgt gaatgacatt gtcgctgtgg gacctgaaca cttttatgcc 540 acaaatgatc actattttgc tgacccttac ttaaaatcct gggaaatgca tttgggatta 600 gcgtggtcat ttgttactta ttatagtccc aatgatgttc gagtagtggc agaaggattt 660 gattttgcta acggaatcaa catctcacca gacggcaagt atgtctatat agctgagttg 720 ctggctcata agatccatgt gtatgaaaag cacgctaatt ggactttaac tccattgaag 780 tccctcgact ttgacaccct tgtggataac atctctgtgg atcctgtgac aggggacctc 840 tgggtgggat gccatcccaa cggaatgcga atcttctact atgacccaaa gaatcctccc 900 ggctcagagg tgcttcgaat ccaggacatt ttatccgaag agcccaaagt gacagtggtt 960 tatgcagaaa atggcactgt gttacagggc agcacggtgg ccgctgtgta caaagggaaa 1020 ctgctgattg gcacagtgtt tcacaaagct ctttactgtg agctg 1065 <210> 12 <211> 398 <212> PRT <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase [Bacillus subtilis subsp. subtilis str. 168]: AQR85543.1 (TA4)) <400> 12 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asn Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Gly Gly Tyr Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 13 <211> 398 <212> PRT <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase: AQR85543.1 (Y364Q) _TA4-1) <400> 13 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asn Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Gln Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 14 <211> 398 <212> PRT <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D) _TA4-2) <400> 14 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asp Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Gln Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 15 <211> 398 <212> PRT <213> Artificial Sequence <220> Synthetic (Asparate aminotransferase: AQR85543.1 (N174D Y364Q) _TA4-6) <400> 15 Met Lys Phe Glu Gln Ser His Val Leu Lys Glu Leu Pro Lys Gln Phe 1 5 10 15 Phe Ala Ser Leu Val Gln Lys Val Asn Arg Lys Leu Ala Glu Gly His 20 25 30 Asp Val Ile Asn Leu Gly Gln Gly Asn Pro Asp Gln Pro Thr Pro Glu 35 40 45 His Ile Val Glu Glu Met Lys Arg Ala Val Ala Asp Pro Glu Asn His 50 55 60 Lys Tyr Ser Ser Phe Arg Gly Ser Tyr Arg Leu Lys Ser Ala Ala Ala 65 70 75 80 Ala Phe Tyr Lys Arg Glu Tyr Gly Ile Asp Leu Asp Pro Glu Thr Glu 85 90 95 Val Ala Val Leu Phe Gly Gly Lys Ala Gly Leu Val Glu Leu Pro Gln 100 105 110 Cys Leu Leu Asn Pro Gly Asp Thr Ile Leu Val Pro Asp Pro Gly Tyr 115 120 125 Pro Asp Tyr Trp Ser Gly Val Thr Leu Ala Lys Ala Lys Met Glu Met 130 135 140 Met Pro Leu Val Lys Asp Arg Ala Phe Leu Pro Asp Tyr Ser Ser Ile 145 150 155 160 Thr Ala Glu Ile Arg Glu Gln Ala Lys Leu Met Tyr Leu Asp Tyr Pro 165 170 175 Asn Asn Pro Thr Gly Ala Val Ala Thr Ser Glu Phe Phe Glu Asp Thr 180 185 190 Val Arg Phe Ala Ala Glu Asn Gly Ile Cys Val Val His Asp Phe Ala 195 200 205 Tyr Gly Ala Val Gly Phe Asp Gly Cys Lys Pro Leu Ser Phe Leu Gln 210 215 220 Thr Glu Gly Ala Lys Asp Ile Gly Ile Glu Ile Tyr Thr Leu Ser Lys 225 230 235 240 Thr Asn Met Ala Gly Trp Arg Val Gly Phe Ala Val Gly Asn Ala 245 250 255 Ser Val Ile Glu Ala Ile Asn Leu Tyr Gln Asp His Met Phe Val Ser 260 265 270 Leu Phe Arg Ala Thr Gln Glu Ala Ala Ala Glu Ala Leu Leu Ala Asp 275 280 285 Gln Thr Cys Val Ala Glu Gln Asn Ala Arg Tyr Glu Ser Arg Arg Asn 290 295 300 Ala Trp Ile Thr Ala Cys Arg Glu Ile Gly Trp Asp Val Thr Ala Pro 305 310 315 320 Ala Gly Ser Phe Phe Ala Trp Leu Pro Val Pro Glu Gly Tyr Thr Ser 325 330 335 Glu Gln Phe Ser Asp Leu Leu Leu Glu Lys Ala Asn Val Ala Val Ala 340 345 350 Ala Gly Asn Gly Phe Gly Glu Tyr Gly Glu Gly Gln Val Arg Val Gly 355 360 365 Leu Leu Thr Ser Glu Glu Arg Leu Lys Glu Ala Ala Tyr Arg Ile Gly 370 375 380 Lys Leu Asn Leu Phe Thr Gln Lys Ser Ile Asp Lys Thr Leu 385 390 395 <210> 16 <211> 349 <212> PRT <213> Artificial Sequence <220> Synthetic (L-lactate dehydrogenase [Alcaligenes eutrophus]: YP_725182.1_) <400> 16 Met Lys Ile Ser Leu Thr Ser Ala Arg Gln Leu Ala Arg Asp Ile Leu 1 5 10 15 Ala Ala Gln Gln Val Pro Ala Asp Ile Ala Asp Asp Val Ala Glu His 20 25 30 Leu Val Glu Ser Asp Arg Cys Gly Tyr Ile Ser His Gly Leu Ser Ile 35 40 45 Leu Pro Asn Tyr Arg Thr Ala Leu Asp Gly His Ser Val Asn Pro Gln 50 55 60 Gly Arg Ala Lys Cys Val Leu Asp Gln Gly Thr Leu Met Val Phe Asp 65 70 75 80 Gly Asp Gly Gly Phe Gly Gln His Val Gly Lys Ser Val Met Gln Ala 85 90 95 Ala Ile Glu Arg Val Arg Gln His Gly His Cys Ile Val Thr Leu Arg 100 105 110 Arg Ser His His Leu Gly Arg Met Gly His Tyr Gly Glu Met Ala Ala 115 120 125 Ala Ala Gly Phe Val Leu Leu Ser Phe Thr Asn Val Ile Asn Arg Ala 130 135 140 Pro Val Val Ala Pro Phe Gly Gly Arg Val Ala Arg Leu Thr Thr Asn 145 150 155 160 Pro Leu Cys Phe Ala Gly Pro Met Pro Asn Gly Arg Pro Pro Leu Val 165 170 175 Val Asp Ile Ala Thr Ser Ala Ile Ala Ile Asn Lys Ala Arg Val Leu 180 185 190 Ala Glu Lys Gly Glu Pro Ala Pro Glu Gly Ser Ile Gly Ala Asp 195 200 205 Gly Asn Pro Thr Thr Asp Ala Ser Thr Met Phe Gly Glu His Pro Gly 210 215 220 Ala Leu Leu Pro Phe Gly Gly His Lys Gly Tyr Ala Leu Gly Val Val 225 230 235 240 Ala Glu Leu Leu Ala Gly Val Leu Ser Gly Gly Gly Thr Ile Gln Pro 245 250 255 Asp Asn Pro Arg Gly Gly Val Ala Thr Asn Asn Leu Phe Ala Val Leu 260 265 270 Leu Asn Pro Ala Leu Asp Leu Gly Leu Asp Trp Gln Ser Ala Glu Val 275 280 285 Glu Ala Phe Val Arg Tyr Leu His Asp Thr Pro Pro Ala Pro Gly Val 290 295 300 Asp Arg Val Gln Tyr Pro Gly Glu Tyr Glu Ala Ala Asn Arg Ala Gln 305 310 315 320 Ala Ser Asp Thr Leu Asn Ile Asn Pro Ala Ile Trp Arg Asn Leu Glu 325 330 335 Arg Leu Ala Gln Ser Leu Asn Val Ala Val Pro Thr Ala 340 345 <210> 17 <211> 349 <212> PRT <213> Artificial Sequence <220> Synthetic (L-lactate dehydrogenase: YP_725182.1 (I48T)) <400> 17 Met Lys Ile Ser Leu Thr Ser Ala Arg Gln Leu Ala Arg Asp Ile Leu 1 5 10 15 Ala Ala Gln Gln Val Pro Ala Asp Ile Ala Asp Asp Val Ala Glu His 20 25 30 Leu Val Glu Ser Asp Arg Cys Gly Tyr Ile Ser His Gly Leu Ser Thr 35 40 45 Leu Pro Asn Tyr Arg Thr Ala Leu Asp Gly His Ser Val Asn Pro Gln 50 55 60 Gly Arg Ala Lys Cys Val Leu Asp Gln Gly Thr Leu Met Val Phe Asp 65 70 75 80 Gly Asp Gly Gly Phe Gly Gln His Val Gly Lys Ser Val Met Gln Ala 85 90 95 Ala Ile Glu Arg Val Arg Gln His Gly His Cys Ile Val Thr Leu Arg 100 105 110 Arg Ser His His Leu Gly Arg Met Gly His Tyr Gly Glu Met Ala Ala 115 120 125 Ala Ala Gly Phe Val Leu Leu Ser Phe Thr Asn Val Ile Asn Arg Ala 130 135 140 Pro Val Val Ala Pro Phe Gly Gly Arg Val Ala Arg Leu Thr Thr Asn 145 150 155 160 Pro Leu Cys Phe Ala Gly Pro Met Pro Asn Gly Arg Pro Pro Leu Val 165 170 175 Val Asp Ile Ala Thr Ser Ala Ile Ala Ile Asn Lys Ala Arg Val Leu 180 185 190 Ala Glu Lys Gly Glu Pro Ala Pro Glu Gly Ser Ile Gly Ala Asp 195 200 205 Gly Asn Pro Thr Thr Asp Ala Ser Thr Met Phe Gly Glu His Pro Gly 210 215 220 Ala Leu Leu Pro Phe Gly Gly His Lys Gly Tyr Ala Leu Gly Val Val 225 230 235 240 Ala Glu Leu Leu Ala Gly Val Leu Ser Gly Gly Gly Thr Ile Gln Pro 245 250 255 Asp Asn Pro Arg Gly Gly Val Ala Thr Asn Asn Leu Phe Ala Val Leu 260 265 270 Leu Asn Pro Ala Leu Asp Leu Gly Leu Asp Trp Gln Ser Ala Glu Val 275 280 285 Glu Ala Phe Val Arg Tyr Leu His Asp Thr Pro Pro Ala Pro Gly Val 290 295 300 Asp Arg Val Gln Tyr Pro Gly Glu Tyr Glu Ala Ala Asn Arg Ala Gln 305 310 315 320 Ala Ser Asp Thr Leu Asn Ile Asn Pro Ala Ile Trp Arg Asn Leu Glu 325 330 335 Arg Leu Ala Gln Ser Leu Asn Val Ala Val Pro Thr Ala 340 345 <210> 18 <211> 349 <212> PRT <213> Artificial Sequence <220> Synthetic (L-lactate dehydrogenase: YP_725182.1 (I48N)) <400> 18 Met Lys Ile Ser Leu Thr Ser Ala Arg Gln Leu Ala Arg Asp Ile Leu 1 5 10 15 Ala Ala Gln Gln Val Pro Ala Asp Ile Ala Asp Asp Val Ala Glu His 20 25 30 Leu Val Glu Ser Asp Arg Cys Gly Tyr Ile Ser His Gly Leu Ser Asn 35 40 45 Leu Pro Asn Tyr Arg Thr Ala Leu Asp Gly His Ser Val Asn Pro Gln 50 55 60 Gly Arg Ala Lys Cys Val Leu Asp Gln Gly Thr Leu Met Val Phe Asp 65 70 75 80 Gly Asp Gly Gly Phe Gly Gln His Val Gly Lys Ser Val Met Gln Ala 85 90 95 Ala Ile Glu Arg Val Arg Gln His Gly His Cys Ile Val Thr Leu Arg 100 105 110 Arg Ser His His Leu Gly Arg Met Gly His Tyr Gly Glu Met Ala Ala 115 120 125 Ala Ala Gly Phe Val Leu Leu Ser Phe Thr Asn Val Ile Asn Arg Ala 130 135 140 Pro Val Val Ala Pro Phe Gly Gly Arg Val Ala Arg Leu Thr Thr Asn 145 150 155 160 Pro Leu Cys Phe Ala Gly Pro Met Pro Asn Gly Arg Pro Pro Leu Val 165 170 175 Val Asp Ile Ala Thr Ser Ala Ile Ala Ile Asn Lys Ala Arg Val Leu 180 185 190 Ala Glu Lys Gly Glu Pro Ala Pro Glu Gly Ser Ile Gly Ala Asp 195 200 205 Gly Asn Pro Thr Thr Asp Ala Ser Thr Met Phe Gly Glu His Pro Gly 210 215 220 Ala Leu Leu Pro Phe Gly Gly His Lys Gly Tyr Ala Leu Gly Val Val 225 230 235 240 Ala Glu Leu Leu Ala Gly Val Leu Ser Gly Gly Gly Thr Ile Gln Pro 245 250 255 Asp Asn Pro Arg Gly Gly Val Ala Thr Asn Asn Leu Phe Ala Val Leu 260 265 270 Leu Asn Pro Ala Leu Asp Leu Gly Leu Asp Trp Gln Ser Ala Glu Val 275 280 285 Glu Ala Phe Val Arg Tyr Leu His Asp Thr Pro Pro Ala Pro Gly Val 290 295 300 Asp Arg Val Gln Tyr Pro Gly Glu Tyr Glu Ala Ala Asn Arg Ala Gln 305 310 315 320 Ala Ser Asp Thr Leu Asn Ile Asn Pro Ala Ile Trp Arg Asn Leu Glu 325 330 335 Arg Leu Ala Gln Ser Leu Asn Val Ala Val Pro Thr Ala 340 345 <210> 19 <211> 333 <212> PRT <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase [Lactobacillus delbrueckii subsp. bulgaricus]: CAI96942.1) <400> 19 Met Thr Lys Ile Phe Ala Tyr Ala Ile Arg Glu Asp Glu Lys Pro Phe 1 5 10 15 Leu Lys Glu Trp Glu Asp Ala His Lys Asp Val Glu Val Glu Tyr Thr 20 25 30 Asp Lys Leu Leu Thr Pro Glu Thr Val Ala Leu Ala Lys Gly Ala Asp 35 40 45 Gly Val Val Val Tyr Gln Gln Leu Asp Tyr Thr Ala Glu Thr Leu Gln 50 55 60 Ala Leu Ala Asp Asn Gly Ile Thr Lys Met Ser Leu Arg Asn Val Gly 65 70 75 80 Val Asp Asn Ile Asp Met Ala Lys Ala Lys Glu Leu Gly Phe Gln Ile 85 90 95 Thr Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ala Ala 100 105 110 Ile Gln Ala Ala Arg Ile Leu Arg Gln Asp Lys Ala Met Asp Glu Lys 115 120 125 Val Ala Arg His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val 130 135 140 Arg Asp Gln Val Val Gly Val Ile Gly Thr Gly His Ile Gly Gln Val 145 150 155 160 Phe Met Gln Ile Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp 165 170 175 Ile Phe Arg Asn Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser 180 185 190 Leu Asp Asp Leu Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro 195 200 205 Asp Val Pro Ala Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys 210 215 220 Met Lys Gln Asp Val Valle Val Asn Val Ser Ser Gly Pro Leu Val 225 230 235 240 Asp Thr Asp Ala Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly 245 250 255 Tyr Ala Met Asp Val Tyr Glu Gly Glu Val Gly Ile Phe Asn Glu Asp 260 265 270 Trp Glu Gly Lys Glu Phe Pro Asp Ala Arg Leu Ala Asp Leu Ile Ala 275 280 285 Arg Pro Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His 290 295 300 Ala Val Arg Asn A Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu 305 310 315 320 Val Glu Gly Lys Glu Ala Glu Thr Pro Val Lys Val Gly 325 330 <210> 20 <211> 333 <212> PRT <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase: CAI 96942.1 (V79T)) <400> 20 Met Thr Lys Ile Phe Ala Tyr Ala Ile Arg Glu Asp Glu Lys Pro Phe 1 5 10 15 Leu Lys Glu Trp Glu Asp Ala His Lys Asp Val Glu Val Glu Tyr Thr 20 25 30 Asp Lys Leu Leu Thr Pro Glu Thr Val Ala Leu Ala Lys Gly Ala Asp 35 40 45 Gly Val Val Val Tyr Gln Gln Leu Asp Tyr Thr Ala Glu Thr Leu Gln 50 55 60 Ala Leu Ala Asp Asn Gly Ile Thr Lys Met Ser Leu Arg Asn Thr Gly 65 70 75 80 Val Asp Asn Ile Asp Met Ala Lys Ala Lys Glu Leu Gly Phe Gln Ile 85 90 95 Thr Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ala Ala 100 105 110 Ile Gln Ala Ala Arg Ile Leu Arg Gln Asp Lys Ala Met Asp Glu Lys 115 120 125 Val Ala Arg His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val 130 135 140 Arg Asp Gln Val Val Gly Val Ile Gly Thr Gly His Ile Gly Gln Val 145 150 155 160 Phe Met Gln Ile Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp 165 170 175 Ile Phe Arg Asn Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser 180 185 190 Leu Asp Asp Leu Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro 195 200 205 Asp Val Pro Ala Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys 210 215 220 Met Lys Gln Asp Val Valle Val Asn Val Ser Ser Gly Pro Leu Val 225 230 235 240 Asp Thr Asp Ala Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly 245 250 255 Tyr Ala Met Asp Val Tyr Glu Gly Glu Val Gly Ile Phe Asn Glu Asp 260 265 270 Trp Glu Gly Lys Glu Phe Pro Asp Ala Arg Leu Ala Asp Leu Ile Ala 275 280 285 Arg Pro Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His 290 295 300 Ala Val Arg Asn A Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu 305 310 315 320 Val Glu Gly Lys Glu Ala Glu Thr Pro Val Lys Val Gly 325 330 <210> 21 <211> 333 <212> PRT <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase: CAI 96942.1 (V79N)) <400> 21 Met Thr Lys Ile Phe Ala Tyr Ala Ile Arg Glu Asp Glu Lys Pro Phe 1 5 10 15 Leu Lys Glu Trp Glu Asp Ala His Lys Asp Val Glu Val Glu Tyr Thr 20 25 30 Asp Lys Leu Leu Thr Pro Glu Thr Val Ala Leu Ala Lys Gly Ala Asp 35 40 45 Gly Val Val Val Tyr Gln Gln Leu Asp Tyr Thr Ala Glu Thr Leu Gln 50 55 60 Ala Leu Ala Asp Asn Gly Ile Thr Lys Met Ser Leu Arg Asn Asn Gly 65 70 75 80 Val Asp Asn Ile Asp Met Ala Lys Ala Lys Glu Leu Gly Phe Gln Ile 85 90 95 Thr Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ala Ala 100 105 110 Ile Gln Ala Ala Arg Ile Leu Arg Gln Asp Lys Ala Met Asp Glu Lys 115 120 125 Val Ala Arg His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val 130 135 140 Arg Asp Gln Val Val Gly Val Ile Gly Thr Gly His Ile Gly Gln Val 145 150 155 160 Phe Met Gln Ile Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp 165 170 175 Ile Phe Arg Asn Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser 180 185 190 Leu Asp Asp Leu Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro 195 200 205 Asp Val Pro Ala Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys 210 215 220 Met Lys Gln Asp Val Valle Val Asn Val Ser Ser Gly Pro Leu Val 225 230 235 240 Asp Thr Asp Ala Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly 245 250 255 Tyr Ala Met Asp Val Tyr Glu Gly Glu Val Gly Ile Phe Asn Glu Asp 260 265 270 Trp Glu Gly Lys Glu Phe Pro Asp Ala Arg Leu Ala Asp Leu Ile Ala 275 280 285 Arg Pro Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His 290 295 300 Ala Val Arg Asn A Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu 305 310 315 320 Val Glu Gly Lys Glu Ala Glu Thr Pro Val Lys Val Gly 325 330 <210> 22 <211> 355 <212> PRT <213> Artificial Sequence <220> Synthetic (Paraoxonase PON1 variant: AAR95986.1_G3C9) <400> 22 Met Ala Lys Leu Thr Ala Leu Thr Leu Leu Gly Leu Gly Leu Ala Leu 1 5 10 15 Phe Asp Gly Gln Lys Ser Ser Phe Gln Thr Arg Phe Asn Val His Arg 20 25 30 Glu Val Thr Pro Val Glu Leu Pro Asn Cys Asn Leu Val Lys Gly Val 35 40 45 Asp Asn Gly Ser Glu Asp Leu Glu Ile Leu Pro Asn Gly Leu Ala Phe 50 55 60 Ile Ser Ser Gly Leu Lys Tyr Pro Gly Ile Met Ser Phe Asp Pro Asp 65 70 75 80 Lys Ser Gly Lys Ile Leu Leu Met Asp Leu Asn Glu Glu Asp Pro Val 85 90 95 Val Leu Glu Leu Gly Ile Thr Gly Asn Thr Leu Asp Ile Ser Ser Phe 100 105 110 Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Thr Val Tyr 115 120 125 Leu Leu Val Val Asn His Pro Asp Ser Ser Ser Thr Val Glu Val Phe 130 135 140 Lys Phe Gln Glu Glu Glu Lys Ser Leu Leu His Leu Lys Thr Ile Arg 145 150 155 160 His Lys Leu Leu Pro Ser Val Asn Asp Ile Val Ala Val Gly Pro Glu 165 170 175 His Phe Tyr Ala Thr Asn Asp His Tyr Phe Ala Asp Pro Tyr Leu Lys 180 185 190 Ser Trp Glu Met His Leu Gly Leu Ala Trp Ser Phe Val Thr Tyr Tyr 195 200 205 Ser Pro Asn Val Val Val Ala Glu Gly Phe Asp Phe Ala Asn 210 215 220 Gly Ile Asn Ile Ser Pro Asp Gly Lys Tyr Val Tyr Ile Ala Glu Leu 225 230 235 240 Leu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thr Leu 245 250 255 Thr Pro Leu Lys Ser Leu Asp Phe Asp Thr Leu Val Asp Asn Ile Ser 260 265 270 Val Asp Pro Val Thr Gly Asp Leu Trp Val Gly Cys His Pro Asn Gly 275 280 285 Met Arg Ile Phe Tyr Tyr Asp Pro Lys Asn Pro Pro Gly Ser Glu Val 290 295 300 Leu Arg Ile Gln Asp Ile Leu Ser Glu Glu Pro Lys Val Thr Val Val 305 310 315 320 Tyr Ala Glu Asn Gly Thr Val Leu Gln Gly Ser Thr Val Ala Ala Val 325 330 335 Tyr Lys Gly Lys Leu Leu Ile Gly Thr Val Phe His Lys Ala Leu Tyr 340 345 350 Cys Glu Leu 355 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (synthetic promoter 8) <400> 23 tttcaattta atcatccggc tcgtataatg tgtgga 36 <210> 24 <211> 88 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pts_RP primer) <400> 24 cggaaccgcc tgcttctgcc ataacatgcg atacaacggc ggttacagca gaatacctgc 60 gataggcagt acggataccg gcagcatc 88 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pts_seq_FP primer) <400> 25 ggcgtcggtt ccgcgaattt c 21 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pts_seq_RP primer) <400> 26 ggtgcagacc aaacggtacc 20 <210> 27 <211> 88 <212> DNA <213> Artificial Sequence <220> Synthetic (delta eda_FP primer) <400> 27 caactgcaca ctcggtacgc agagtcactt ccagaacgcg cattacaccg cgtgttccag 60 tttttttacc acgataaccg gtacaacc 88 <210> 28 <211> 88 <212> DNA <213> Artificial Sequence <220> Synthetic (delta eda_RP primer) <400> 28 ggttgtaccg gttatcgtgg taaaaaaact ggaacacgcg gtgtaatgcg cgttctggaa 60 gtgactctgc gtaccgagtg tgcagttg 88 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (delta eda_seq_FP primer) <400> 29 ccgatggcaa aagcgttgg 19 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (delta eda_seq_RP primer) <400> 30 cgcgccagct tagtaatgc 19 <210> 31 <211> 90 <212> DNA <213> Artificial Sequence <220> Synthetic (delta poxB_RP primer) <400> 31 ggcaggggtg aaacgcatct ggggagtcac aggcgactct ctgaactaac catcgagtgg 60 atgtccaccc gccacgaaga agtggcggcc 90 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (delta poxB_seq_FP primer) <400> 32 tggaataacg cagcagttg 19 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (delta poxB_seq_RPprimer) <400> 33 ggtcttagtg acagtcttaa tc 22 <210> 34 <211> 87 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pta_FP primer) <400> 34 gaccagcgtc agccttggcg tgatccgtgc aatggaacgc aaataactca gccgcgtacc 60 ggtggcgatg cgcccgatca gactacg 87 <210> 35 <211> 87 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pta_RP primer) <400> 35 cgtagtctga tcgggcgcat cgccaccggt acgcggctga gttatttgcg ttccattgca 60 cggatcacgc caaggctgac gctggtc 87 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pta_seq_FP) <400> 36 ggcgttcgtc tgagcgtttt c 21 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pta_seq_RP) <400> 37 ggattcttgc agcttcgcc 19 <210> 38 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ackA_FP) <400> 38 gaaatttgcc atcatcgatg cagtaaatgg tgaagagtac ctttgaaagc acgtatcaaa 60 tggaaaatgg acggcaataa acaggaagc 89 <210> 39 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ackA_RP) <400> 39 gcttcctgtt tattgccgtc cattttccat ttgatacgtg ctttcaaagg tactcttcac 60 catttactgc atcgatgatg gcaaatttc 89 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ackA_seq_FP) <400> 40 tctggtttag ccgaatgttt cc 22 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ackA_seq_RP) <400> 41 atgttcagtt cttctaccgg 20 <210> 42 <211> 88 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ldhA_RP) <400> 42 gcaacaggtg aacgagtcct ttggctttga gctggaattt ttttaactgc caatggctgc 60 gaagcggtat gtattttcgt aaacgatg 88 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ldhA_seq_FP) <400> 43 ttttccggtg tcagcggg 18 <210> 44 <211> 16 <212> DNA <213> Artificial Sequence <220> Synthetic (delta ldhA_seq_RP) <400> 44 gactttctgc tgacgg 16 <210> 45 <211> 90 <212> DNA <213> Artificial Sequence <220> Synthetic (delta adhE_RP) <400> 45 gtaaaaaaag cccagcgtga atatgccagt ttcactcaag agcaataact gctgcagatg 60 ctcgaatccc actcgcgaaa atggccgttg 90 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (delta adhE_seq_FP) <400> 46 ggagctgtat gcggctttaa c 21 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (delta adhE_seq_RP) <400> 47 gtagacaaaa tcttccgcgc cg 22 <210> 48 <211> 88 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pflB_RP) <400> 48 ccaaaggtga ctggcagaat gaagtaaacg tccgtgactt catttaaagt ccttcctggc 60 tggcgctact gaagcgacca ccaccctg 88 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pflB_seq_FP) <400> 49 tgcagagaag tgaactgtgc c 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (delta pflB_seq_RP) <400> 50 cagaaaaact acactccgta cg 22 <210> 51 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta iclR_FP) <400> 51 cccggcttgt tgcgccagtt ccgtgagtgc cacactgcca ttcagccacg cgttaaagac 60 tgaacctgtc cagtcgctgg tgcggtggc 89 <210> 52 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta iclR_RP) <400> 52 gccaccgcac cagcgactgg acaggttcag tctttaacgc gtggctgaat ggcagtgtgg 60 cactcacgga actggcgcaa caagccggg 89 <210> 53 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta iclR_seq_FP) <400> 53 gcgcgtttgg gcgagatc 18 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> Synthetic (delta iclR_seq_RP) <400> 54 ctgaaattac tggagtgg 18 <210> 55 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_FP) <400> 55 ggctttctgt gcaaagcgca ccacatcaaa ctgtttcagc gctgttagac ccacaccggg 60 cagccaaatt cagcgggcaa acgcagcag 89 <210> 56 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_RP) <400> 56 ctgctgcgtt tgcccgctga atttggctgc ccggtgtggg tctaacagcg ctgaaacagt 60 ttgatgtggt gcgctttgca cagaaagcc 89 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_seq_FP) <400> 57 cctgttccag atgggcataa tc 22 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (delta lysA_seq_RP) <400> 58 gggtctacga tgcgcaaatt attcg 25 <210> 59 <211> 90 <212> DNA <213> Artificial Sequence <220> Synthetic (delta thrB_RP) <400> 59 cttatcggca aagcgtccga ggttgttgag actgaatgtc tctgttatgc accatcaaca 60 ggtgtcaccg ccgccccgag cacatcaaac 90 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (delta thrB_seq_FP) <400> 60 ttgctcggag atgtagtcac gg 22 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> Synthetic (delta thrB_seq_RP) <400> 61 gcgatactgc gccggtaaaa tag 23 <210> 62 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta metA_FP) <400> 62 gaagaaaacg tctttgtgat gacaacttct cgtgcgtctg gttaattaac ctgatgccga 60 agaagattga aactgaaaat cagtttctg 89 <210> 63 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta metA_RP) <400> 63 cagaaactga ttttcagttt caatcttctt cggcatcagg ttaattaacc agacgcacga 60 gaagttgtca tcacaaagac gttttcttc 89 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (delta metA_seq_FP) <400> 64 ctggtcagga aattcgtcca c 21 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (delta metA_seq_RP) <400> 65 cgaatcaacg ctgccggaaa g 21 <210> 66 <211> 89 <212> DNA <213> Artificial Sequence <220> Synthetic (delta lacI_RP) <400> 66 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttgacga tggcggagct 60 gaattacatt cccaaccgcg tggcacaac 89 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> Synthetic (delta lacI_seq_FP) <400> 67 gatatttatg ccagccagcc 20 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> Synthetic (delta lacI_seq_RP) <400> 68 ccacgtttct gcgaaaacgc ggg 23 <210> 69 <211> 56 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Ptrc-acs-FP) <400> 69 gtttaatcgg tacccgggga tcgcggccgc cgcgcccttc ctgccagtca attttc 56 <210> 70 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Ptrc-acs-RP) <400> 70 cttttaagaa ggagatatac atatgagcca aattcacaaa cacacc 46 <210> 71 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (Ptrc-acs-FP) <400> 71 ggtgtgtttg tgaatttggc tcatatgtat atctccttct taaaag 46 <210> 72 <211> 51 <212> DNA <213> Artificial Sequence <220> Synthetic (Ptrc-acs-RP) <400> 72 cggcgtgcgt ttatttttat ccttgtcatc gactgcacgg tgcaccaatg c 51 <210> 73 <211> 51 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Ptrc-acs-FP) <400> 73 gcattggtgc accgtgcagt cgatgacaag gataaaaata aacgcacgcc g 51 <210> 74 <211> 48 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Ptrc-acs-RP) <400> 74 gggcgcgcgc cattctccgg tcgactctag atcatgccgt catcccac 48 <210> 75 <211> 27 <212> DNA <213> Artificial Sequence <220> Synthetic (US-ldhA-FP) <400> 75 atcgcggccg ctgtctgttt tgcggtc 27 <210> 76 <211> 34 <212> DNA <213> Artificial Sequence <220> Synthetic (US-IdhA-RP) <400> 76 ctggagaaag tcttatgtaa tcttgccgct cccc 34 <210> 77 <211> 34 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-ldhA-FP) <400> 77 ggggagcggc aagattacat aagactttct ccag 34 <210> 78 <211> 33 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-ldhA-RP) <400> 78 ctagaggatc ccaagcagaa tcaagttcta ccg 33 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (Gn-ldhA-FP) <400> 79 gatggtacgg cgattgggat g 21 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (Gn-ldhA-RP) <400> 80 gccagggaga aaaaatcag 19 <210> 81 <211> 42 <212> DNA <213> Artificial Sequence <220> Synthetic (US-iclR-FP) <400> 81 ctgcgcggac gctgaggatc ccggtgatcc cgtcctctca cg 42 <210> 82 <211> 39 <212> DNA <213> Artificial Sequence <220> Synthetic (US-iclR-RP) <400> 82 ttcgaacccc agagtcccgc ctgccgctcg taggtcctg 39 <210> 83 <211> 43 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-iclR-FP) <400> 83 gcaggaccta cgagcggcag gcgggactct ggggttcgaa atg 43 <210> 84 <211> 51 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-iclR-RP) <400> 84 tgcctgcagg tcgactctag attatttgtt aactgttaat tgtccttgtt c 51 <210> 85 <211> 18 <212> DNA <213> Artificial Sequence <220> Synthetic (Gn-iclR-FP) <400> 85 ctgaacagca ggtcgtcc 18 <210> 86 <211> 18 <212> DNA <213> Artificial Sequence <220> Synthetic (Gn-iclR-RP) <400> 86 gcgtcgaaac cttcgatg 18 <210> 87 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (SP1 promoter) <400> 87 aaaaagagta ttgacttcgc atctttttgt acctataatg tgtgga 46 <210> 88 <211> 47 <212> DNA <213> Artificial Sequence <220> Synthetic (SP2 promoter) <400> 88 aaaaaattta tttgcttatt aattcatccg gctcgtatat gtgtgga 47 <210> 89 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (SP3 promoter) <400> 89 ttgacatcag gaaaattttt ctgtataatg tgtgga 36 <210> 90 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (SP4 promoter) <400> 90 ttgacaatta atcatccggc tcgtaattta tgtgga 36 <210> 91 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (SP5 promoter) <400> 91 aaaaaattta tttgctttcg catctttttg tacctataag tgtgga 46 <210> 92 <211> 47 <212> DNA <213> Artificial Sequence <220> Synthetic (SP6 promoter) <400> 92 aaaaaattta tttgctttcg catctttttg tacctgtaat gtgtgga 47 <210> 93 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (SP7 promoter) <400> 93 ttcactttta atcatccggc tcgtataatg tgtgga 36 <210> 94 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (SP8 promoter) <400> 94 tttcaattta atcatccggc tcgtataatg tgtgga 36 <210> 95 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (SP9 promoter) <400> 95 ttccctatta atcatccggc tcgtataatg tgtgga 36 <210> 96 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT_Aspatate transaminase_M.loti_ANN59010.1_F primer) <400> 96 gaccatggaa ttcatggaag agtttcacaa ggtc 34 <210> 97 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT_Aspatate transaminase_M.loti_ANN59010.1_R primer) <400> 97 acagccaagc ttttaccggt gggcggacag c 31 <210> 98 <211> 31 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aspAT Aspatate transaminase M. loti ANN57047.1_F primer) <400> 98 gaccatggaa ttcatgctcc acacgatttc g 31 <210> 99 <211> 28 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aspAT Aspatate transaminase M. loti ANN57047.1_R primer) <400> 99 agtttggatc ctcacttggc gaggaacg 28 <210> 100 <211> 32 <212> DNA <213> Artificial Sequence <220> Synthetic (aspAT Aspatate transaminase A. tumefaciens ACM26415.1_F primer) <400> 100 gaccatggaa ttcatggaag agtttcataa ag 32 <210> 101 <211> 27 <212> DNA <213> Artificial Sequence <220> Synthetic (aspAT Aspatate transaminase A. tumefaciens ACM26415.1_R primer) <400> 101 agtttggatc cttaagaacg atgagcg 27 <210> 102 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspAT Aspatate transaminase B. subtilis AQR85543.1_F primer) <400> 102 ttcagaattc aaaagatctt ttaagaagga gatatacata tgcagagcaa aggcg 55 <210> 103 <211> 32 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aspAT Aspatate transaminase B. subtilis AQR85543.1_R primer) <400> 103 agtttggatc cttaatggtg atggtgatgg tg 32 <210> 104 <211> 32 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aspAT Aspatate transaminase E. coli AVI55449.1_F primer) <400> 104 gaccatggaa ttcatgactg aaccccgttc ag 32 <210> 105 <211> 31 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aspAT Aspatate transaminase E. coli AVI55449.1_R primer) <400> 105 acagccaagc ttttagtccg ccttcgccgg c 31 <210> 106 <211> 29 <212> DNA <213> Artificial Sequence <220> Synthetic (bcAT branched chain transaminase D. mccartyi AQY72500.1_F primer) <400> 106 gaaggagata tacatatggc tagcatgac 29 <210> 107 <211> 35 <212> DNA <213> Artificial Sequence <220> Synthetic (bcAT branched chain transaminase D. mccartyi AQY72500.1_R primer) <400> 107 ctttgttagc agccggatct cagtggtggt ggtgg 35 <210> 108 <211> 34 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (bcAT branched chain transaminase E. coli AVI53929.1_F primer) <400> 108 aagaaggaga tatacatatg gctagcatga ctgg 34 <210> 109 <211> 32 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (bcAT branched chain transaminase E. coli AVI53929.1_R primer) <400> 109 ccggatctca gtggtggtgg tggtggtgct cg 32 <210> 110 <211> 35 <212> DNA <213> Artificial Sequence <220> Synthetic (bcAT branched chain transaminase E. spp AOL 13801.1_F primer) <400> 110 taagaaggag atatacatat ggctagcatg actgg 35 <210> 111 <211> 37 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (bcAT branched chain transaminase E. spp AOL13801.1_R primer) <400> 111 gtgctcgagt tgattaactt gatctaacca gccgtat 37 <210> 112 <211> 34 <212> DNA <213> Artificial Sequence <220> Synthetic (aroAT acromatic transaminase E. coli AUV21492.1_F primer) <400> 112 taagaaggag atatacatat gtttcaaaaa gttg 34 <210> 113 <211> 28 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aroAT acromatic transaminase E. coli AUV21492.1_R primer) <400> 113 gtgctcgagt tgattaagca gataatcg 28 <210> 114 <211> 32 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aroAT acromatic transaminase E. coli AVI54196.1_F primer) <400> 114 gaccatggaa ttcatggctg acactcgccc tg 32 <210> 115 <211> 31 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Synthetic (aroAT acromatic transaminase E. coli AVI54196.1_R primer) <400> 115 aacagccaag cttttattcc gcgttttcgt g 31 <210> 116 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. denitrificans AGI22743.1_F primer) <400> 116 cagaattcaa aagatctttt aagaaggaga tatacatatg actgaacccc g 51 <210> 117 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. denitrificans AGI22743.1_R primer) <400> 117 agatccttac tcgagtttgg atccttagtc cgccttcgcc 40 <210> 118 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. putida AE015451.2_F primer) <400> 118 cagaattcaa aagatctttt aagaaggaga tatacatatg gccaacccag gttcg 55 <210> 119 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaAT alanine transaminase P. putida AE015451.2_R primer) <400> 119 ctcgagtttg gatccttact tacgagtcag gcc 33 <210> 120 <211> 54 <212> DNA <213> Artificial Sequence <220> Synthetic (alaC alanine transaminase E. coli AAN66442.1_F primer) <400> 120 ttcagaattc aaaagatctt ttaagaagga gatatacata tggctgacac tcgc 54 <210> 121 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1_R primer) <400> 121 cgagtttgga tccttattcc gcgttttcgt g 31 <210> 122 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1 A140P-Y275D_F primer) <400> 122 ttcagaattc aaaagatctt ttaagaagga gatatacata tggctgacac tcgc 54 <210> 123 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (alaC alanine transaminase E. coli AAN66442.1 A140P-Y275D_R primer) <400> 123 cgagtttgga tccttattcc gcgttttcgt g 31 <210> 124 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspat aspatate transaminase P. denitrificans AGI22891.1_F primer) <400> 124 ttcagaattc aaaagatctt ttaagaagga gatatacata tgctcggacc cggcg 55 <210> 125 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aspat aspatate transaminase P. denitrificans AGI22891.1_R primer) <400> 125 tcgagtttgg atcctcagcg attctggtgc gcg 33 <210> 126 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase P. denitrificans AGI22971.1_F primer) <400> 126 gtggtggtgg tggtggtgct cgagcagttt caggcgcagc gc 42 <210> 127 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (aroAT acromatic transaminase P. denitrificans AGI22971.1_R primer) <400> 127 aactttaaga aggagatata catatgatga gcaagttctg gagtccc 47 <210> 128 <211> 39 <212> DNA <213> Artificial Sequence <220> Synthetic (aroAT acromatic transaminase P. fluorescens AUM68313.1_F primer) <400> 128 gtggtggtgg tggtggtgct cgaggagttc gccgagggc 39 <210> 129 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (aroAT acromatic transaminase P. fluorescens AUM68313.1_R primer) <400> 129 actttaagaa ggagatatac atatgatgag taaattctgg agcccg 46 <210> 130 <211> 30 <212> DNA <213> Artificial Sequence <220> Synthetic (TA Library F) <400> 130 ctcactatag gggaattgtg agcggataac 30 <210> 131 <211> 26 <212> DNA <213> Artificial Sequence <220> Synthetic (TA Library R) <400> 131 gctagttatt gctcagcggt ggcagc 26 <210> 132 <211> 30 <212> DNA <213> Artificial Sequence <220> Synthetic (TA K14 F) <400> 132 gaaggaattg cctnnkcaat tcttcgcttc 30 <210> 133 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA K14 R) <400> 133 gaagcgaaga attgmnnagg caattccttc 30 <210> 134 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA G40 F) <400> 134 caatctggga cagnnkaatc cagatcagc 29 <210> 135 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA G40 R) <400> 135 gctgatctgg attmnnctgt cccagattg 29 <210> 136 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA N178 F) <400> 136 gaattatccg aatnnkccga ctggagctg 29 <210> 137 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA N178 R) <400> 137 cagctccagt cggmnnattc ggataattc 29 <210> 138 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA Y364 F) <400> 138 gaatatggcg agggcnnkgt cagagtcg 28 <210> 139 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (TA Y364 R) <400> 139 cgactctgac mnngccctcg ccatattc 28 <210> 140 <211> 30 <212> DNA <213> Artificial Sequence <220> Synthetic (lactate dehydrogenases (ldHO) Alcaligenes eutrophus (Ae) YP725182.1_F primer) <400> 140 ataacatatg aagatctccc tcaccagcgc 30 <210> 141 <211> 54 <212> DNA <213> Artificial Sequence <220> Synthetic (lactate dehydrogenases (ldHO) Alcaligenes eutrophus (Ae) YP725182.1_R primer) <400> 141 taataggatc ctcagtgatg gtgatggtga tgggccgtgg ggacggccac gttg 54 <210> 142 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate / L-lactate dehydrogenase (ldH-2) Cupriavidus basilensis (Cb) WP 043344208.1_F primer) <400> 142 ataacatatg aagataacac tgcaatc 27 <210> 143 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate / L-lactate dehydrogenase (ldH-2) Cupriavidus basilensis (Cb) WP 043344208.1_R primer) <400> 143 taataggatc ctcagtggtg atgatggtga tggcccgccg gcagtgccac gccaagttc 59 <210> 144 <211> 29 <212> DNA <213> Artificial Sequence <220> Synthetic (malate / L-lactate dehydrogenase (ldH-2) Achromobacter xylosoxidans (Ax) WP 006389860.1_F primer) <400> 144 ataacatatg aagatctcca ttacccaag 29 <210> 145 <211> 47 <212> DNA <213> Artificial Sequence <220> Synthetic (malate / L-lactate dehydrogenase (ldH-2) Achromobacter xylosoxidans (Ax) WP 006389860.1_R primer) <400> 145 taataggatc ctcagtggtg atgatggtga tgaggcaacg cgtcagc 47 <210> 146 <211> 29 <212> DNA <213> Artificial Sequence <220> Synthetic (malate / L-lactate dehydrogenase (ldh-2) Burkholderia glumae (Bg) YP 002909484.1_F primer) <400> 146 ataacatatg cagatatccc tcgacgatg 29 <210> 147 <211> 68 <212> DNA <213> Artificial Sequence <220> Synthetic (malate / L-lactate dehydrogenase (ldh-2) Burkholderia glumae (Bg) YP 002909484.1_R primer) <400> 147 taataggatc cctagtggtg atgatggtga tgggcccgtg cggccggcgg caccacgccg 60 agttcgtc 68 <210> 148 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate / L-lactate dehydrogenase (ldh-2) Escherichia fergusonii (Ef) WP 002431747.1_F primer) <400> 148 ataacatatg tatgggtaca gataccttc 29 <210> 149 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (malate / L-lactate dehydrogenase (ldh-2) Escherichia fergusonii (Ef) WP 002431747.1_R primer) <400> 149 taataggatc cttagtggtg atgatggtga tgatgctgat tcctgaggat gtaac 55 <210> 150 <211> 29 <212> DNA <213> Artificial Sequence <220> Synthetic (D-2-hydroxyacid dehydrogenase (ldh-2) Escherichia coli (Ec) WP024240865.1_F primer) <400> 150 ataacatatg atgtcattac aaattgctg 29 <210> 151 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (D-2-hydroxyacid dehydrogenase (ldh-2) Escherichia coli (Ec) WP024240865.1_R primer) <400> 151 taataggatc ctcagtggtg atgatggtga tgtccagcta atgctgattc ctg 53 <210> 152 <211> 32 <212> DNA <213> Artificial Sequence <220> Synthetic (D-2-hydroxyacid dehydrogenase (ldhA) Lactobacillus financial (Lm) WP 003689565.1_F primer) <400> 152 ataacatatg acaagaataa ttgcttatca tg 32 <210> 153 <211> 58 <212> DNA <213> Artificial Sequence <220> Synthetic (D-2-hydroxyacid dehydrogenase (ldhA) Lactobacillus financial (Lm) WP 003689565.1_R primer) <400> 153 taataggatc cttagtggtg atgatggtga tgttctccct tgaaacttat ttcatgtg 58 <210> 154 <211> 27 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase (ldhA) Escherichia coli K-12 (Eck) NP 415898.1_F primer) <400> 154 ataacatatg aaactcgccg tttatag 27 <210> 155 <211> 51 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenase (ldhA) Escherichia coli K-12 (Eck) NP 415898.1_R primer) <400> 155 taataggatc cttagtggtg atgatggtga tgaaccagtt cgttcgggca g 51 <210> 156 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH1_FP) <400> 156 ctatatcagc cacggcctgt cgagtctgcc caactaccgc accgccctcg 50 <210> 157 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH1_RP) <400> 157 cgagggcggt gcggtagttg ggcagactcg acaggccgtg gctgatatag 50 <210> 158 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH2_FP) <400> 158 ctatatcagc cacggcctgt cgactctgcc caactaccgc accgccctcg 50 <210> 159 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH2_RP) <400> 159 cgagggcggt gcggtagttg ggcagagtcg acaggccgtg gctgatatag 50 <210> 160 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH3_FP) <400> 160 ctatatcagc cacggcctgt cgaatctgcc caactaccgc accgccctcg 50 <210> 161 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH3_RP) <400> 161 cgagggcggt gcggtagttg ggcagattcg acaggccgtg gctgatatag 50 <210> 162 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH4_FP) <400> 162 ctatatcagc cacggcctgt cggacctgcc caactaccgc accgccctcg 50 <210> 163 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH4_RP) <400> 163 cgagggcggt gcggtagttg ggcaggtccg acaggccgtg gctgatatag 50 <210> 164 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH5_FP) <400> 164 ctatatcagc cacggcctgt cgaatctgcc caactaccgc accgccctcg 50 <210> 165 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH5_RP) <400> 165 cgagggcggt gcggtagttg ggcagattcg acaggccgtg gctgatatag 50 <210> 166 <211> 27 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldh) Escherichia coli K-12 NP 415898.1_F primer) <400> 166 ataacatatg aaactcgccg tttatag 27 <210> 167 <211> 51 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldh) Escherichia coli K-12 NP 415898.1_R primer) <400> 167 taataggatc cttagtggtg atgatggtga tgaaccagtt cgttcgggca g 51 <210> 168 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases Lactobacillus helveticus WP 003628108.1 1_F primer) <400> 168 ataacatatg acaaaggttt ttgc 24 <210> 169 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases Lactobacillus helveticus WP 003628108.1 1_R primer) <400> 169 taataggatc ctcagtggtg atgatggtga tgaaacttgt tcttgttcaa agcaac 56 <210> 170 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate / D-glycerate dehydrogenase (ldhD) Pediococcus acidilactic < / RTI > X70925.1_F primer) <400> 170 ataacatatg aagattattg cttatgg 27 <210> 171 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate / D-glycerate dehydrogenase (ldhD) Pediococcus acidilactici X70925.1_R primer) <400> 171 taataggatc ctcagtggtg atgatggtga tgctcaaact taacttcatt ctttg 55 <210> 172 <211> 27 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldHD) Pseudomonas aeruginosa NP_249618.1_F primer) <400> 172 ataacatatg cgcatcctgt tcttcag 27 <210> 173 <211> 50 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldHD) Pseudomonas aeruginosa NP_249618.1_R primer) <400> 173 taataggatc cctagtggtg atgatggtga tgggcccgga cccgattgcg 50 <210> 174 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Clostridium perfringens 13 NP_561446.1_F primer) <400> 174 ataacatatg atgataaaat tagtatgtta t 31 <210> 175 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (D-lactate dehydrogenases (ldhD) Clostridium perfringens 13 NP_561446.1_R primer) <400> 175 taataggatc cttagtggtg atgatggtga tgatgatgct attgcatttt tacaagtatt 60 tc 62 <210> 176 <211> 26 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus reuteri ZP_03974385.1_F primer) <400> 176 ataacatatg aaaggaatgg gaaaac 26 <210> 177 <211> 52 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus reuteri ZP_03974385.1_R primer) <400> 177 taataggatc ctcagtggtg atgatggtga tgcattctta tttcatttcg tg 52 <210> 178 <211> 26 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldHD) Leuconostoc mesenteroides cremoris ZP_03913173.1_F primer) <400> 178 ataacatatg aagatttttg cttacg 26 <210> 179 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldHD) Leuconostoc mesenteroides cremoris ZP_03913173.1_R primer) <400> 179 taataggatc cttagtggtg atgatggtga tgatattcaa cagcaatagc tgg 53 <210> 180 <211> 26 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldhD) Oenococcus oeni ZP_01544962.1_F primer) <400> 180 ataacatatg aaaatttatg cttatg 26 <210> 181 <211> 55 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldhD) Oenococcus oeni ZP_01544962.1_R primer) <400> 181 taataggatc cttagtggtg atgatggtga tggaatttaa cgagattctt gtctc 55 <210> 182 <211> 28 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus delbrueckii subsp. bulgaricus CAI96942.1_F primer) <400> 182 ataacatatg actaaaattt ttgcttac 28 <210> 183 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (D-lactate dehydrogenases (ldhD) Lactobacillus delbrueckii subsp. bulgaricus CAI96942.1_R primer) <400> 183 taataggatc cttagtggtg atgatggtga tggaaactcc agttaaggtt ggc 53 <210> 184 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH6_FP) <400> 184 catcactaag atgagcctgc gtaactccgg tgttgacaac atcgacatgg cta 53 <210> 185 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH6_RP) <400> 185 tagccatgtc gatgttgtca acaccggagt tacgcaggct catcttagtg atg 53 <210> 186 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (LDH7_FP) <400> 186 catcactaag atgagcctgc gtaacaacgg tgttgacaac atcgacatgg cta 53 <210> 187 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH7_RP) <400> 187 tagccatgtc gatgttgtca acaccgttgt tacgcaggct catcttagtg atg 53 <210> 188 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH8_FP) <400> 188 catcactaag atgagcctgc gtaacaccgg tgttgacaac atcgacatgg cta 53 <210> 189 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH8_RP) <400> 189 tagccatgtc gatgttgtca acaccggtgt tacgcaggct catcttagtg atg 53 <210> 190 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH9_FP) <400> 190 catcactaag atgagcctgc gtaacgacgg tgttgacaac atcgacatgg cta 53 <210> 191 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH9_RP) <400> 191 tagccatgtc gatgttgtca acaccgtcgt tacgcaggct catcttagtg atg 53 <210> 192 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH10_FP) <400> 192 catcactaag atgagcctgc gtaacaaggg tgttgacaac atcgacatgg cta 53 <210> 193 <211> 53 <212> DNA <213> Artificial Sequence <220> Synthetic (LDH10_RP) <400> 193 tagccatgtc gatgttgtca acacccttgt tacgcaggct catcttagtg atg 53 <210> 194 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp5-epd-FP) <400> 194 atctaagcgg ccgcggaatt atgcaattcg tggtac 36 <210> 195 <211> 71 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp5-epd-RP) <400> 195 aaaaaattta tttgctttcg catctttttg tacctataag tgtggaatga ccgtacgcgt 60 agcgataaat g 71 <210> 196 <211> 18 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-epd-FP) <400> 196 gccagtttag tatcgacc 18 <210> 197 <211> 28 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-epd-RP) <400> 197 acaaaagcat gatcctgttg aagatgcg 28 <210> 198 <211> 73 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp5-epd-FP) <400> 198 catttatcgc tacgcgtacg gtcattccac acttataggt acaaaaagat gcgaaagcaa 60 ataaattttt ttc 73 <210> 199 <211> 37 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp5-epd-RP) <400> 199 tagtaatcta gaggagttgg catctttctg cgatttc 37 <210> 200 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp5-dxs-FP) <400> 200 atctaagcgg ccgctcgaca tttcattgtc gttgag 36 <210> 201 <211> 71 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp5-dxs-RP) <400> 201 caaaaaattt atttgctttc gcatcttttt gtacctataa gtgtggaatg agttttgata 60 ttgccaaata c 71 <210> 202 <211> 25 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-dsx-FP) <400> 202 gtaaagctta ccggaaagca gctgt 25 <210> 203 <211> 23 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-dsx-RP) <400> 203 agcaactcga agcctgcgtt aag 23 <210> 204 <211> 76 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp5-dxs-FP) <400> 204 tccacactta taggtacaaa aagatgcgaa agcaaataaa ttttttcggc accctggcgt 60 tttcccaacg ttgcag 76 <210> 205 <211> 39 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp5-dsx-RP) <400> 205 tagtaatcta gaggtcatat gttcggcgtt agcacaaac 39 <210> 206 <211> 32 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp5-pdxj-FP) <400> 206 atctaagcgg ccgcggcttg cggcaaaggt cg 32 <210> 207 <211> 70 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp5-pdxj-RP) <400> 207 aaaaaattta tttgctttcg catctttttg tacctataag tgtggaatgg ctgaattact 60 gttaggcgtc 70 <210> 208 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-pdxj-FP) <400> 208 ctttcacgtt gtgataggtc ag 22 <210> 209 <211> 70 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-pdxj-RP) <400> 209 aaaaaattta tttgctttcg catctttttg tacctataag tgtggaatgg ctgaattact 60 gttaggcgtc 70 <210> 210 <211> 57 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp5-pdxj-FP) <400> 210 gacgcctaac agtaattcag ccattccaca cttataggta caaaaagatg cgaaagc 57 <210> 211 <211> 24 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp5-pdxj-RP) <400> 211 cgatctttaa gagtaactcc gatg 24 <210> 212 <211> 34 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp4-lldp-FP) <400> 212 atctaagcgg ccgcgattgg aatgcccatc gcac 34 <210> 213 <211> 61 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp4-lldp-RP) <400> 213 ttgacaatta atcatccggc tcgtaattta tgtggaatga atctctggca acaaaactac 60 g 61 <210> 214 <211> 24 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-lldp-FP) <400> 214 ccatctgacc aatctcaaag ctgt 24 <210> 215 <211> 24 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp5-lldp-RP) <400> 215 ttgtgaacaa agcacctggt cgcg 24 <210> 216 <211> 57 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp4-lldp-FP) <400> 216 tccacataaa ttacgagccg gatgattaat tgtcaattgg tagggccaat tcttgtg 57 <210> 217 <211> 37 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp4-lldp-RP) <400> 217 tagtaatcta gagaaagaag tcgaaaaaaa cgaaatc 37 <210> 218 <211> 36 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp4-ducA-FP) <400> 218 atctaagcgg ccgcgttgat gattgcatag ataacc 36 <210> 219 <211> 70 <212> DNA <213> Artificial Sequence <220> Synthetic (US-Psp4-ducA-RP) <400> 219 ttgacaatta atcatccggc tcgtaattta tgtggaatgc aggttgctgg cggtctggac 60 tatctggttc 70 <210> 220 <211> 24 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp4-ducA-FP) <400> 220 ccatctgacc aatctcaaag ctgt 24 <210> 221 <211> 24 <212> DNA <213> Artificial Sequence <220> Synthetic (Psp4-ducA-RP) <400> 221 ttgtgaacaa agcacctggt cgcg 24 <210> 222 <211> 64 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp4-ducA -FP) <400> 222 tccacataaa ttacgagccg gatgattaat tgtcaatgtt ttttaacaag ttgatattag 60 attg 64 <210> 223 <211> 37 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-Psp4-ducA-RP) <400> 223 tagtaatcta gagaaagaag tcgaaaaaaa cgaaatc 37 <210> 224 <211> 37 <212> DNA <213> Artificial Sequence <220> Synthetic (US-dsdx-FP) <400> 224 atctaagcgg ccgcggtatc cagcgacgat tttagcg 37 <210> 225 <211> 38 <212> DNA <213> Artificial Sequence <220> Synthetic (US-dsdx-RP) <400> 225 gtacgtctct ttgcgcttac atatctcacc ttcccctg 38 <210> 226 <211> 21 <212> DNA <213> Artificial Sequence <220> Synthetic (G-dsdx-FP) <400> 226 gcgtgtaaag tcacctaatg c 21 <210> 227 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (G-dsdx-RP) <400> 227 gggcatcagc agacatatg 19 <210> 228 <211> 38 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-dsdx-FP) <400> 228 caggggaagg tgagatatgt aagcgcaaag agacgtac 38 <210> 229 <211> 35 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-dsdx-RP) <400> 229 tagtaatcta gacgctcata atgccgattg ataac 35 <210> 230 <211> 35 <212> DNA <213> Artificial Sequence <220> Synthetic (US-kgtP-FP) <400> 230 atctaagcgg ccgcggagct gatccgcgag catac 35 <210> 231 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (US-kgtP-RP) <400> 231 acggcaggag acataatggc atgtagtgac gggtcagttg ccagac 46 <210> 232 <211> 22 <212> DNA <213> Artificial Sequence <220> Synthetic (G-kgtP-FP) <400> 232 tggcgtctgg atctggaaaa cg 22 <210> 233 <211> 19 <212> DNA <213> Artificial Sequence <220> Synthetic (G-kgtP-RP) <400> 233 tgtgtaagcg cagcgatgc 19 <210> 234 <211> 46 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-kgtP-FP) <400> 234 gtctggcaac tgacccgtca ctacatgcca ttatgtctcc tgccgt 46 <210> 235 <211> 34 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-kgtP-RP) <400> 235 tatagtaatc tagagaagct gttttggcgg atga 34 <210> 236 <211> 35 <212> DNA <213> Artificial Sequence <220> Synthetic (US-actP-FP) <400> 236 atctaagcgg ccgccttatc tttattaagg taaac 35 <210> 237 <211> 54 <212> DNA <213> Artificial Sequence <220> Synthetic (US-actP-RP) <400> 237 agtcctgcat gaggtacaag catcatgtaa tctctcccct tccccggtcg tctg 54 <210> 238 <211> 20 <212> DNA <213> Artificial Sequence <220> Synthetic (G-actP-FP) <400> 238 ctgtgggatt tcgatagtat 20 <210> 239 <211> 23 <212> DNA <213> Artificial Sequence <220> Synthetic (G-actP-RP) <400> 239 agcaacctgg gcgatacctc gac 23 <210> 240 <211> 54 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-actP-FP) <400> 240 cagacgaccg gggaagggga gagattacat gatgcttgta cctcatgcag gact 54 <210> 241 <211> 33 <212> DNA <213> Artificial Sequence <220> Synthetic (DS-actP-RP) <400> 241 tagtaatcta gaaagctgct tgaagagaag cag 33

Claims

글루코스를 탄소원으로 사용하여 광학적으로 순수한 2,4-디히드록시-부틸레이트(2,4-dihydroxybutyrate) 또는 2-히드록시-감마-부티로락톤(2-hydroxy gamma butyrolactone)을 생산하는 미생물에 하기 (1) 내지 (4) 중 선택된 하나 이상의 단계를 수행하여 상기 미생물을 변이시키는 단계를 포함하는, 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤 생산 미생물 변이체의 제조 방법:
(1) ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, 및 iclR 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실, 또는 acs, ppc, 및 metL 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계,
(2) epd, dxs, 및 pdxj 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현시키는 단계,
(3) lpd 및 ducA 중 하나 이상의 유전자의 과발현시키는 단계, 및
(4) kgtP, dsdx, 및 actP 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실시키는 단계.To a microorganism producing optically pure 2,4-dihydroxybutyrate or 2-hydroxy gamma butyrolactone using glucose as a carbon source, Dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variant, comprising the step of performing at least one selected from the following (1) to (4) Manufacturing method:
(1) ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, and iclR deleting a gene of one or more genes selected from the group consisting of, or acs, ppc, and from the group consisting of metL gene at least one group selected Overexpressing the gene, or both,
(2) overexpressing at least one gene selected from the group consisting of epd, dxs, and pdxj genes,
(3) over-expressing one or more genes of lpd and ducA , and
(4) deletion of one or more genes selected from the group consisting of kgtP, dsdx, and act P genes.

제1항에 있어서, 상기 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물은 대장균(E. coli), 효모(Yeast), 및 코리네박테리움(Corynebacterium)으로 이루어진 군에서 선택된 하나 이상인, 방법.The method according to claim 1, wherein the microorganism producing the 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone is E. coli , yeast, (Corynebacterium).

제1항에 있어서, 상기 미생물을 변이시키는 단계는 (1) 단계, 및 (2) 내지 (4) 단계 중 선택된 하나 이상의 단계를 포함하는 것인, 방법.The method according to claim 1, wherein the step of mutating the microorganism comprises one or more steps selected from the group consisting of (1) and (2) to (4).

제1항에 있어서, 상기 미생물을 변이시키는 단계는 (1) 내지 (4) 단계를 전부 포함하는 것인, 방법.The method according to claim 1, wherein the step of mutating the microorganism includes all steps (1) to (4).

제1항 내지 제4항 중 어느 한 항에 있어서, 상기 (1) 단계는 ptsG, eda, adhE, pf1B, lysA, thrB, metA, LacI, ldhA, 및 iclR 유전자 전부를 결실시키고, acs, ppc, 및 metL 유전자 전부를 과발현시키는 단계를 포함하는 것인, 방법.The method according to any one of claims 1 to 4, wherein (1) comprises: ptsG, eda, adhE, pf1B, lysA, thrB, metA, LacI, ldhA, and iclR and deleting a gene altogether, acs, ppc, And overexpressing all of the metL gene.

제1항 내지 제4항 중 어느 한 항에 있어서, 상기 (2) 단계는 epd, dxs, 및 pdx 유전자 전부를 과발현시키는 단계를 포함하는 것인, 방법.5. The method according to any one of claims 1 to 4, wherein said step (2) comprises overexpressing all of the epd, dxs , and pdx genes.

제1항 내지 제4항 중 어느 한 항에 있어서, 상기 (3) 단계는 lpd, 및 ducA 유전자 전부를 과발현시키는 단계를 포함하는 것인, 방법.5. The method according to any one of claims 1 to 4, wherein step (3) comprises overexpressing all of the lpd and ducA genes.

제1항 내지 제4항 중 어느 한 항에 있어서, 상기 (4) 단계는 kgtP, dsdx, 및 actP 유전자 전부를 결실시키는 단계를 포함하는 것인, 방법.5. The method according to any one of claims 1 to 4, wherein said step (4) comprises deleting all of the kgtP , dsdx , and actP genes.

제1항에 있어서, 상기 미생물 변이체 제조방법은
호모세린으로부터 4-히드록시-2-옥소-부틸레이트(4-hydroxy-2-oxo-butyrate)의 전환을 촉진하는 단계를 추가로 포함하는 것인, 방법.2. The method according to claim 1,
Further comprising the step of promoting the conversion of 4-hydroxy-2-oxo-butyrate from homoserine.

제9항에 있어서, 상기 호모세린으로부터 4-히드록시-2-옥소-부틸레이트의 전환을 촉진하는 단계는 트랜스아미네이즈(transaminase)를 처리하여 호모세린(homoserine)의 알파 위치 아민기를 제거하는 단계를 포함하는 것인, 방법.10. The method of claim 9, wherein the step of promoting the conversion of 4-hydroxy-2-oxo-butyrate from the homoserine comprises treating transaminase to remove alpha-amine groups of homoserine &Lt; / RTI >

제10항에 있어서, 상기 트랜스아미네이즈는 피루브산(pyruvate)을 아미노기 어셉터(acceptor)로 사용하는 효소인, 방법.11. The method of claim 10, wherein the transaminase is an enzyme that uses pyruvate as an amino acceptor.

제11항에 있어서, 상기 트랜스아미네이즈는 서열번호 12의 아미노산 서열로 이루어진 효소, 서열번호 13의 아미노산 서열로 이루어진 효소, 서열번호 14의 아미노산 서열로 이루어진 효소, 및 서열번호 15의 아미노산 서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상을 포함하는 것인, 방법.The transaminase according to claim 11, wherein the transaminase comprises an enzyme consisting of the amino acid sequence of SEQ ID NO: 12, an enzyme consisting of the amino acid sequence of SEQ ID NO: 13, an enzyme consisting of the amino acid sequence of SEQ ID NO: 14, &Lt; RTI ID = 0.0 > enzymes. &Lt; / RTI >

제9항에 있어서, 상기 미생물 변이체 제조방법은 4-히드록시-2-옥소-부틸레이트를 2,4-디히드록시-부틸레이트로 전환을 촉진하는 단계를 추가로 포함하는 것인, 방법.10. The method of claim 9, wherein the method further comprises the step of promoting conversion of 4-hydroxy-2-oxo-butyrate to 2,4-dihydroxy-butyrate.

제13항에 있어서, 상기 2,4-디히드록시-부틸레이트는 (2S)-2,4-디히드록시-부틸레이트 및 (2R)2,4-디히드록시-부틸레이트로 이루어진 군에서 선택된 하나 이상인, 방법.14. The process of claim 13, wherein the 2,4-dihydroxy-butyrate is selected from the group consisting of (2S) -2,4-dihydroxy-butyrate and (2R) 2,4-dihydroxy- Selected one or more.

제13항에 있어서, 상기 전환을 촉진하는 단계는 L-4-히드록시-2-옥소-리덕테이즈(L-hydroxy-2-oxo-reductase) 또는 D-4-히드록시-2-옥소-리덕테이즈(D-hydroxy-2-oxo-reductase)를 사용하는 단계를 포함하는 것인, 방법.14. The method of claim 13, wherein the step of promoting the conversion is L-hydroxy-2-oxo-reductase or D-4-hydroxy- Wherein the method comprises using D-hydroxy-2-oxo-reductase.

제15항에 있어서, 상기 L-4-히드록시-2-옥소-리덕테이즈는 서열번호 16의 아미노산 서열로 이루어진 효소, 서열번호 17의 아미노산 서열로 이루어진 효소 및 서열번호 18의 아미노산 서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상을 포함하는 것인, 방법. 16. The method according to claim 15, wherein the L-4-hydroxy-2-oxo-reductase comprises an enzyme consisting of the amino acid sequence of SEQ ID NO: 16, an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence of SEQ ID NO: &Lt; RTI ID = 0.0 > enzymes. &Lt; / RTI >

제15항에 있어서, 상기 D-4-히드록시-2-옥소-리덕테이즈는 서열번호 19의 아미노산 서열로 이루어진 효소, 서열번호 20의 아미노산 서열로 이루어진 효소, 및 서열번호 21의 아미노산 서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상을 포함하는 것인, 방법.15. The method according to claim 15, wherein the D-4-hydroxy-2-oxo-reductase comprises an enzyme consisting of the amino acid sequence of SEQ ID NO: 19, an enzyme consisting of the amino acid sequence of SEQ ID NO: 20 and an amino acid sequence of SEQ ID NO: Wherein the enzyme comprises one or more enzymes selected from the group consisting of enzymes that have been made.

제13항에 있어서, 상기 방법은 2,4-디히드록시-부틸레이트에 락톤화를 촉진하여 2-히드록시-감마-부티로락톤의 생산을 촉진하는 단계를 추가로 포함하는 것인, 방법.14. The method of claim 13, wherein the method further comprises the step of promoting lactonization to 2,4-dihydroxy-butyrate to promote the production of 2-hydroxy-gamma-butyrolactone. .

제18항에 있어서, 상기 락톤화의 촉진은 서열번호 22의 아미노산 서열로 이루어진 락토네이즈(lactonase)의 발현을 촉진하여 수행되는 것인, 방법.19. The method of claim 18, wherein the promotion of lactonization is performed by promoting the expression of lactonase comprising the amino acid sequence of SEQ ID NO: 22.

제18항에 있어서, 상기 2-히드록시-감마-부티로락톤은 (2S)-2-히드록시 감마 부티로락톤 및 (2R)-2-히드록시 감마 부티로락톤으로 이루어진 군에서 선택된 하나 이상인, 방법.19. The method of claim 18, wherein the 2-hydroxy-gamma-butyrolactone is at least one selected from the group consisting of (2S) -2-hydroxy gamma butyrolactone and (2R) -2-hydroxy gamma butyrolactone , Way.

2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물의 게놈(genome)에 (1) 내지 (4) 중 어느 하나 이상의 유전자 변이가 도입된, 2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤 생산 미생물 변이체:
(1) ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, 및 iclR 유전자로 이루어진 군에서 선택된 하나 이상의 결실, 또는 acs, ppc, 및 metL 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계;
(2) epd, dxs, 및 pdxj 유전자로 이루어진 군에서 선택된 하나 이상의 유전자의 과발현,
(3) lpd 및 ducA 중 하나 이상의 유전자의 과발현,
(4) kgtP, dsdx, 및 actP 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실.(1) to (4) are introduced into the genome of a microorganism that produces 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone, 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variant:
(1) at least one gene selected from the ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, and the group consisting of from the group consisting of iclR gene in the selected one or more deletions, or acs, ppc, and metL gene Overexpression, or both;
(2) overexpression of one or more genes selected from the group consisting of epd, dxs , and pdxj genes,
(3) overexpression of one or more genes of lp d and ducA ,
(4) deletes one or more genes selected from the group consisting of kgtP, dsdx , and actP genes.

2,4-디히드록시-부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물의 게놈(genome)에서,
ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, iclR, kgtP, dsd, 및 actP 유전자가 결실되고,
acs, ppc, metL, epd, dxs, pdxj, lpd, 및 ducA 유전자가 과발현된, 미생물 변이체.In the genome of microorganisms that produce 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone,
The ptsG, eda, adhE, pf1B, lysA, thrBC, metA, LacI, ldhA, iclR, kgtP, dsd, and actP gene is deleted,
acs, ppc, metL, epd, dxs, pdxj, lpd, and ducA genes.

제21항에 있어서, 상기 미생물 변이체는 기탁번호 KCCM12281P의 균주인, 미생물 변이체.22. The microorganism variant according to claim 21, wherein the microorganism variant is a strain of KCCM12281P.

제21항에 있어서, 상기 미생물 변이체는 기탁번호 KCCM12282P의 균주인, 미생물 변이체.22. The microorganism variant according to claim 21, wherein the microorganism variant is a strain of KCCM12282P.

제21 내지 제24항 중 어느 한 항에 있어서, 상기 미생물 변이체는 하기 (1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 벡터를 추가로 포함하는 것인, 미생물 변이체:
(1) 서열번호 13의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 14 및 서열번호 15의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 트랜스아미네이즈 변이효소 암호화 유전자,
(2) 서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 L-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자,
(3) 서열번호 20의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 21의 아미노산열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 D-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자, 및
(4) 서열번호 22의 아미노산서열로 이루어진 효소를 암호화하는 유전자.25. The microorganism variant according to any one of claims 21 to 24, wherein the microbial mutant further comprises one or more genes selected from the following (1) to (4) or a recombinant vector comprising the same:
(1) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 13, and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 15, or one or more transaminase mutant enzyme encoding genes selected from the group consisting of SEQ ID NO:
(2) at least one L-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: Enzyme-encoding genes,
(3) at least one D-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 20 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: An enzyme-encoding gene, and
(4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.

제21항에 있어서, 상기 미생물 변이체는 호모세린, 4-히드록시-2-옥소 부티레이트, 2,4-디히드록시-부틸레이트, 및 2-히드록시-감마-부티로락톤로 이루어진 군에서 선택된 하나 이상을 과량 생산하는 것인, 미생물 변이체.22. The composition of claim 21, wherein the microbial variant is selected from the group consisting of homoserine, 4-hydroxy-2-oxobutyrate, 2,4-dihydroxy-butyrate, and 2-hydroxy-gamma-butyrolactone Microbial variants, which produce more than one.

제21항 내지 제24항 및 제26항 중 어느 한 항의 미생물 변이체를 배양하는 단계를 포함하는, 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone) 또는 2,4-디히드록시 부틸레이트(2,4-dihydroxy butanoic acid)의 생산방법.(2-hydroxy gamma butyrolactone) or 2,4-dihydroxybutyrate, which comprises culturing the microorganism variant of any one of claims 21 to 24 and 26, (2,4-dihydroxy butanoic acid).

제27항에 있어서, 상기 미생물 변이체는 하기 (1) 내지 (3) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 벡터를 추가로 포함하는 것인, 생산 방법:
(1) 서열번호 13의 아미노산서열로 이루어진 효소를 암호화하는 유전자, 서열번호 14의 아미노산서열로 이루어진 효소를 암호화하는 유전자, 및 서열번호 15의 아미노산서열로 이루어진 효소로 이루어진 군에서 선택된 하나 이상의 트랜스아미네이즈 변이효소 암호화 유전자,
(2) 서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 L-히드록시-2-옥소-리덕테이즈 변이효소암호화 유전자,
(3) 서열번호 20의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 21의 아미노산열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 D-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자, 및
(4) 서열번호 22의 아미노산서열로 이루어진 효소를 암호화하는 유전자.28. The production method according to claim 27, wherein the microbial mutant further comprises one or more genes selected from the following (1) to (3) or a recombinant vector comprising the same:
(1) a gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 13, a gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 14, and an enzyme consisting of the amino acid sequence of SEQ ID NO: Natri mutational enzyme coding gene,
(2) at least one L-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: Enzyme-encoding genes,
(3) at least one D-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 20 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: An enzyme-encoding gene, and
(4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.

제27항에 있어서, 상기 배양하는 단계는 질소원으로 효모 추출물 및 암모늄 염을 포함하는 배지에서 수행하는 것인, 생산방법.28. The production method according to claim 27, wherein said culturing is carried out in a culture medium containing yeast extract and ammonium salt as a nitrogen source.

제27항에 있어서, 상기 배양하는 단계 이후에, 당과 메티오닌(methionine), 라이신(lysine), 트레오닌(threonine), 및 이소류신(isoleucine)으로 이루어진 군에서 선택된 하나 이상의 아미노산을 첨가하여 유가식으로 발효하는 단계를 추가로 포함하는, 생산방법. 28. The method according to claim 27, wherein after the step of culturing, at least one amino acid selected from the group consisting of sugar, methionine, lysine, threonine, and isoleucine is added, &Lt; / RTI >

제30항에 있어서, 발효단계에서 순수 광학 이성질체인 (2S)- 2,4-디히드록시 부틸레이트 또는 (2R)- 2,4-디히드록시 부틸레이트를 생산한 후, pH를 1.0 내지 3.0 로 낮추어 화학적으로 (2S)-2-히드록시-감마-부티로락톤 또는 (2R)-2-히드록시-감마-부티로락톤으로 전환하는 단계를 추가로 포함하는 것인, 생산방법.31. The method according to claim 30, wherein, in the fermentation step, (2S) -2,4-dihydroxybutyrate or (2R) -2,4-dihydroxybutyrate, which is a pure optical isomer, To (2S) -2-hydroxy-gamma-butyrolactone or (2R) -2-hydroxy-gamma-butyrolactone chemically.

제21항 내지 제24 및 제26 중 어느 한 항의 미생물 변이체 또는 그 배양물을 포함하는, 2-히드록시 감마 부티로락톤(2-hydroxy gamma butyrolactone) 또는 2,4-디히드록시 부틸레이트(2,4-dihydroxy butanoic acid)의 생산용 조성물.(2-hydroxy gamma butyrolactone) or 2,4-dihydroxybutyrate (2-hydroxybutyralactone), which comprises the microorganism variant or culture thereof of any one of claims 21 to 24 and 26, , 4-dihydroxy butanoic acid).

제32항에 있어서, 상기 미생물 변이체는 하기 (1) 내지 (3) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 벡터를 추가로 포함하는 것인, 생산용 조성물:
(1) 서열번호 13의 아미노산으로 이루어진 효소를 암호화하는 염기서열로 이루어진 유전자, 서열번호 14의 아미노산으로 이루어진 효소를 암호화하는 염기서열로 이루어진 유전자, 및 서열번호 15의 아미노산으로 이루어진 효소를 암호화하는 염기서열로 이루어진 유전자로 이루어진 군에서 선택된 하나 이상의 트랜스아미네이즈 변이효소 암호화 유전자,
(2) 서열번호 17의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 18의 아미노산서열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 L-히드록시-2-옥소-리덕테이즈 변이효소암호화 유전자,
(3) 서열번호 20의 아미노산서열로 이루어진 효소를 암호화하는 유전자 및 서열번호 21의 아미노산열로 이루어진 효소를 암호화하는 유전자로 이루어진 군에서 선택된 하나 이상의 D-히드록시-2-옥소-리덕테이즈 변이효소 암호화 유전자, 및
(4) 서열번호 22의 아미노산서열로 이루어진 효소를 암호화하는 유전자.The production composition according to claim 32, wherein the microbial mutant further comprises one or more genes selected from the following (1) to (3) or a recombinant vector comprising the same:
(1) a gene consisting of a nucleotide sequence encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 13, a gene consisting of a nucleotide sequence encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 14, and a nucleotide sequence encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: A gene encoding a transaminase mutant enzyme encoding gene selected from the group consisting of a gene consisting of a nucleotide sequence,
(2) at least one L-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 17 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: Enzyme-encoding genes,
(3) at least one D-hydroxy-2-oxo-reductase mutation selected from the group consisting of a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 20 and a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: An enzyme-encoding gene, and
(4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.

서열번호 13의 아미노산 서열로 이루어진, 트랜스아미네이즈 변이효소.A transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO:

서열번호 14의 아미노산 서열로 이루어진, 트랜스아미네이즈 변이효소.A transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 14.

서열번호 15의 아미노산 서열로 이루어진, 트랜스아미네이즈 변이효소.A transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 15.

서열번호 17의 아미노산 서열로 이루어진, L-히드록시-2-옥소-리덕테이즈 변이효소.17. An L-hydroxy-2-oxo-lyudatease mutant enzyme comprising the amino acid sequence of SEQ ID NO:

서열번호 18의 아미노산 서열로 이루어진, L-히드록시-2-옥소-리덕테이즈 변이효소.18. An L-hydroxy-2-oxo-lyudatease mutant enzyme comprising the amino acid sequence of SEQ ID NO: 18.

서열번호 20의 아미노산 서열로 이루어진, D-히드록시-2-옥소-리덕테이즈 변이효소.A D-hydroxy-2-oxo-reductase mutant enzyme consisting of the amino acid sequence of SEQ ID NO: 20.

서열번호 21의 아미노산 서열로 이루어진, D-히드록시-2-옥소-리덕테이즈 변이효소. A D-hydroxy-2-oxo-lyudatease mutant enzyme consisting of the amino acid sequence of SEQ ID NO: 21.

서열번호 22의 아미노산 서열로 이루어진, 락토네이즈 변이효소.A lactonase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 22.