WO2019013573A2 - Method for preparing 2-hydroxy-gamma-butyrolactone or 2,4-dihydroxy-butyrate - Google Patents

Method for preparing 2-hydroxy-gamma-butyrolactone or 2,4-dihydroxy-butyrate Download PDF

Info

Publication number
WO2019013573A2
WO2019013573A2 PCT/KR2018/007923 KR2018007923W WO2019013573A2 WO 2019013573 A2 WO2019013573 A2 WO 2019013573A2 KR 2018007923 W KR2018007923 W KR 2018007923W WO 2019013573 A2 WO2019013573 A2 WO 2019013573A2
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
seq
amino acid
acid sequence
hydroxy
Prior art date
Application number
PCT/KR2018/007923
Other languages
French (fr)
Korean (ko)
Other versions
WO2019013573A9 (en
WO2019013573A3 (en
Inventor
박성훈
김용환
이성국
심증엽
유태현
수만라마
응웬하이남
Original Assignee
울산과학기술원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 울산과학기술원 filed Critical 울산과학기술원
Publication of WO2019013573A2 publication Critical patent/WO2019013573A2/en
Publication of WO2019013573A3 publication Critical patent/WO2019013573A3/en
Publication of WO2019013573A9 publication Critical patent/WO2019013573A9/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42Hydroxy-carboxylic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02Oxygen as only ring hetero atoms
    • C12P17/04Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y203/00Acyltransferases (2.3)
    • C12Y203/01Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01046Homoserine O-succinyltransferase (2.3.1.46)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y207/00Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01039Homoserine kinase (2.7.1.39)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y207/00Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02004Aspartate kinase (2.7.2.4)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y301/00Hydrolases acting on ester bonds (3.1)
    • C12Y301/08Phosphoric triester hydrolases (3.1.8)
    • C12Y301/08001Aryldialkylphosphatase (3.1.8.1), i.e. paraoxonase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y401/00Carbon-carbon lyases (4.1)
    • C12Y401/01Carboxy-lyases (4.1.1)
    • C12Y401/0102Diaminopimelate decarboxylase (4.1.1.20)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y401/00Carbon-carbon lyases (4.1)
    • C12Y401/01Carboxy-lyases (4.1.1)
    • C12Y401/01031Phosphoenolpyruvate carboxylase (4.1.1.31)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y602/00Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01Acid-Thiol Ligases (6.2.1)
    • C12Y602/01001Acetate-CoA ligase (6.2.1.1)

Definitions

  • the present invention relates to a newly proposed biosynthetic pathway for the production of 2-hydroxy gamma butyrolactone (HGBL) and its precursor, 2, 4-dihydr oxybutanoic acid, present.
  • HGBL 2-hydroxy gamma butyrolactone
  • 2, 4-dihydr oxybutanoic acid present.
  • HGBL 2-hydroxy gamma butyrolactone
  • it is an important intermediate that can be used as resin for photoresist, material for pharmaceutical raw materials and material for coating metal surface.
  • U.S. Pat. Nos. 4,994,597 and 5,087,751 disclose 3,4-dihydroxybutyric acid derivatives. Such an acid production method is distinguished from the present invention in which the hydrolysis of metal cyanide with 3, 4-dihydroxybutyl chloride is related to hydrolysis.
  • the acid is an intermediate of 3 -hydroxybutyrolactone.
  • (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-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 bales, treatment of various nervous systems and metabolic disorders.
  • 1-carnitine a naturally occurring vitamin and a component used in many applications, including health food additives and tonic bales, treatment of various nervous systems and metabolic disorders.
  • the market for carnitine is several hundred tons. It is produced in pure form in the form of d and 1. However, there is no direct synthetic route that has commercial value yet.
  • (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 with a 4-linked L-nuclear source. These substances are not known.
  • a common lactone preparation process is known from the following patents:
  • the present invention relates to a process for the production of 2,4-dihydroxy-butyrate (2,4- 1 (1 < th > 13 6) or 2-hydroxy- Dihydroxy-butyrate or a salt thereof, which comprises the step of carrying out at least one step selected from the following (1) to (4) in a microorganism producing lactone (2-hydroxy gamma butyrolactone) Hydroxy-gamma-butyrolactone-producing microorganism variant.
  • 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 that produces 2,4-dihydroxy-butyrate or 2-hydroxy-gamma- Dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variants.
  • 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.
  • the present invention also relates to a pharmaceutical composition
  • 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.
  • the present invention also relates to a polypeptide comprising the amino acid sequence of SEQ ID NO:
  • 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.
  • the present invention also provides a L-hydroxy-2-oxo-reductase mutant consisting of the amino acid sequence of SEQ ID NO: 17 and consisting of the amino acid sequence of L-hydroxy-2-oxo-reductase mutant Enzyme is provided.
  • the present invention also provides a D-hydroxy-2-oxo-reductase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 20, a D-hydroxy-2-oxo-reductase enzyme comprising the amino acid sequence of SEQ ID NO: Provide mutated enzymes.
  • the present invention also provides a lactonase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 22.
  • pure isomers of optically active isoforms can be produced by reacting 2-hydroxygalactosidic acid butyrolactone with two isomers, or its precursor, 2,4-dihydroxy butanoic acid to provide.
  • 2-hydroxygalactosidic acid butyrolactone with two isomers, or its precursor, 2,4-dihydroxy butanoic acid to provide.
  • 2,4-dihydroxy butanoic acid which is a precursor of 2-hydroxy gamma butyrolactone
  • 2,4-dihydroxy butanoic acid is isolated from 2,4-dihydroxy butanoic acid when it is produced in the form of an optically active pure isomer or a mixture thereof After purification, it may be a method of producing the final 2-hydroxy gamma butyrolactone in the state of being optically active pure isomers or a mixture thereof by a chemical method.
  • 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.
  • the production method may be to use a transaminase characterized by the use of a-ketoglutarate (a_KG) as an amino acceptor to receive an amino group from homoserine.
  • a_KG a-ketoglutarate
  • the production method includes a pathway for regenerating the amino acceptor a-KG from glutamic acid produced from the ⁇ -KG and simultaneously using aspartate transaminase for the production of aspartic acid, a homoserine biosynthesis precursor .
  • the production method is a method wherein the oxygen bonded to the 2-carbon of 4-hydr oxy-2-ox-butanoic acid in the repellent pathway shown in FIG. 1 is reduced in a stereo- L-lactate dehydrogenase, D-lactate dehydrogenase, and black are used as the enzymes which convert to hydroxy group.
  • Fig. 1 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 mutation of a gene having a lactonase activity (P0N1) derived from a human. Lt; / RTI >
  • the strain may be a homoserine and a producing strain and a homoserine producing strain.
  • 2-hydroxy gamma butyrolactone which has optical activity on position 2 carbon from homoserine through the pathway shown in Figure 1,
  • a precursor which is an organic acid precursor as shown in FIG. 4
  • a gene mutation strain produced to efficiently produce homoserine from sugar is provided.
  • the strain may be a homoserine and a producing strain and a homoserine producing strain.
  • the microorganism for producing the mutant strain may be one or more microorganisms selected from the group consisting of Escherichia coli, yeast coryzae, and the like.
  • the microorganism may be a microorganism that is improved by using one or more of the following methods, individually or in combination:
  • ppc phosphoenol pyruvate carboxylase
  • the produced homoserine does not convert to homoserine phosphate, but removes the thrB gene coding for homoserine kinase.
  • the strain may be characterized in that the biosynthesis pathway of vitamin B6 is enhanced so as to promote biosynthesis of transaminase and pyridoxal-5'-phosphate as a cofactor in order to increase the activity of transaminase.
  • 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.
  • This method is particularly useful for the production of 2-hydroxygalactosyl butyrate and / or its precursor, 2,4-dihydroxy butanoic acid, by adding yeast extract and ammonium salt as a nitrogen source, And then culturing the cells using a medium supplemented with amino acids such as methionine, lysine, threonine, and isoleucine.
  • the cultivation was carried out in the presence of glucose, methionine, lysine, threonine, and isoleucine in the middle of fermentation for the production of high concentration 2-hydroxy gamma butyrolactone and / or its precursor 2,4-dihydroxy butanoic acid (E.g., a mixture of methionine, lysine, threonine, and isoleucine).
  • the present invention relates to the production of optically pure 2,4-dihydroxybutyrate or 2-hydroxy gamma-butyrolactone using glucose as a carbon source (1) 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variant, comprising the step of carrying out one or more steps selected from the group consisting of: to provide.
  • the kind of the carbon source is not particularly limited,
  • optically pure refers to (2S) -2, 4-dihydroxy-butyrate or (2R) -2,4- dihydroxybutyrate or (2S)
  • 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%.
  • the microorganism producing the 2,4-dihydroxybutyrate or 2-hydroxy gamma-butyrolactone is Although there is no particular limitation on the kind, E. coli 0? (E. coli), yeast (Yeast), and Corynebacterium (Corynebacterium), preferably E. coli.
  • 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.
  • the step of mutating the microorganism may be carried out, and the step (2) to (4)
  • 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.
  • MiA, adhE, and /? Gene may be removed to prevent the production of by-products such as lactic acid, ethane, and formic acid. Removal of these genes prevents by-product formation and increases the carbon flux to homoserine,
  • the / cH? Gene among the genes that can be removed in the above step (1) is removed in order to enhance the activity of the glyoxylate shunt, which is one of the methods for promoting acetal reuse.
  • the removal can be carried out by a conventional method, You can remove it with the pop-in pop-out method.
  • 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.
  • PEP phosphoenolpyruvate
  • glucose is transported into cells by other transport 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.
  • the eda gene is a gene coding for KHG / KDG aldolase.
  • the ED pathway The use of the EMP (Embden-Meyerhof-Parnas pathway) pathway is facilitated and the carbon flux to oxaloacetate can be applied.
  • a method for removing the gene a conventional method can be used, but the MAGE method can be preferably used.
  • the lacl gene removes the lacl inhibitor for the utilization of the Lac promoter (promoter utilization).
  • the gene can be removed by a conventional method, have.
  • the overexpression of the gene in the step (1) may be performed by a conventional method. For example, a large amount of a vector containing a gene in a microbial cell may be introduced, or an over-expression promoter substitution method or the like may be used.
  • overexpression of the 3CS gene may increase the expression of the acs gene in order to minimize the production of acetic acid.
  • pTA-ack and poxB genes can also be deleted, but the deletion of these genes, especially the deletion of genes, often adversely affects cell growth.
  • the method of increasing the expression of the acs gene can be used without limitation in a method commonly used in the related art.
  • the promoter of the 3CS 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 a re-promoter.
  • Overexpression of the ppc gene in step (1) is intended to increase the expression of the ppc gene encoding phosphoenol pyruvate carboxylase, which is the key enzyme of the anapl-erotic pathway, to inhibit acetate production and improve the carbon flux to homoserine.
  • a gene expression increasing 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.
  • 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.
  • a medium copy plasmid pUCPK plasmid.
  • two lac promoters and trc promoters can be used to control metL ⁇ expression size.
  • the step (2) is carried out to increase the production of Vitamin B6.
  • the PLP Pyridoxal-5-Phosphate
  • the rate of PLP biosynthesis is regulated by the proteins encoded by the epd, dxs, pdxJ genes and the like.
  • one or more of these three genes may be overexpressed to improve the rate of PLP biosynthesis.
  • a conventional method can be used.
  • step (2) above preferably overexpresses all epd, dxs, and pdxj genes.
  • the increase in the expression level of ldp, ifcu ⁇ in step (3) increases the extracellular delivery rate of 2,4-dihydroxy-butyrate (DHB)
  • DDB 2,4-dihydroxy-butyrate
  • promoters can be substituted, and overexpression of the membrane protein interferes with cell growth, so that a mid-level synthetic promoter SP5 (Synthet ic promoter 5) or the like is preferably used.
  • SP5 Synthet ic promoter 5
  • the step (3) comprises the steps of lpd, and all of the ducA gene
  • step (4) is performed to inhibit intracellular re-introduction of 2, 4-dihydroxy-butyrate (DHB) Any one or more of the genes encoding the protein, kgtP, dsdx and actP, can be removed.
  • the primer sequences usable at this time are shown in Table 18. < tb >< TABLE >
  • the 4 step may be to remove all of the 5 kgtP, dsdx, and ac gene.
  • the mutant strains prepared by carrying out the mutant strains prepared in the above step (1) through the steps (1) and (2) in Table 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).
  • 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 >
  • 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, and specifically, transaminase And removing the alpha-position amine group of the homoserine.
  • the transaminase can be obtained by introducing a large amount of a vector containing a gene encoding the gene into a microorganism and transforming the transaminase, And a method of replacing the promoter of the gene encoding with the over-expression promoter.
  • 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: 13 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.
  • 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
  • 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
  • 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: .
  • the method for producing a microorganism variant may further include a step of promoting conversion of 4-hydroxy-2-oxo-butyrate to 2,4-dihydroxy-butyrate .
  • 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-butylate.
  • a large amount of a vector containing a gene encoding a reductase that reduces 4-hydroxy-2-oxo-butylate is introduced into a microorganism and transformed, or a promoter of a gene encoding reductase is replaced with an over-expression promoter Method or the like can be used.
  • the 2,4-dihydroxy-butyrate produced by the reduction reaction is 2,4-dihydroxy- (2S) -2, 4-dihydroxy-butyrate and (2R) -2, 4-dihydroxy-butyrate being the pure optical isomers
  • 2,4-dihydroxy- (2S) -2, 4-dihydroxy-butyrate and (2R) -2, 4-dihydroxy-butyrate being the pure optical isomers
  • One or more, racemates, optical isomeric waxes may be prepared,
  • the kind of the optical isomer of 2,4-dihydroxy-butyrate may be selected depending on the intended use of the compound.
  • 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: 18 preferably 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 May be an enzyme consisting of an amino acid sequence.
  • 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
  • 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:
  • 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:
  • the D-hydroxy-2-oxo-reductase comprises an enzyme consisting of the amino acid sequence of SEQ ID NO: 19,
  • 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
  • the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 21 may be composed of the nucleotide sequence of SEQ ID NO:
  • 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 performed by promoting the expression of l actonase.
  • the expression promoting method may be carried out by introducing a vector containing a lactonase gene into an excessive cell to increase the amount of lactonase, A variety of methods can be used, such as replacing the promoter expressing the gene with an over-expression promoter.
  • the promoting of the lactonization may be carried out by promoting the expression of l actonase 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: 22 May comprise the nucleotide sequence of SEQ ID NO: 11.
  • the resulting 2-hydroxy-gamma-butyrolactone is at least one compound selected from the group consisting of pure optical isomers (2S) -2-hydroxy gamma butyrolactone and (2R) -2 : hydroxy gamma butyrolactone number and, preferably, (2S) - 2-hydroxy-gamma -butyrolactone or (2R) in-lock may tonil 2 _-hydroxy-gamma -butyrolactone.
  • (2R) -2-hydroxy gamma butyrolactone may have an optical purity of 90% to 100%, preferably 95% to 100%, more preferably 99% to 100%.
  • the genome of the microorganism producing the 2,4-dihydroxy-butyrate or the 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.
  • the microbial mutants are preferably pt s, eda, l ad, thrB, metA, lysA, adhE, pf IB, IdhA, and ic lR gene is deleted, overexpression of the acs gene, and ppc It is possible to produce a microorganism variant in which the gene is distinguished.
  • the microorganism variant may be the EcW13 strain shown in Table 15, preferably the strain of Accession No. KCCM12281P.
  • the microorganism variant is selected from the group consisting of ptsG, eda, adhE (2, 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone) , microbial mutants in which pflB, lysA, thrBC, metA, Lac I, IdhA, iclR, kgtP, dsd and aci genes are deleted and acs, ppc, metL, epd, dxs, pdxj, Idp and ciucA genes are overexpressed ,
  • the microorganism variant may be a strain of EcW20, which was tested in the following Examples, and the strain of KCCM12282P.
  • the microbial mutant may further comprise one or more genes selected from the following (1) to (4) or a recombinant vector comprising the same.
  • 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
  • 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,
  • 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: And '
  • 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.
  • a method for producing a microorganism which comprises culturing the above-described microorganism variant as described above, wherein 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutyrate -dihydroxy butanoic acid. < / RTI >
  • the microorganism variant used in the production method may be one or more genes selected from the following (1) to (4) or a recombinant vector containing the same It may be additionally included.
  • 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,
  • 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.
  • 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 as the 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 lowered to a pH of 1.0 to 3.0, preferably a pH of 1.0 to 2.0, Butyrolactone. ≪ / RTI >
  • (2S) -2, 4-dihydroxybutyrate After producing (2S) -2, 4-dihydroxybutyrate, the pH is lowered to 1.0 to 3.0 to obtain (2S) -2-hydroxybutyrate having an optical purity of 95% to 100%, preferably 97% Hydroxy-gamma-butyrolactone, and (2R) -2, 4- (2R) -2-hydroxy-gamma-butyrolactone having an optical purity of 95% to 100%, preferably 97% to 100% by lowering the pH to 1.0 to 3.0 after producing dihydroxybutyrate .
  • One example of the present invention is a microorganism which comprises 2-hydroxygamma butyrolactone or 2, 4-dihydroxybutyrate (2 , 4-dihydroxy butanoic acid).
  • the microorganism variant may further comprise one or more genes selected from the following (1) to (4) or a recombinant vector comprising the same.
  • 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.
  • 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.
  • An example of the present invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: Thereby providing a transaminase mutant enzyme.
  • the gene coding for the transaminase mutant enzyme may comprise the nucleotide sequence of SEQ ID NO: 4.
  • 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.
  • 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.
  • 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:
  • 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 has the amino acid sequence of SEQ ID NO: 10
  • 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.
  • the present invention relates to a mutant which overexpresses 2-hydroxy gamma butyrolactone (HGBL) and its precursor, 2,4-dihydroxybutanoic acid, and a 2-hydroxy gamma butyrolactone (HGBL) 4-dihydroxybutanoic acid.
  • HGBL 2-hydroxy gamma butyrolactone
  • 2-hydroxy-gamma-butyrolactone can be made into a pure optical isomer of (R) or (S) form.
  • 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 ine.
  • FIG. 2 shows a pathway for biosynthesis of 4-hydroxy-2-oxo-butanoate from homoserine ine using homoserine dehydrogenase.
  • FIG. 3 shows a pathway for biosynthesis of 4-hydroxy-2-oxo-butanoate using homoserine transaminase and aspartate transaminase from homoserine ine.
  • FIG. 4 shows a strategy for producing a large amount 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 result of electrophoresis of the strain-cleaved excision product producing the TA1, TA2, TA3 and TA6 enzymes under denaturing conditions, indicating that 4 types of TA ⁇ TA2, TA3 and TA6 are enzymatically produced only in insoluble form.
  • FIG. 6B is a block diagram of an embodiment of the present invention, wherein TA4, TA5, TA7, YA8, TA9, TAIO, TAll, TA12, TA13,
  • TA5, TA7, YA8, TA9, TAIO, TA11, TA12, TA12, and TA12 were obtained as a result of centrifugation of the lysate of strains producing 11 kinds of enzymes such as TA15 and TA15, TA13, TA14 and TA15 enzymes are obtained in a soluble state.
  • FIG. 7A shows eleven transaminase enzymes expressed in soluble form
  • FIG. 7B shows the results of purification of the TA4 enzyme, which exhibits the highest activity for homoserine, by pure separation using affinity chromatography, and electrophoresis Results.
  • 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 of 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.
  • FIG. 10 shows the 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 the PYM0L viewer (http: // www. org), respectively.
  • PDB ID: 1 ASM E. coli aspartic acid amino acid transferase
  • FIG. 11 shows four amino acids (I lel7, Gly38, Asnl94, Arg386) which are interacting with carboxylic acid (carboxylic acid of aspartic acid residue) of maleic acid in E. coli aspartic acid amino acid transferase (1ASM) , And the TA4 model structure as compared with that of the aspartic acid residue in the TA4 enzyme
  • 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 activity of the eight LDH enzymes shown in Table 9 using pyruvate and 0HB as substrates.
  • Ae_ldhA shows a three-dimensional structure of Ae_ldhA by homology modeling using a crystal structure (PDB ID: 2G8Y) of E. coli lactate dehydrogenase as a template
  • PDB ID: 2G8Y crystal structure
  • E. coli lactate dehydrogenase E. coli lactate dehydrogenase
  • FIG. 15A shows the structure of the skin leucine and HOB structure (Pubchem Database) for the Ae_ldhA mutant design by performing a docking simulation using the triangular matching method at the enzyme active site of Ae_ld l, And skin lean acid.
  • FIG. 15B shows the result of examining the interaction between amino acid residues of the enzyme and HOB using a docking simulation.
  • FIG. 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 ⁇ 1 of NADH to NAD for 1 min.
  • FIG. 17 shows the results of analysis of the (D) -lactate dehydrogenase enzyme activity in the nine strains shown in Table 12 using pyruvate and 0HB 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.
  • 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, glutamic acid dehyde-3- phosphate; 4PE, 4-phospho-D-erythronate; 3P4K, 3Phoxy ⁇ 4 pho s phohydr oxy- alpha-ketobutyrate; 4PT, 4 ⁇ phosphohydroxy ⁇ L ⁇ threonine; 2A3B, 3 ⁇ hydroxy ⁇ lamin, acetone phosphate; DXS, deoxyxylulose-5-phosphate.
  • EPD erythrose-4-phosphate dehydrogenase
  • PdxB 4-phospho-D-erythronate
  • SerC 3-phosphoserine ine aminotransferases
  • 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. 24A shows the results of a flow-through bioassay of EcW20 (pDHB-L) strain for the production of L-form 2, 4-dihydroxybutyric acid.
  • 24B shows the results of a flow-through biotite assay of the EcW20 (pDHB-D) strain for the production of D-form 2, 4-dihydroxybutyric acid.
  • Figure 25 shows the pACYC_Ponl plasmid map expressing lactonase necessary to convert 2,4-dihydroxybutyr ic acid to HGBL. And (G3C9) genes were expressed by the tac promoter, and the produced proteins were designed to have the necessary lead sequence to move to the cell membrane.
  • FIG. 26A shows a two-stage bioreactor culture for the production of L-DHB and HGBL As a result, the amount of 2, 4-DHB and HGBL produced and biomass of L-form, which are produced with time in the case of using glucose as a substrate, are shown.
  • FIG. 26B shows the result of D-form 2, 4-DHB and HGBL production with time as a result of the biotransformant culture for the production of D-DHB and HGBL, .
  • the yield of D-HGBL was very low, below 0.1 g / L, because the activity of Ponl enzyme was very low for 2,4-DHB.
  • Step 1 Removal of alpha position amine from homoserine
  • Step 2 Reduction reaction (oxygen reduction bound to carbon # 2)
  • Step 3 Lactoni zat ion
  • the first reaction may be catalyzed by a homoserine ine deaminase or a homoserine transaminase enzyme, and the homoserine diamine-catalyzed reaction is shown in FIG. 2
  • Serine deaminase which shows high activity in serine
  • amino acid oxidase which is very active but slightly active in many other amino acids
  • These enzymes can be used directly or after being mutated to have high activity.
  • the mutant alanine aminotransferase prepared to have activity against homoserine ine Similar amino transferases can be used. Also
  • Amino acceptors that receive an amino group from homoser ine include a-ketoglutarate
  • oHCG which is an amino acceptor from glutamic acid produced from ⁇ -KG, is regenerated and homoserine ine biosynthesis It is desirable to use aspartate transaminase for the production of the precursor aspartic acid.
  • dehydrogenase and D-lactate dehydrogenase can be used.
  • the hydroxy group is bound to the 2-position carbon in the (R) or (S) form and the structure of the 2-hydroxy gamma butyrolactone
  • the paraoxonase shown in FIG. 1 can be used by mutating a gene (P0N1) having a lactonase activity from a human, and it is preferable to express it in a non-cytoplasmic periplasm since P0N1 is reversible and lactone can be produced only at an acidic pH.
  • P0N1 a gene having a lactonase activity from a human
  • glyoxylate shunt gene Removal of the / c / gene to suppress expression
  • apspartate kinase enzyme is allowed to express the mutated enzyme to prevent feedback inhibition by threonine and lysine;
  • strains engineered to efficiently biosynthesize HOB from homoserine can produce 2,4-dihydroxy-butanoic acid (dHBA) and 2-hydroxy gamma butyrolactone (HGBL) by additionally expressing homoserine dehydrogenase and / or P0N1.
  • dHBA 2,4-dihydroxy-butanoic acid
  • HGBL 2-hydroxy gamma butyrolactone
  • biologic reactions that convert dHBA to HGBL using P0N1 all are less efficient, so dHBA
  • HGBL Biologically produced, purified and chemically converted to HGBL.
  • the homoserine biosynthetic pathway suggested in Fig. When the homozygous transformation of the proposed homoserine in FIG. 1 is performed, a microorganism that has been engineered to produce dHBA or HGBL can be cultured as a main carbon source to produce dHBA and HGBL as target products.
  • yeast extract and ammonium salt are added to the culture medium as a nitrogen source, and the amino acid,
  • FIG. 4 schematically shows a strategy for producing a large amount of homoserine using glucose, and a strategy for biosynthesis of 2-hydroxy gamma butyrolactone and its precursor, 2,4-dihydroxybutanoic acid.
  • the green arrow indicates the antagonism to be strengthened
  • the red arrow indicates the mechanism of inhibiting enzyme production or enzymatic activity
  • the apricot X mark indicates the elimination or elimination of the repelling antagonist, .
  • the homoserine pathway is known to synthesize aspartate amino acids.
  • lysine, methionine (met) and threonine (thr) production pathways were removed.
  • lysine biosynthesis namely, lysA encoding diaminopimelate decarboxylase, metA encoding homoserinesuccinyl transferase, and homoserine kinase (homoserine kinase) ) And thrB (threonine synthase encoding) (Table 1).
  • 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.
  • PEP phosphoenol pyruvate
  • glucose is transported into cells by other transport proteins such as GalP, and prevention of Carbon Cat abolite repression and prevention of by-product formation by overflow metabolism can be expected.
  • the ED pathway was deleted by deletion of the eda gene coding for KHG / KDG aldolase. MAGE method was used.
  • Escherichia coli W3110 strain and BL2KDE3 were purchased from Korean Collection for Type Cultures (KCTC).
  • Escherichia coli TOPIO strain is a plasmid cloning and maintenance. Multiplex automated genome engineering (MAGE) and pop-in pop-out methods were used for gene deletion.
  • MAGE Multiplex automated genome engineering
  • pop-in pop-out methods were used for gene deletion.
  • the present inventors developed a mutant E. coli Homoserine and production ⁇ Note the type shown in Table 2 below.
  • Dielectric isolation kit was purchased from Promega (Madison, WI, USA). High-f idelity pfu-a polymerase was purchased from Invitrogen (Seoul, Korea). DNA cleavage enzymes and DNA enhancers were obtained from New England Bio-LAb (Beverly, Mass., 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 (Bee ton-Dickinson, NJ, USA). All other reagents and enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • MAGE Multiplex Automated Genome Engineering
  • beta- protein which plays a role of recombinase, was introduced into pSIM5 plasmid and inserted into E. coli by electroporation method.
  • the synthetic ssDNA oligos were constructed so that a homology arm, 5 '-terminal homolgy arm and 3' terminal homology arm, overlap with the target gene sequence 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 mutation in the target gene through homologous recombinat ion.
  • ssDNA oligo was mixed with the cell suspension, placed in an elctro cuvette and electroporated. Then, the resulting suspension was transferred to a culture tube, and 5 mL of LB was added thereto and cultured at 30 ° C. for 3 hours (first cycle). Such The procedure was repeated 6-10 times (6-10 cycles) and 100 uL of cell culture was plated on agar platel and incubated overnight. The generated colonies were screened by PCR method and mutant strains deficient in the genes were identified and secured.
  • Table 3 shows the types of primers used for gene removal or cloning.
  • AldhA_RP gcaacaggtgaacgagt cctt tggctttgagct gaat tttt tTAActgccaatggctgcgaagcgg
  • Alysa_RP ctgctgcgtttgcccgctgaatttggctgcccggtgtgggtcTAAcagcgctgaaacagtttgatgt
  • AthrB_RP cttatcggcaaagcgtccgaggttgttgagactgaatgtctctgTTAtgcaccatcaacaggtgtca
  • AmetA_RP Cagaaactgattttcagtttcaatcttcttcggcatcaggttaattaaccagacgcacgagaagttg
  • AlacI_RP cttatcagaccgtt tcccgcgtggtgaaccaggccagccacgttTGAcgatggcggagctgaattac 66 attcccaaccgcgtggcacaac
  • Ptrc-acs-RP Cggcgtgcgtttattttatccttgtcatcgactgcacggtgcaccaatgc 72
  • the pKOV plasmid with sacB-Km cassette was used (Table 4). Specifically, a fragment with an upstream and downstream region of 600-700 bp of the target gene was amplified by PCR using the corresponding primers from the E. coli W3110 chromosome (Table 3). The sequence of this fragment was inserted into the pKOV plasmid using the early Gene Art Seamless Cloning and Assembly Kit (Invitrogen, USA). Then, recombinant pKOV lasmid 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.
  • Plasmids used for gene deletion, promoter substitution, gene overexpression by pop-in pop-out or MAGE method are shown in Table 4 below.
  • open reading frame (0RF) of the target gene was amplified by PCR using the appropriate primer (Table 3).
  • a restriction enzyme cloning was the plasmid, or the like that is pUCPK, P ET-T7p, pBbAlk, P Trc-99a. This plasmid was then introduced into the corresponding host strain.
  • trc promoter Overexpression, trc promoter, medium copy, K50, T50 promoter, high copy, T50 promoter, high copy, pSIM5 MAGE, ⁇ recombinant protein, Cm25 Addgene, USA pUCPK Overexpression, lac promoter, Addgene, USA pTrc-99a Overexpression, trc promoter, low copy, K50 Addgene, USA pKOV Pop " in-Pop out, Cm25 Addgene, USA p iCPK-wetl Ptrc-metL Thi s study
  • Transaminate (TA) enzymes play an important role in life and have been studied extensively for a long time, but the TA enzyme specific for homoserine ine has not yet been revealed in vivo.
  • many enzymes 17 of the known TA enzymes aspartate transaminase, alanine transaminase, branched chain transaminase, and aromatic transaminase, were selected for activity against homoserine (Table 6). These enzymes were obtained from E. coli, Enterobacter spp., Baci 1 lus subtilis, Mesorhizobium loti, Agrobacterium tumefaciens, Pseudomonas denitri ficans, P. puti da and P. // i / oresce2s '
  • E. coli BL2KDE3 expresses the enzyme of Table 6 in the host.
  • LB medium was used and kanamycin 50 mg / L was used for the maintenance and preservation of plasmids.
  • PET as an expression vector and T7 as a promoter.
  • the coding sequence was amplified from the genomic DNA of the microorganism, cloned into the E. coli Top 10 strain using the seamless cloning and assembly kit (Invitrogen) with the pET vector containing His-tag Respectively.
  • the recombinant strain producing TA for enzyme production is 50 and cultured in LB medium containing mg / L kanamycin. Liquid volume 20 mL, 250 mL flasks were used and cultured at 20 ° C and 200 rpm.
  • 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 solution (pH 7.0) and the binding complete solution (20 mM Phosphate buffer solution, 0.5 M NaCl, 20 mM imidazole). Then, 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.
  • 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 black non-naturing conditions (FIGS. 6A and 6B). Then, glycerol was added at 20% and stored at -80 ° C.
  • 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, the enzyme activity was not observed. Therefore, the TA activity was investigated using the reaction of (i), pyruvate. The product, alanine, was determined by HPLC after being modified with 0PA.
  • TA activity was measured using a 50 mM phosphate buffer solution (pH 7.0), 0.1 mM cofactor pyridoxal phosphate (PLP), 10 mM homoserine, and an appropriate amount of TA enzyme.
  • the solution was incubated at 37 ° C for about 5 minutes and then added with 10 mM pyruvate. After 10 minutes of reaction, 12 mM perchloric acid was added to stop the reaction and centrifuged at 10,000 g for 5 minutes. A 5 ⁇ L sample was then taken to obtain 12.5 ⁇ ! OPA solution (25 mg OPA, 50 yL 2-mercaptoethanol, pH 9.5 0.5 mM saturated sodium borate solution, 4.5 mL methanol) was incubated at room temperature and filtered.
  • OPA solution 25 mg OPA, 50 yL 2-mercaptoethanol, pH 9.5 0.5 mM saturated sodium borate solution, 4.5 mL methanol
  • the alanine modified by 0PA was quantitatively analyzed by HPLC on a Zorbax eclipse XBD-C18 column.
  • the alanine derivative modified with 0PA 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.
  • TA4 enzyme Since TA4 enzyme showed the highest activity, this enzyme was mutated to obtain an enzyme with increased activity.
  • a mutant enzyme library was constructed and highly active enzyme mutants were obtained using high throughput screening (HTS) method related to cell growth.
  • HTS high throughput screening
  • 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.
  • 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.
  • M9 minimal medium with no nitrogen source 10 mM homoserine as a nitrogen source, and a small amount (0.5 mM) of threonine and methionine were added to induce microbial growth, respectively. As shown in FIG.
  • 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.
  • 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.
  • PDB ID: 1 ASM E. coli aspartic acid amino acid transferase
  • the structure and sequence of the 1ASM can be found at https: // www. Refer to rcsb.org/structure/lASM.
  • 1ASM structure has pyruvate phosphodiester (p yr idoxal-5 '-phosphate (PLP) as coenzyme and maleic acid as substrate analogue.
  • PEP p yr idoxal-5 '-phosphate
  • This model was created using MOE (Molecular Operating Environment) and evaluated through PROCHECK and ProSA online structure analysis. The protein structure was confirmed using a PYM0L viewer (http://www.pymo 1. org) (FIG. 10).
  • TA4 enzyme mutant libary was constructed.
  • TA4 library was constructed by randomly modifying four amino acids of TA4 (Lysl4, Gly40, Asnl78, Try364) expected to interact with the carboxylic acid of aspartic acid residues by means of the assemble PCR method.
  • the primers used are shown in Table 8. eu.
  • the restriction enzyme Xbal and Xhol sites the PCR products from primers 1 and 2 were cloned into a pET30b plasmid.
  • the resulting TA4 library was transformed with E. coli E ) 10 and the plasmid And purified. Table 8 below shows the primer sequences used in the construction of the TA4 mutant enzyme library.
  • m a (Adenine, adenine) or c (Cytosine, cytosine)
  • n a (adenine, adenine) or g (guanine, guanine) or c (cytosine, cytosine) or t (thymine, thymine)
  • k g (guanine, guanine) or t (thymine, thymine)
  • the prepared library was transformed with E. coli BL2 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 solution, and then inoculated to 0 M 0.05 minimal medium (including 50 um / mL kanamycin) with 20 mM homoserine as a nitrogen source. after. After culturing at 37 rpm for 3 hours at 200 rpm, TA4 enzyme was induced by adding 0.05 mM IPTG. When the 0D value reached 0.5, the culture was diluted 100-fold and then re-cultured.
  • Such a culture-dilution cycle was repeated 10 times After repetition, the culture was spread on an LB agar medium containing 50 ug / mL kanamycin, and 50 colonies were grown on a well grown M9 medium. A total of five mutants were obtained from these gene sequences and named TA4-1 to TA4-5, respectively.
  • 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).
  • 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.
  • OH 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.
  • the 0HB reductase belongs to 2-hydroxy acid dehydrogenase, including lactate dehydrogenase (EC1.1.1.27, EC1.1.1.28), ma late dehydrogenase (EC1.1.37, EC1.1.82, EC1.1.299) And branched chain (D), (L) -2-hydroxyacid dehydrogenase (ECl.ll 272, ECl.1.1.345) are well known.
  • the activity of 0HB is expected to be high from Alcaligenes eutrophus H16, Cupriavidus basi lensis, Achromobacter xylosoxidans, Burkholderiaglumae, Escherichia fergusonii, Escherichia coli, Lactobaci 1 lusmali and Escherichia coli K-12 to produce L-form isomer (L) -lactate dehydrogenase enzymes were selected (Table 9). (L) -l actate dehydrogenase candidate enzymes for OHB 2S reductase enzyme screening are shown in Table 9.
  • E. coli BL2KDE3 star strain was used as a host strain for E. coli expression and E. coli DH5a strain was used as a host for cloning and preservation of polar powder.
  • LB medium containing kanamycin 50 pg / 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 attached Hi s-tag to the C-terminus. The PCR fragment was inserted into a pET vector (Table 10), cloned into the E. coli strain DH5a, and sequenced into E. coli BL2KDE3 star.
  • the pG-Tf2 plasmid expressing Chaperon was further introduced.
  • the 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 ug / mL kanamycin.
  • the incubation temperature was 30 ° C, stirring speed was 100 rpm, and 250 mL medium was added to a 1 L flask.
  • the cell concentration reached 0.5 0D, 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 solution (pH 7.0), suspended in binding buffer (20 mM pH 7.0 phosphate buffer solution, 0.5 M NaCl, 10 mM imidazole) 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 enzyme activity was analyzed using pyruvate and 0HB as substrates.
  • 0HB was synthesized from homoserine since it was not commercially available. That is, 125 mM homoserine solution to pH 7.8, 100 mL Tris prepared by wancheung solution of 1.25 U / raL snake venom (L ) - from the amino acid oxidase enzyme with 4400 U / mL of 37 ° C and then combined common and catalase enzyme 90 For a while. Then, the solution was filtered with an Ultracentrifugal filter (10 kDa, Amicon) filter to obtain 0HB.
  • an Ultracentrifugal filter (10 kDa, Amicon
  • the enzyme solution was prepared by mixing 60 mM Hepes buffer (pH 7.0), 50 mM NaCl, 5 mM MgCl 2 , 5 mM fructose-1, 6-bi sphosphate and an appropriate amount of enzyme at 37 ° C for 2 min. After that, 0.1 mM NAD (P) H and an appropriate amount of 0HB black were added pyruvate to initiate the reaction. Enzyme activity was calculated using the rate of decrease of NAD (P) H and the extinction coefficient.
  • Table 10 below describes plasmids for OHB L-reductase screening.
  • racemic DHB was synthesized by chemically degrading racemic 2-hydroxy gamma butyrolactone (HGBL) (Sigma-Aldrich, MO, USA) in position 2 OH at R-form and S_form.
  • HGBL 2-hydroxy gamma butyrolactone
  • Racemic HGBL was dissolved in metahn, 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.
  • 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 colorimetr ic 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 0HB products reacted with Ae-LDH was performed simultaneously using a chiral column and a colorimetric assay kit.
  • Ae_ldhA model uses Molecular Operating Environment (M0E) Through PR0CHECK and ProSA online structure analysis,
  • 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 the mutated Ae_ldhA (pET24ma_Ae-ldh0, refer to Zhang et al., "NADH-dependent lactate dehydrogenase from lei genes eutrophus H16 reduces 2-oxoadi ate to 2-hydroxyadipate" Biotechnology and Bioprocess Engineering, 19: 1048-1057 (2014)) 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 Mi 11 ipore Co., Darmstadt, Germany), it was stored at -80 ° C.
  • LDH2_FP ctatatcagccacggcctgtcgactctgcccaactaccgcaccgccctcg 158
  • LDH3_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 160
  • LDH3_RP cgagggcggtgcggtagt tgggcagat tcgacaggccgtggctgatatag 161
  • LDH4_RP cgagggcggtgcggtagt tgggcaggtccgacaggccgtggctgatatag 163
  • LDH5_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 164
  • LDH5_RP cgagggcggtgcggtagggggcagat tcgacaggccgtggctgatatag 165
  • the activity of the enzyme was measured by observing the degree of oxidation of NADH at 340 nm and the activity of the enzyme (1 U) was determined by measuring the activity of the enzyme required to oxidize 1 ymol of NADH to NAD Respectively. All of the mutant enzymes showed reduced activity against pyruvate and 0HB.
  • 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 the activity against 0HB was decreased.
  • Example 4 Screening of 2R-D-reductase enzyme of 2-0xo-4-hydroxy butyric acid (OHB) and preparation of mutant enzyme
  • OHB 2R-reductase enzyme was screened for D-lactate dehydrogenase.
  • Lactobacillus listeria, L. jensenii, Oenococcus oenii, L. iantum, L. reuteri and L. casei, which are highly active against OHB, have been identified as E. coli, Pediococcus acidi lacti, Pseudomonas aeruginosa, Leuconostoc mesenteroides cremoris,
  • the expected (D) -lactate dehydrogenase enzymes were selected (Table 12). Table 12 shows candidate enzymes for screening dehydrogenase of the OHB D-reductase enzyme.
  • E. coli BL2KDE3 star strain was used as a host strain for the expression of the enzyme, and E. coli DH5a 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 primer shown in Table 10 below And then Hi s-tag was attached to the C-terminal. The PCR fragment was inserted into a pET vector, cloned into E. coli strain DH5a, and sequenced into E. coli BL2KDE3 star.
  • the pG-Tf2 plasmid which expresses the chaperon.
  • D-LDH enzyme For the production of D-LDH enzyme, recombinant BL21 (pET-Xx-LDH) strain was aerobically cultured in LB medium containing 50 Ug / mL kanamycin. The incubation temperature was 30 Q C, stirring speed was 100 rpm, and 250 mL medium was added to a 1 L flask. When the cell concentration reached 0.5 0D, 0.1 mM IPTG was added and an additional 10 hours of pleasure. The cells were then centrifuged, washed three times with 25 mM phosphate buffer solution (pH 7.0), suspended in binding buffer (20 mM pH 7.0 phosphate buffer solution, 0.5 M NaCl, 10 mM imidazole) . The cell lysate was centrifuged at 41: 25,000 g for 30 minutes to remove the insoluble portion, and the soluble portion was collected and used for enzyme activity analysis or additional enzyme separation.
  • D-LDH enzyme activity was measured in the same manner as in the case of L-LDH.
  • pyruvate and 0HB were used as substrates. Like L-LDH, most of the enzymes had 3-10-fold higher activity on pyruvate than on 0HB (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 ⁇ mol / mg protein / min.
  • Lb-LDH showed the highest activity of 0.8 umol / mg protein / min.
  • the selectivity for pyruvate and 0HB was about 1: 0.2.
  • L-LDH was selected to produce D-reductase mutant enzyme having high activity at 0HB.
  • 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 of His296, Arg235, and Glu264 were present in the antagonistic activity site with pyruvate, and additionally two hydrophobic amino acid residues Val78 and TyrlOl were present around the active site. Since these hydrophobic residues are likely to interfere with the reaction with 0HB, they have been replaced with hydrophilic amino residues (Ser, Thr, Asn, Asp, Lys).
  • 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 (Am icon, Merck Millipore Co., Darmstadt, Germany), it was stored at -80 ° C.
  • Table 13 below shows the primer sequences used for the site-directed mutagenesis of Lb-LDH.
  • LDH6_FP cat cactaagatgagcctgcgtaactccggtgt tgacaacatcgacatggct 184
  • LDH7_FP cat cactaagatgagcctgcgtaacaacggtgt tgacaacatcgacatggcta 186
  • LDH8_FP cat cactaagatgagcctgcgtaacaccggtgt tgacaacatcgacatggcta 188
  • LDH9_FP catcactaagatgagcctgcgtaacgacggtgttgacaacatcgacatggcta 190
  • LDH10_FP catcactaagatgagcctgcgtaacaagggtgttgacaacatcgacatggcta 192
  • transaminase and OHB reductase should be optimally expressed.
  • the increase of TA activity should increase the expression of pyridoxal-5-phosphate (PLP) which is a cofactor of TA antagonist as well as the expression of TA enzyme itself, that is, the biosynthesis of vitamin B6.
  • PPP pyridoxal-5-phosphate
  • the extracellular delivery rate of DHB should be increased.
  • DHB production strains were prepared by further modifying strains prepared for homoserine production in Example 1 above.
  • PLP is biosynthesized through a DXP-dependent pathway.
  • the rate of PLP biosynthesis is regulated by proteins encoded by epd, dxs, pdxJ gene, etc.
  • 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 expression of epd, dxs ' , and pdxJ genes to increase vitamin B6 biosynthesis. As a result, three strains with enhanced PLP biosynthesis were obtained (Table
  • 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.
  • 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.
  • G- actP agcaacctgggcgatacctcgac
  • Example 6 Production and fermentation of optically pure S-form or L-form 2, 4-dihydroxybutyric acid (S-black R-DHB) 6-1. Genetically recombinant plasmids for production of DHB
  • 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 Ndel and EcoRI site of pBAD plasmid.
  • Two new plasmids were obtained: pBAD_TA4-l_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.
  • pDHB-D pBAD_TA4-1_LDHD3
  • DHB production pathway plasmids pDHB-L and pDHB-D were respectively introduced into the homoserine producing strains developed in Example 1 for DHB production.
  • a homozygous homozygous mutant strain, EcW20 was used to replace the 10 gene deletions pts, Aeda, MacI, AthrB, AmetA, MysA, AadhE, ApfJB, MdhA and two promoter substitutions (APacs: -PSP8), and the plasmid pUCPK-P rd »e for overexpression of the metL gene.
  • 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.
  • 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; MgS0 4 7_H 2 0, 2 g; KH 2 P0 4, 2 g; (NH 4) 2 S0 4, 10 g; L-methionine, 0.2 g; L L-isoleucine, 0.05 g, trace metal solution, 10 ml
  • the concentration of glucose was 10 g / L, Kanamycin and ampicillin were 50 mg / L .
  • Arabinose was added at different concentrations in the range of 0 - 1 g / L after 3 hours of incubation.
  • the EcW20 strain having no DHB production plasmid produced no DHB at all.
  • 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.
  • 1 g / L of arabinose was added, cell growth and glucose consumption decreased and DHB production decreased 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.
  • D-DHB production was investigated in the same way through the EcW20 (pDHB-D) strain in which pDHBD was introduced.
  • pDHB-D the EcW20 (pDHB-D) strain in which pDHBD was introduced.
  • L-DHB we have a DHB production plasmid .
  • the non-EcW20 strain produced no DHB at all.
  • 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.
  • 1 g / L of arabinose was added, cell growth, glucose consumption, and DHB production were reduced to 0.17 g / L.
  • Bioreactor experiments were performed using EcW20 (pDHB-L) and EcW20 (pDHB_D) strains (Fig. 24A).
  • the medium 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 speed of 1 wm and the agitation speed was appropriately 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.
  • the solid line of the black circles indicates the cell concentration
  • the white circles indicate the product DHB
  • the inverted triangles indicate the glucose concentration.
  • the final cell concentration was 10 g / L and the final L-DHB concentration was 20 g / L.
  • the final cell concentration of D-DHB was 8 g / L and the final L-DHB concentration was 14 g / L.
  • l act onase enzyme is required.
  • 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 the cell membrane or peripolar space. Since this enzyme is derived from an animal, appropriate mutation is necessary for microbial expression.
  • the ponl gene mutant (G3C9) was used and the s ignal sequence was attached at the N-terminus to be expressed in the periplank c space.
  • the ponl (G3C9) gene was synthesized and then cloned into a pACYC-Duet plasmid having a low copy number of s-f Jl (SEQ ID NO: 16).
  • PCR amplification FP, tagacacatatggct aaactgacagcg; RP, tacat act cagat t acagct cacagt aaagagc 111 g
  • FP tagacacatatggct aaactgacagcg
  • RP tacat act cagat t acagct cacagt aaagagc 111 g
  • a tac promoter was used for the expression of ponl (G3C9) (Fig. 18).
  • This plasmid was named pACYC_Ponl.
  • the obtained Ponl pl asmi d was 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), and plasmids for producing DHB in homoserine were used for pDHB-L and pDHB- D was used (Fig. 21). These strains were named EcW16 (pHGBL-L) and EcW16 (pHGBL-D), respectively. 7-2 Production of HGBL for Biological Control
  • HGBL production Two - stage cultivation was performed for HGBL production.
  • DHB was produced in one culture and then HGBL was produced by lowering pH in the second stage.
  • 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.
  • D-DHB 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 ponl expression and the production of additional proteins, particularly ponl membrane proteins, were presumed to be responsible for this. In addition, L-DHB was obtained at a higher concentration than D-DHB because the activity of 2-oxo-reductase was significantly higher than that of D-form as described above.
  • 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.
  • 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).
  • the gain D-HGBL was subjected to the same experiments (Fig. 26b) that is conducted after 48 hours incubation the first stage for 24 hours in a two-stage incubation 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 ponl enzyme is active only in DHB of L-form.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Furan Compounds (AREA)

Abstract

The present invention suggests: a newly shown biosynthesis pathway using the production of homoserine as an intermediate stage for the preparation of 2-hydroxy gamma butyrol acetone (HGBL) and a precursor thereof, 2,4-dihydroxybutanoic acid; and a gene recombinant strain having the pathway. HGBL having a hydroxyl group substituted at the 2nd position thereof can be used as an important intermediate, which is usable as a resin for photoresist, a raw material for a medical product, and a material for coating a metal surface.

Description

【명세서】  【Specification】
【발명의 명칭】  Title of the Invention
2-히드록시 감마 부티로락톤 또는 2, 4-디히드록시 -부티레이트 의 제조 방법  Process for producing 2-hydroxy-gamma butyrolactone or 2,4-dihydroxy-butyrate
【기술분야】  TECHNICAL FIELD
본 발명은 2-hydroxy gamma butyro lactone (HGBL) 및 이의 전구체인 2 , 4-d i hydr oxybut ano i c 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 relates to a newly proposed biosynthetic pathway for the production of 2-hydroxy gamma butyrolactone (HGBL) and its precursor, 2, 4-dihydr oxybutanoic acid, present. 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.
【배경기술】  BACKGROUND ART [0002]
미국특허 4,994,597 및 5,087,751 (I匪 e)은 3,4—디히드록시부티르산 유도체를 발표한다. 이러한 산 제조방법은 금속시안화물과 3 ,4-디히드록시 부틸 클로라이드의 반웅과 가수분해가 관련된 본 발명과 구별된다. 상기 산은 3-히드록시부티로락톤의 중간물질이다. U.S. Pat. Nos. 4,994,597 and 5,087,751 (I 匪 e) disclose 3,4-dihydroxybutyric acid derivatives. Such an acid production method is distinguished from the present invention in which the hydrolysis of metal cyanide with 3, 4-dihydroxybutyl chloride is related to hydrolysis. The acid is an intermediate of 3 -hydroxybutyrolactone.
(S)-3-히드록시부티로락톤은 콜레스테를 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 보층물, 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 1 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 bales, treatment of various nervous systems and metabolic disorders. The market for carnitine is several hundred tons. It is produced in pure form in the form of d and 1. 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-핵소스를 갖는 출발물질을 사용할 필요가 있으므로 상기 공정으로 제조될 수 없다. 이러한 물질은 알려져 있지 않다. 일반적인 락톤 제조방법은 다음 특허로 알려져 있다: (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 with a 4-linked L-nuclear source. These substances are not known. A common lactone preparation process is known from the following patents:
U, .S. Patent No. 3 ,024; ,250 to Klein et al . , U, .S. Patent No. 3, 024 ; , 250 to Klein et al. ,
U, .S. Patent No. 3; ,868, ,370 to Smith, U, .S. Patent No. 3 ; , 868,, 370 to Smith,
U. .S. Patent No. 3; ,997; ,569 to Powel 1 -, U.S. Patent No. 3 ; , 997 ; , 569 to Powel 1 -,
U. .S. Patent No. A. ,105, ,674 to De Thomas et al .  U.S. Patent No. A., 105, 674 to De Thomas et al.
U. ,s. Patent No. 4: ,155: ,919 to Ramioui lie et al U., p. Patent No. 4 : , 155 : , 919 to Ramioui lie et al
U, ,s. Patent No. 4; ,772„ ,729 to Rao, U ,, s. Patent No. 4 ; , 772 ", 729 to Rao,
U. ,s. Patent No. ,. ,940, ,805 to Fisher et al . , U., p. Patent No. , . , 940, 805 to Fisher et al. ,
U. ,s. Patent No. 5; ,292, ,939 to Hoi 1 ingsworth, U., p. Patent No. 5 ; , 292,, 939 to Hoi 1 ingsworth,
U. ,s. Patent No. 5, ,319, ,110 to Hoi 1 ingsworth,  U., p. Patent No. 5,, 319,, 110 to Hoi 1 ingsworth,
U. ,s. Patent No. 5, ,374, ,773 to Hoi 1 ingsworth,  U., p. Patent No. 5,, 374,, 773 to Hoi 1 ingsworth,
U. ,s. Patent No. 5, ,502, ,217 to Fuchikami et al .  U., p. Patent No. 5, 502, 217 to Fuchikami et al.
이들 특허는 다양한 락톤 제조 공정을 발표한다. 그러나 이들 모두는 These patents announce various lactone manufacturing processes. But all of them
2-hydroxy 위치의 gammabutyrolactone의 제조 공정에 대해서는 언급하지 않고 있다. The process for the preparation of gammabutyrolactone in the 2-hydroxy position is not mentioned.
【발명의 상세한 설명】  DETAILED DESCRIPTION OF THE INVENTION
[기술적 과제】  [Technical Problem]
본 발명은 글루코스를 탄소원으로 사용하여 2번 위치 탄소에 광학활성을 갖는 2,4-디히드록시-부틸레이트(2,4- 1 (1 « 13 6) 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 the production of 2,4-dihydroxy-butyrate (2,4- 1 (1 < th > 13 6) or 2-hydroxy- Dihydroxy-butyrate or a salt thereof, which comprises the step of carrying out at least one step selected from the following (1) to (4) in a microorganism producing lactone (2-hydroxy gamma butyrolactone) Hydroxy-gamma-butyrolactone-producing microorganism variant.
(1) ptsG, eda, adhE, pflB, lysA, thrBC, metA, Lad, IdhA, 및 /c ?유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실, 또는 acs, ppc, 및 metL유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계,  (1) deletion of one or more genes selected from the group consisting of ptsG, eda, adhE, pflB, lysA, thrBC, metA, Lad, IdhA and / Overexpressing one or more genes, or both,
(2) epd, dxs, 및 pdxj유전자로 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) Ipc녜 ducA중 하나 이상의 유전자의 과발현시키는 단계, 및 (4) kgtP, dsdx, 및 ac 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실시키는 단계. (3) overexpressing one or more genes of Ipc < RTI ID = 0.0 > and / or & (4) deletion of one or more genes selected from the group consisting of kgtP, dsdx, and ac 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 that produces 2,4-dihydroxy-butyrate or 2-hydroxy-gamma- Dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variants.
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  The present invention also relates to a polypeptide comprising the amino acid sequence of SEQ ID NO:
트랜스아미네이즈 변이효소, 서열번호 14의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소, 및 서열번호 15의 아미노산 서열로 이루어진 트랜스아미네이즈 변이효소를 제공한다. 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 provides a L-hydroxy-2-oxo-reductase mutant consisting of the amino acid sequence of SEQ ID NO: 17 and consisting of the amino acid sequence of L-hydroxy-2-oxo-reductase mutant Enzyme is provided.
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, a D-hydroxy-2-oxo-reductase enzyme comprising the amino acid sequence of SEQ ID NO: Provide mutated enzymes.
또한 본 발명은 서열번호 22의 아미노산 서열로 이루어진, 락토네이즈 변이효소를 제공한다.  The present invention also provides a lactonase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 22.
【기술적 해결방법】  [Technical Solution]
homoserine으로부터 생합성하는 경로를 통하여 광학활성을 갖는 순수 이성질체 흑은 2가지 이성질체가 흔합된 2ᅳ hydroxy ga隱 a butyrolactone과 혹은 이의 전구체인 2,4-dihydroxy butanoic acid를 생산하는 방법을 제공한다. 상기 방법의 일 예가 도 1에 모식적으로 예시되어 있다. Through pure biosynthesis from homoserine, pure isomers of optically active isoforms can be produced by reacting 2-hydroxygalactosidic acid butyrolactone with two isomers, or its precursor, 2,4-dihydroxy butanoic acid to provide. 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 흑은 이들의 흔합물인 상태로 생산하는 하는 경우 2,4-dihydroxy butanoic acid를 분리정제한 후 화학적인 방법으로 광학 활성 순수 이성질체 혹은 이들의 흔합물인 상태로 최종 2— hydroxy gamma butyrolactone를 생산하는 방법일 수 있다.  In one example, 2,4-dihydroxy butanoic acid, which is a precursor of 2-hydroxy gamma butyrolactone, is isolated from 2,4-dihydroxy butanoic acid when it is produced in the form of an optically active pure isomer or a mixture thereof After purification, it may be a method of producing the final 2-hydroxy gamma butyrolactone in the state of being optically active pure isomers or a mixture thereof by a chemical method.
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 a -ketoglutarate ( a_KG)를 사용하는 것을 특징으로 하는 transaminase를 사용하는 것일 수 있다.  In one example, the production method may be to use a transaminase characterized by the use of a-ketoglutarate (a_KG) as an amino acceptor to receive an amino group from homoserine.
또한 상기 생산 방법은 상기 α-KG로부터 생산되는 glutamic acid로부터 amino acceptor인 a— 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 for regenerating the amino acceptor a-KG from glutamic acid produced from the α-KG and simultaneously using aspartate transaminase for the production of aspartic acid, a homoserine biosynthesis precursor .
다른 예에서, 상기 생산 방법은 도 1에 제시한 반웅 경로 중 4- hydr oxy-2-ox-but ano i c acid의 2번 탄소에 결합된 산소를 stereo-specific 하게 흑은 non-specific 하게 환원시켜 hydroxy기로 전환하는 효소로는 L- lactate dehydrogenase와 D-lactate dehydrogenase , 흑은 이들의 흔합 효소를 사용하는 것을 특징으로 하는 경로를 포함할 수 있다.  In another example, the production method is a method wherein the oxygen bonded to the 2-carbon of 4-hydr oxy-2-ox-butanoic acid in the repellent pathway shown in FIG. 1 is reduced in a stereo- L-lactate dehydrogenase, D-lactate dehydrogenase, and black are used as the enzymes which convert to hydroxy group.
다른 예는, 도 1에 제시한 paraoxonase는 인간유래로 lactonase 활성을 갖는 유전자 (P0N1)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. 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. 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 mutation of a gene having a lactonase activity (P0N1) derived from a human. Lt; / RTI > The strain may be a homoserine and a producing strain and a homoserine producing strain. Another example is 2-hydroxy gamma butyrolactone, which has optical activity on position 2 carbon from homoserine through the pathway shown in Figure 1, In order to efficiently produce 2,4-dihydroxy butanoic acid, a precursor which is an organic acid precursor, as shown in FIG. 4, a gene mutation strain produced to efficiently produce homoserine from sugar is provided. 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.  The microorganism for producing the mutant strain may be one or more microorganisms selected from the group consisting of Escherichia coli, yeast coryzae, and the like.
상기 미생물은 다음과 같은 방법들 중에서 선택된 하나 이상을 개별적으로 흑은 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:
l.phosphoenol pyruvate (PEP)를 oxaloacetic acid (0AA)로 효율적으로 전환하는 phosphoenol pyruvate carboxylase (ppc) 유전자의 과발현;  Overexpression of phosphoenol pyruvate carboxylase (ppc) gene, which efficiently converts lphosphoenol pyruvate (PEP) to oxaloacetic acid (0AA);
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. lyoxylate shunt의 활성을 높이기 위해 glyoxylate shunt 유전자 발현을 억재하는 iclR유전자의 제거 ;  3. Elimination of the iclR gene, which inhibits glyoxylate shunt gene expression, to increase the activity of the lyoxylate 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. Improves 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. 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 remove lysine production;
6. methionine 생산을 제거하기 위해 homoserine succinyltransferase를 coding 하는 metA유전자를 제거;  6. Remove the metA gene encoding homoserine succinyltransferase to remove methionine production;
7. 생산된 homoserine이 homoserine phosphate로 전환되지 않도톡 homoserine kinase를 coding하는 thrB유전자를 제거 . 상기 제작된 homoserine 과생산 균주에 앞서 설명한 생산 방법의 경로가 도입되어 당으로부터 2번 위치 탄소에 광학활성을 갖는 2-hydroxy gamma butyro 1 act ne과 혹은 이의 전구체인 2, 4-di hydroxy butanoic ' acid를 생산하도록 제작된 균주가 제공된다. 이 때, 상기 균주는 transaminase의 활성을 증대시키기 위하여 transaminase와 cofactor인 pyridoxal-5 '- phosphate 의 생합성이 촉진되도록 vitamin B6 생합성 경로를 강화시킨 것을 특징으로 하는 것일 수 있다. 7. The produced homoserine does not convert to homoserine phosphate, but removes the thrB gene coding for homoserine kinase. The pathway of the above-described production method was introduced into the prepared homoserine and the production strain, and 2-hydroxy gamma butyro 1 act ne, which is optically active at position 2 carbon from the sugar, and its precursor, 2,4-dihydroxy butanoic acid Lt; RTI ID = 0.0 > a < / RTI > At this time, the strain may be characterized in that the biosynthesis pathway of vitamin B6 is enhanced so as to promote biosynthesis of transaminase and pyridoxal-5'-phosphate as a cofactor 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 흑은 2가지 이성질체가 흔합된 2-hydroxy gamma butyro lactone 및 /또는 이의 전구체인 2, 4-di hydroxy butanoic acid를 생산하는 방법이 제공된다.  Also, a method for producing 2-hydroxy gamma butyrolactone and / or its precursor, 2,4-dihydroxy butanoic acid, which is pure isomeric black having two optically active isomers, is provided from the culture solution by culturing the strain described above do.
상기 방법은, 2-hydroxy ga隱 a butyro lactone 및 /또는 이의 전구체인 2, 4-di hydroxy 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.  This method is particularly useful for the production of 2-hydroxygalactosyl butyrate and / or its precursor, 2,4-dihydroxy butanoic acid, by adding yeast extract and ammonium salt as a nitrogen source, And then culturing the cells using a medium supplemented with amino acids such as methionine, lysine, threonine, and isoleucine.
상기 배양은 생물 반웅기를 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 흔합물 (예컨대, methionine, lysine, threonine, 및 isoleucine의 흔합물)을 첨가하여 수행되는 것일 수 있다.  The cultivation was carried out in the presence of glucose, methionine, lysine, threonine, and isoleucine in the middle of fermentation for the production of high concentration 2-hydroxy gamma butyrolactone and / or its precursor 2,4-dihydroxy butanoic acid (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 butyro lactone)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 production of optically pure 2,4-dihydroxybutyrate or 2-hydroxy gamma-butyrolactone using glucose as a carbon source (1) 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variant, comprising the step of carrying out one or more steps selected from the group consisting of: to provide.
( 1) ptsG, eda, adhE, pflB, lysA, thrB, metA, Lad, IdhA, 및 iclR 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실, 또는 acs, (1) deletion of one or more genes selected from the group consisting of ptsG, eda, adhE, pflB, lysA, thrB, metA, Lad, IdhA and iclR genes,
PPC 및 metL유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계, Overexpressing one or more genes selected from the group consisting of PPC and metL genes, or both,
(2) epd, dxs, 및 ρ τ유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현시키는 단계,  (2) overexpressing one or more genes selected from the group consisting of epd, dxs, and ρ τ genes,
(3) lpd및 ckicA중 하나 이상의 유전자의 과발현시키는 단계, 및 (3) over-expressing one or more genes of lpd and ckicA, and
(4) kgtP, dsdx, 및 aci 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 aci genes.
상기 탄소원은 그 종류를 특별히 한정하지 않으나,  The kind of the carbon source is not particularly limited,
글루코스 (Glucose)인 것이 적절하다. It is appropriate that it is 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) 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一감마—부티로락톤 (2— hydroxy gamma butyrol actone)을 생산하는 미생물은 그 종류를 특별히 한정하지 않으나, 대장균 0?. coli) , 효모 (Yeast ) , 및 코리네박테리움 (Corynebacter i um) 으로 이루어진 군에서 선택된 하나 이상일 수 밌으며, 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 Although there is no particular limitation on the kind, E. coli 0? (E. coli), yeast (Yeast), and Corynebacterium (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. 바람직하게는, 상기 미생물을 변이시키는 단계는 ) 단계, 및 (2) 내지 (4) 단계 중 선택된 하나 이상의 단계를 포함해 수행할 수 있으며, 가장 바람직하게는 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. Preferably, the step of mutating the microorganism may be carried out, and the step (2) to (4)
(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, Homoser inesuccinyl Transferase 를 코딩하는 metk, 그리고 Homoser ine 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, it is possible to remove one or more of lysA encoding diaminopimelate decoxyase, metk encoding Homoserine inesuccinyl Transferase, and thrBC encoding Homoserine ine Kinase and Threonine Synthase, respectively, which are genes encoding homoserine acid basic enzyme,
상기 (1) 단계에서 제거가능한 유전자 중 MiA, adhE, ;?/ 유전자는 부산물인 젖산, 에탄을, 포름산 등의 생산을 막기 위해 제거하는 것일 수 있다. 상기 유전자들의 제거는 부산물 생성을 막고 homoserine으로 가는 탄소 flux를 증가시킨다,  Among the genes that can be removed in step (1), MiA, adhE, and /? Gene may be removed to prevent the production of by-products such as lactic acid, ethane, and formic acid. Removal of these genes prevents by-product formation and increases the carbon flux to homoserine,
상기 (1) 단계에서 제거가능한 유전자 중 /cH?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.  The / cH? Gene among the genes that can be removed in the above step (1) is removed in order to enhance the activity of the glyoxylate shunt, which is one of the methods for promoting acetal reuse. The removal can be carried out by a conventional method, You can remove it with the pop-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 Catabol ite 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 transport 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 을려줄 수 있다. 상기 유전자의 제거 방법은 통상적인 방법을 사용할 수 있으나, 바람직하게는 MAGE 방법을 사용할 수 있다. Among the genes that can be removed in the above step (1), the eda gene is a gene coding for KHG / KDG aldolase. When the gene is deleted, the ED pathway The use of the EMP (Embden-Meyerhof-Parnas pathway) pathway is facilitated and the carbon flux to oxaloacetate can be applied. 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 lacl 유전자는 Lac 프로모터 활용 (promoter utilization)을 위해 lacl 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 the above step (1), the lacl gene removes the lacl inhibitor for the utilization of the Lac promoter (promoter utilization). The gene can be removed by a conventional method, have.
상기 (1) 단계에서 유전자의 과발현은 통상의 방법을 사용할 수 있다. 예를들어 미생물 세포에 유전자가 포함된 백터를 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 large amount of a vector containing a gene in a microbial cell may be introduced, or an over-expression promoter substitution method or the like may be used.
구체적으로 , 상기 (1) 단계에서 3CS 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 유전자의 결실이 세포성장에 나쁜 영향을 주는 경우가 많아 이를 배제하였다.  Specifically, in step (1), overexpression of the 3CS gene may increase the expression of the acs gene in order to minimize the production of acetic acid. pTA-ack and poxB genes can also be deleted, but the deletion of these genes, especially the deletion of genes, often adversely affects cell growth.
acs 유전자의 발현을 증대시키는 방법은 관련업계에서 통상적으로 사용하는 방법을 제한없이 사용할 수 있지만, 바람직하게는 3CS 발현 유전자의 프로모터를 유전자 과발현 프로모터로 바꾸어줄 수 있으며, lac 프로모터, tac 프로모터, trc 프로모터 등을 사용할 수 있으며, 바람직하게는 re프로모터로 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 3CS 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 a re-promoter.
상기 (1) 단계에서 ppc 유전자의 과발현은 acetate 생성을 막고 호모세린으로의 탄소 flux를 향상시키기 위하여 anapl erotic 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 encoding phosphoenol pyruvate carboxylase, which is the key enzyme of the anapl-erotic pathway, to inhibit acetate production and improve the carbon flux to homoserine. A gene expression increasing 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, ΑΚΠ 및 AKIII 중에서 ΑΚΠ는 aspartate kinase 와 serine dehydrogenase 활성을 동시에 가지고 있어 ΑΚΙΠ 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 the above step (1) This is to optimize the pathway after aspartic acid, known as bottleneck. It is known that three aspartate kinases AKI, AKII and AKIII play an important role in homoserine production. Of these AKII and AKIII, only two enzymes are known to exhibit good activity at high homoserine concentrations. Among AKI and AKIII, Aspartate kinase and serine dehydrogenase activity are known to be more advantageous than ΑΚΙΠ. Therefore, the mutant strain of the present invention can overexpress the metL gene coding for the AKII enzyme.
상기 metL유전자의 과발현 방법은 통상의 방법을 사용할 수 있으며, 바람직하게는 medium copy plasmid인 pUCPK plasmid를 이용하여 과발현시킬 수 있다. 이 때 metL^ 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 control metL ^ expression size.
상기 결실키기거나 과발현시키는 유전자를 표 1에 정리해 나타냈다. 본 발명의 일 구체예에 따르면, 상기 (1) 단계는 ptsG, eda, adhE, pflB, lysA, thrB, etA, Lac I, IdhA, 및 /c/?유전자 전부를 결실시키고, acs, ppc, 및 metL유전자 전부를 과발현시키는 단계를 포함하는 것일 수 있다.  The genes for over-expressing or over-expressing the genes are summarized in Table 1. According to one embodiment of the present invention, the step (1) comprises deleting all of the ptsG, eda, adhE, pflB, lysA, thrB, etA, Lac I, IdhA and / lt; RTI ID = 0.0 > metL < / RTI > gene.
본 발명의 일 예에 따르면, 상기 (2) 단계는 Vitamin B6 생산 증가를 위해 수행하는 것으로, 대장균의 경우 PLP(Pyridoxal— 5-Phosphate)는 DXP(l-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 one embodiment of the present invention, the step (2) is carried out to increase the production of Vitamin B6. In the case of Escherichia coli, the PLP (Pyridoxal-5-Phosphate) is dependent on D-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. For the over-expression method, a conventional method can be used. Preferably, it is appropriate 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 pdxj유전자 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, ifcu^의 발현양 증가는 2,4-디히드록시 -부틸레이트 (DHB)의 세포 외 이송속도를 증대시키기 위해 수행하는 것으로, 유기산 막 이송 단백질, 특히 l act i c aci d 와 저분자량의 carboxyl i c aci d 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, ifcu ^ in step (3) increases the extracellular delivery rate of 2,4-dihydroxy-butyrate (DHB) In this study, we selected importer and exporter as a query among organic acid membrane transfer protein, especially l act ic acid and low molecular weight carboxyl acid transfer protein. Then, two most promising exporters, ldp and ldp, / DcuA < / RTI > protein.
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(Synthet i c promoter 5) 등을 사용하는 것이 바람직하다.  As a method for increasing the expression of these proteins, conventional methods can be used, preferably promoters can be substituted, and overexpression of the membrane protein interferes with cell growth, so that a mid-level synthetic promoter SP5 (Synthet ic promoter 5) or the like is preferably used.
바람직하게는, 상기 (3) 단계는 lpd, 및 ducA유전자 전부를  Preferably, the step (3) comprises the steps of lpd, and all of the ducA gene
과발현시킬 수 있다. Overexpression.
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 actP중 어느 하나 이상을 제거할 수 있다. 이 때 사용할 수 있는 프라이머 (pr imer ) 서열을 표 18에 나타냈다. 바람직하게는, 상기 (4) 단계는 kgtP, dsdx, 및 ac 5유전자 전부를 제거시킬 수 있다. 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 encoding the protein, kgtP, dsdx and actP, can be removed. The primer sequences usable at this time are shown in Table 18. < tb >< TABLE > Preferably, the 4 step may be to remove all of the 5 kgtP, dsdx, and ac gene.
상기 ( 1)단계를 사용해 제조한 변이균주를 표 2(EcWl 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 mutant strains prepared in the above step (1) through the steps (1) and (2) in Table 2 (EcWl to EcW13) 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-οχο- 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 (homoser ine)의 알파 위치 아민기를 제거해 4-히드록시 -2-옥소-부틸레이트의 전환을 촉진하는 것을 의미하며, 구체적으로, 트랜스아미네이즈 ( transaminase)를 처리하여 호모세린 (homoser ine)의 알파 위치 아민기를 제거하는 단계를 포함하는 것일 수 있다. 상기 트랜스아미네이즈는 이를 암호화하는 유전자가 포함된 백터를 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, and specifically, transaminase And removing the alpha-position amine group of the homoserine. The transaminase can be obtained by introducing a large amount of a vector containing a gene encoding the gene into a microorganism and transforming the transaminase, And a method of replacing the promoter of the gene encoding with the over-expression 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 아루어진 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: 13 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 example of the present invention, the method for producing a microorganism variant may further include a 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 백터를 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-butylate. A large amount of a vector containing a gene encoding a reductase that reduces 4-hydroxy-2-oxo-butylate is introduced into a microorganism 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 환원반웅에 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 흔합물을 제조할 수도 있으며,According to one embodiment of the present invention, the 2,4-dihydroxy-butyrate produced by the reduction reaction is 2,4-dihydroxy- (2S) -2, 4-dihydroxy-butyrate and (2R) -2, 4-dihydroxy-butyrate being the pure optical isomers One or more, racemates, optical isomeric waxes may be prepared,
(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. (2S) -2, 4-dihydroxy-butyrate or (2R) -2, 4-dihydroxy-butyrate, respectively. The kind of the optical isomer of 2,4-dihydroxy-butyrate may be selected depending on the intended use 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 in order to promote the conversion of D-hydroxy-2-oxo-reductase to 4-dihydroxy-butyrate.
본 발명의 바람직한 일 예에 따르면, 상기 L-히드록시 -2-옥소- 리덕테이즈는 서열번호 16의 아미노산 서열로 이루어진 효소, 서열번호  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,
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. 17 and an enzyme consisting of the amino acid sequence of SEQ ID NO: 18, preferably 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 May be an enzyme consisting of an amino acid sequence. 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  According to another embodiment of the present invention, the D-hydroxy-2-oxo-reductase comprises an enzyme consisting of the amino acid sequence of SEQ ID NO: 19,
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 호소, 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 아미노산서열로 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. An enzyme consisting of an amino acid sequence of SEQ ID NO: 20, and an enzyme consisting of an amino acid sequence of SEQ ID NO: 21, preferably an amino acid sequence of SEQ ID NO: 20, and / An enzyme comprising an amino acid sequence. 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: And the gene encoding the enzyme consisting of the amino acid sequence of SEQ ID NO: 21 may be composed of 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 ( l actonase)의 발현을 촉진하여 수행되는 것일 수 있으며, 상기 발현 촉진 방법은 락토네이즈 유전자를 포함하는 백터를 과량 세포에 도입해 락토네이즈 양을 증가시키거나 락토네이즈 유전자를 발현하는 프로모터를 과발현 프로모터를 치환하는 등 다양한 방법을 사용할 수 있다. 바람직하게는, 상기 락톤화의 촉진은 서열번호 22의 아미노산 서열로 이루어진 락토네이즈 ( l actonase)의 발현을 촉진하여 수행되는 것일 수 있으며, 상기 서열번호 22의 아미노산 서열로 이루어진 락토네이즈를 암호화하는 유전자는 서열번호 11의 염기서열로 이루어진 것일 수 있다. 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. 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 performed by promoting the expression of l actonase. The expression promoting method may be carried out by introducing a vector containing a lactonase gene into an excessive cell to increase the amount of lactonase, A variety of methods can be used, such as replacing the promoter expressing the gene with an over-expression promoter. Preferably, the promoting of the lactonization may be carried out by promoting the expression of l actonase 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: 22 May comprise the nucleotide sequence of SEQ ID NO: 11. The resulting 2-hydroxy-gamma-butyrolactone is at least one compound selected from the group consisting of pure optical isomers (2S) -2-hydroxy gamma butyrolactone and (2R) -2 : hydroxy gamma butyrolactone number and, preferably, (2S) - 2-hydroxy-gamma -butyrolactone or (2R) in-lock may tonil 2 _-hydroxy-gamma -butyrolactone.
상기 방법에 의해 생산된 (2S)-2-히드록시 감마 부티로락톤 또는 (2S) -2-hydroxy gamma butyrolactone produced by the above method or
(2R)-2-히드록시 감마 부티로락톤은 광학 순도가 90% 내지 100%, 바람직하게는 95% 내지 100%, 더욱 바람직하게는 99% 내지 100%일 수 있다. 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. (2R) -2-hydroxy gamma butyrolactone may have an optical purity of 90% to 100%, preferably 95% to 100%, more preferably 99% to 100%. According to one embodiment of the present invention, the genome of the microorganism producing the 2,4-dihydroxy-butyrate or the 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, pflB, lysA, thrBZ , met A, Lad, IdhA, 및 /c/y?유전자로 이루어진 군에서 선택된 하나 이상의 결실, 또는 acs, ppc, 및 metL유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계; (1) one or more deletions selected from the group consisting of ptsG, eda, adhE, pflB, lysA, thrBZ, metA, Lad, IdhA, and / c / Overexpressing one or more genes selected, Or both;
(2) epd, dxs, 및 pdxi 유전자로 이루어진 군에서 선택된 하나 이상의 유전자의 과발현,  (2) overexpression of one or more genes selected from the group consisting of epd, dxs, and pdxi genes,
(3) Idp및 ducA중 하나 이상의 유전자의 과발현,  (3) overexpression of one or more genes of Idp and ducA,
(4) kgtP, dsdx, 및 ac尸유전자로 이루어진 군에서 선택된 하나 이상의 유전자의 결실.  (4) deletion of one or more genes selected from the group consisting of kgtP, dsdx, and ac 尸 genes.
본 발의 일 예에 따르면, 상기 미생물 변이체는 바람직하게는 pt s , eda , l ad , thrB , metA , lysA , adhE , pf IB , IdhA , 및 i c lR 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. 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, pflB, lysA, thrBC, metA, Lac I, IdhA, iclR, kgtP, dsd, 및 aci 유전자가 결실되고, acs, ppc, metL, epd, dxs, pdxj, Idp, 및 ciucA 유전자가 과발현된, 미생물 변이체를 제조할 수 있으면, 상기 미생물 변이체는 하기 실시예에서 실험한 EcW20 균주로 기탁번호 KCCM12282P 의 균주일 수 있다. According to the foot of example, the microbial mutants are preferably pt s, eda, l ad, thrB, metA, lysA, adhE, pf IB, IdhA, and ic lR gene is deleted, overexpression of the acs gene, and ppc It is possible to produce a microorganism variant in which the gene is distinguished. The microorganism variant may be the EcW13 strain shown in Table 15, preferably the strain of Accession No. KCCM12281P. According to another embodiment of the present invention, the microorganism variant is selected from the group consisting of ptsG, eda, adhE (2, 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone) , microbial mutants in which pflB, lysA, thrBC, metA, Lac I, IdhA, iclR, kgtP, dsd and aci genes are deleted and acs, ppc, metL, epd, dxs, pdxj, Idp and ciucA genes are overexpressed , The microorganism variant may be a strain of EcW20, which was tested in the following Examples, and the strain of KCCM12282P.
본 발명의 일 예에 따르면, 상기 미생물 변이체는 하기 ( 1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 백터를 추가로 포함할 수 있다.  According to one embodiment of the present invention, the microbial mutant 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, (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 (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) 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: And '
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 butanoi c acid)의 생산방법을 제공한다.  According to an embodiment of the present invention, there is provided a method for producing a microorganism which comprises culturing the above-described microorganism variant as described above, wherein 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutyrate -dihydroxy butanoic acid. < / RTI >
상기 생산방법에 사용하는 상기 미생물 변이체는 하기 ( 1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 백터를 추가로 포함하는 것일 수 있다. The microorganism variant used in the production method may be one or more genes selected from the following (1) to (4) or a recombinant vector containing the same It may be additionally included.
(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,
(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 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 ( i soleucine)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 as the 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 lowered to a pH of 1.0 to 3.0, preferably a pH of 1.0 to 2.0, Butyrolactone. ≪ / 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-hydroxybutyrate having an optical purity of 95% to 100%, preferably 97% Hydroxy-gamma-butyrolactone, and (2R) -2, 4- (2R) -2-hydroxy-gamma-butyrolactone having an optical purity of 95% to 100%, preferably 97% to 100% by lowering the pH to 1.0 to 3.0 after producing dihydroxybutyrate .
본 발명의 일 예는, 상기에서 설명한 미생물 변이체 또는 그 배양물을 포함하는, 2-히드록시 감마 부티로락톤 (2-hydroxy ga隱 a butyro lactone) 또는 2 , 4-디히드록시 부틸레이트 (2 ,4-dihydroxy butanoi c acid)의 생산용 조성물을 제공한다.  One example of the present invention is a microorganism which comprises 2-hydroxygamma butyrolactone or 2, 4-dihydroxybutyrate (2 , 4-dihydroxy butanoic acid).
상기 미생물 변이체는 하기 ( 1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 백터를 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,
(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 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 변이효소 암호화하는 유전자는 서열번호 4의 염기서열로 이루어진 것일 수 있다. 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 is a polypeptide comprising the amino acid sequence of SEQ ID NO: Thereby providing a transaminase mutant enzyme. The gene coding for the transaminase mutant enzyme may comprise the nucleotide sequence of SEQ ID NO: 4. 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 아미노산서열로 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  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 has the amino acid sequence of SEQ ID NO: 10
염기서열로 이루어진 것일 수 있다. Base sequence.
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.
[발명의 효과】  [Effects of the Invention】
본 발명은 2-hydroxy gamma butyro l actone (HGBL) 및 이의 전구체인 2, 4-dihydroxybutanoi c aci d 를 과발현하는 변이 균주 및 이를 이용한 2- hydroxy gamma butyro l ac tone (HGBL) 및 이의 전구체인 2,4- dihydroxybutanoi c 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 웅용될 수 있다. The present invention relates to a mutant which overexpresses 2-hydroxy gamma butyrolactone (HGBL) and its precursor, 2,4-dihydroxybutanoic acid, and a 2-hydroxy gamma butyrolactone (HGBL) 4-dihydroxybutanoic acid. 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. These isomers as well as intermediates for pharmaceuticals, pesticides, seasonings and perfumes, especially photoresists for semiconductors, intermediates for coating materials on metal surfaces Can be usefully exploited.
【도면의 간단한 설명】  BRIEF DESCRIPTION OF THE DRAWINGS
도 1은 homoser ine 으로부터 광학적으로 순수한 2— hydroxy gamma butyro lactone 및 이의 유기산 전구체인 2,4-dihydroxybutanoic acid를 생합성하는 신규 생합성 경로를 제시하고 있다.  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 ine.
도 2는 homoser ine 으로부터 homoser ine dehydrogenase를 이용하여 4-hydroxy-2-oxo-butanoate를 생합성하는 경로를 제시하고 있다.  FIG. 2 shows a pathway for biosynthesis of 4-hydroxy-2-oxo-butanoate from homoserine ine using homoserine dehydrogenase.
도 3은 homoser ine 으로부터 homoser ine transaminase 및 aspartate transaminase를 이용하여 4一 hydroxy— 2— oxo— butanoate를 생합성하는 경로를 제시하고 있다.  FIG. 3 shows a pathway for biosynthesis of 4-hydroxy-2-oxo-butanoate using homoserine transaminase and aspartate transaminase from homoserine ine.
도 4는 포도당을 이용하여 homoser ine을 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.  FIG. 4 shows a strategy for producing a large amount 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.
도 6a는 TA1, TA2, TA3, TA6 효소를 생산하는 균주 파쇄엑 전체를 denaturing 조건에서 전기영동한 결과로, TA^ TA2, TA3, 및 TA6 4 종류는 insoluble 한 형태로만 효소가 생성됨을 나타낸다.  FIG. 6A shows the result of electrophoresis of the strain-cleaved excision product producing the TA1, TA2, TA3 and TA6 enzymes under denaturing conditions, indicating that 4 types of TA ^ TA2, TA3 and TA6 are enzymatically produced only in insoluble form.
도 6b는 TA4, TA5, TA7, YA8, TA9, TAIO, TAll, TA12, TA13, TA14 및 FIG. 6B is a block diagram of an embodiment of the present invention, wherein TA4, TA5, TA7, YA8, TA9, TAIO, TAll, TA12, TA13,
TA15 등 총 11종 효소를 생산하는 균주의 파쇄액을 원심분리하여 상등액을 얻고 상등액 내 존재하는 단백질을 denaturing 조건에서 전기영동한 결과로 TA4, TA5, TA7, YA8ᅳ TA9, TAIO, TAll, TA12, TA13, TA14 및 TA15 효소가 soluble 한 상태로 얻어진다는 것을 보여준다. TA5, TA7, YA8, TA9, TAIO, TA11, TA12, TA12, and TA12 were obtained as a result of centrifugation of the lysate of strains producing 11 kinds of enzymes such as TA15 and TA15, TA13, TA14 and TA15 enzymes are obtained in a soluble state.
도 7a는 soluble 한 형태로 발현된 11종의 트랜스아미네이즈 효소 증 FIG. 7A shows eleven transaminase enzymes expressed in soluble form
TA4, TA5, TAIO, TAll, TA15 등 5개의 효소만이 호모세린 (homoser ine)에 대하여 활성을 나타내었고 이 중 TA4가 가장 높은 활성을 가진다는 것을 나타낸다. Only five enzymes such as TA4, TA5, TAIO, TAll and TA15 exhibited activity against homoserine, indicating that TA4 has the highest activity.
도 7b는 호모세린에 대하여 가장 높은 활성을 나타낸 TA4 효소를 affinity chromatography로 순수분리하여 정제한 결과를 전기영동한 결과이다. FIG. 7B shows the results of purification of the TA4 enzyme, which exhibits the highest activity for homoserine, by pure separation using affinity chromatography, and electrophoresis Results.
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 질소원으로사용된다는 것을보여준다.  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 of 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.
도 9는 대장균 내 존재하는 아스파르트산 아미노산 전이효소 (PDB ID : 1ASM)와 TA4효소의 아미노산서열을 비교한 결과이다.  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.
도 10은 대장균 아스파르트산 아미노산 전이효소 (PDB ID : 1ASM)의 크리스탈 구조를 주형으로 사용하여, 상동성 모델링을 통해 TA4의 3차원 구조를 구축하고, PYM0L뷰어 (http : //www .pymo 1 . org)를 사용하여 확인한 단백질 구조를 나타낸다.  10 shows the 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 the PYM0L viewer (http: // www. org), respectively.
도 11은 대장균 아스파르트산 아미노산 전이효소 ( 1ASM) 구조에서 말레산의 카르복실산 (아스파르트산 잔기의 카르복실산)과상호작용을 하고 있는 4개의 아미노산 ( I lel7 , Gly38 , Asnl94 , Arg386)을 확인하고, 이와 비교한 TA4모델 구조를 통해서 TA4효소 내 아스파르트산 잔기의  FIG. 11 shows four amino acids (I lel7, Gly38, Asnl94, Arg386) which are interacting with carboxylic acid (carboxylic acid of aspartic acid residue) of maleic acid in E. coli aspartic acid amino acid transferase (1ASM) , And the TA4 model structure as compared with that of the aspartic acid residue in the TA4 enzyme
카르복실산과 상호작용을 할 것으로 예상되는 4개의 아미노산 (Lysl4 , Gly40 Asnl78 , Try364)을 선정해 나타낸 것이다. (Lysl4, Gly40 Asnl78, Try364), which are expected to interact with carboxylic acids.
도 12는 TA4-1 내지 TA4-6의 변이 효소의 활성을 나타낸다. 이중 TA4-1은 Y364Q, TA4-2는 N174D의 아미노산서열 변이를 갖고 있으며 높은 활성을 보여 주었다. TA4-6는 TA4-1 및 TA4-2가 갖는 변이를 모두 갖는 효소로 가장높은 활성인 20 U/mg protein의 활성을 나타내었다.  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.
도 13은 표 9에 나타낸 8가지의 LDH효소의 효소활성을 pyruvate 와 0HB를 기질로사용하여 분석한 결과를 나타낸다.  FIG. 13 shows the results of analysis of enzymatic activity of the eight LDH enzymes shown in Table 9 using pyruvate and 0HB as substrates.
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 M0E(Molecular Operating Environment )를 사용하여 제작했으며, PR0CHECK 및 ProSA 온라인 구조 분석을 통해 평가 및 PYM0L뷰어 (http://w丽. pymol.org)를 사용하여 확인한 단백질 구조를 나타낸 것이다. 14 shows a three-dimensional structure of Ae_ldhA by homology modeling using a crystal structure (PDB ID: 2G8Y) of E. coli lactate dehydrogenase as a template As a result, the Ae_ldhA model was constructed using Molecular Operating Environment (M0E), and evaluated using the PROCHECK and ProSA online structure analysis and the protein structure identified using the PYM0L viewer (http: // www. Pymol.org) will be.
도 15a는 Ae_ldhA 변이체 디자인을 위해 피부르산과 HOB 구조 (Pubchem Database)의 구조를 Ae_ld l의 효소 활성 위치에 Triangular Matching 방법을 사용하여 도킹 시물레이션 (docking simulation)을 한 후, 이를 이용하여 효소의 아미노산 잔기와 피부르산과의 상호작용올 검토한 결과를 나타낸다.  FIG. 15A shows the structure of the skin leucine and HOB structure (Pubchem Database) for the Ae_ldhA mutant design by performing a docking simulation using the triangular matching method at the enzyme active site of Ae_ld l, And skin lean acid.
도 15b는 도킹 시뮬레이션 (docking simulation)을 이용하여 효소의 아미노산 잔기와 HOB와의 상호작용을 검토한 결과를 나타낸다.  FIG. 15B shows the result of examining the interaction between amino acid residues of the enzyme and HOB using a docking simulation.
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 μη)1의 NADH를 NAD로 산화시키는데 필요한 효소의 양으로 정의하였다.  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 μηη 1 of NADH to NAD for 1 min.
도 17은 표 12에 나타낸 9가지의 균주 내 (D)-lactate dehydrogenase 효소활성을 pyruvate 와 0HB를 기질로 사용하여 분석한 결과를 나타낸다. 도 18은 PDB databank에 나와있는 Lb-LDH 효소의 결정구조를 이용하여 engineering 해야 할 아미노산 잔기를 나타낸 것이다.  17 shows the results of analysis of the (D) -lactate dehydrogenase enzyme activity in the nine strains shown in Table 12 using pyruvate and 0HB 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.
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 um이의 NADH를 NAD+로 산화시키는데 필요한 효소의 양으로 정의하였다. 변이 효소의 활성을 야생 효소의 활성과 비교하여 상대 활성으로 나타내었다.  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 specific activity (1 U) of the enzyme was determined by adding 1 μM 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.
도 20a는 대장균에서 PLP가 생합성되는 DXP 의존 경로에 작용하는 생합성 유전자의 구조를 나타낸다.  20A shows the structure of a biosynthetic gene acting on a DXP-dependent pathway in which PLP is biosynthesized in E. coli.
도 20b는 대장균에서 PLP가 생합성되는 DXP 의존 경로의 생합성 경로를 나타낸다. 대사산물의 약어는 다음과 같다. G6P; glusoe-6- phosphate; E4P, erythrose-4-phosphate; GA3P, g 1 ycer a 1 dehyde-3- phosphate ; 4PE , 4-phospho-D-erythronate; 3P4K, 3ᅳ hydroxyᅳ 4_ pho s phohydr oxy- alpha-ketobutyrate ; 4PT, 4ᅳ phosphohydroxyᅳ Lᅳ threonine; 2A3B, 3ᅳ hydroxyᅳ l_amin으 acetone phosphate ; DXS, deoxyxylulose-5- phosphate . 이탤릭체는 효소이름을 나타낸다: EPD, erythrose-4-phosphate dehydrogenase; PdxB , 4-phospho-D-erythronate; SerC , 3-phosphoser ine aminotransferas; PdxA, 4-phosphohydroxy-L-threonine dehydrogenase ; PdxJ , PNP synthase ; Dxs , 1-deoxyxylulose 5-phosphate synthase; PdhH, PNP oxidase . 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, glutamic acid dehyde-3- phosphate; 4PE, 4-phospho-D-erythronate; 3P4K, 3Phoxy ᅳ 4 pho s phohydr oxy- alpha-ketobutyrate; 4PT, 4 ᅳ phosphohydroxy ᅳ L ᅳ threonine; 2A3B, 3 ᅳ hydroxy ᅳ lamin, acetone phosphate; DXS, deoxyxylulose-5-phosphate. Italic indicates the enzyme name: EPD, erythrose-4-phosphate dehydrogenase; PdxB, 4-phospho-D-erythronate; SerC, 3-phosphoserine ine aminotransferases; 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 각각사용한 경우를 나타낸 것이다.  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.
도 22는 플라스크 배양을 통한 L-DHB 생산 결과로, 유전자 재조합 pBAD_TA4-l_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-l_LDH2 (pDHB—L)플라스미드를 갖지 않는 EcW20균주를 나타낸다.  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-l_LDH2 (pDHB-L) plasmid was introduced at various arabinose concentrations / L) was measured. 'Control' represents the EcW20 strain which does not have the pBAD_TA4-I_LDH2 (pDHB-L) plasmid.
도 23은 플라스크 배양을 통한 D-DHB 생산 결과로, 유전자 재조합 pBAD_TA4-l_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-l_LDH8 (pDHB-D)플라스미드를 갖지 않는 EcW20 균주를 나타낸다.  23 shows the concentration (g / L) of DNB produced after culturing the EcW20 (pDHB_D) strain in which recombinant pBAD_TA4-l_LDH8 (pDHB-D) plasmid was introduced at various arabinose concentrations as a result of D-DHB production through flask culture ) Were measured. 'Control' represents an EcW20 strain that does not have the pBAD_TA4-l_LDH8 (pDHB-D) plasmid.
도 24a는 L— form 2 , 4-dihydroxybutyr i c acid 생산을 위한 EcW20(pDHB-L) 균주의 유가식 생물반웅기 실험결과를 나타낸다.  FIG. 24A shows the results of a flow-through bioassay of EcW20 (pDHB-L) strain for the production of L-form 2, 4-dihydroxybutyric acid.
도 24b는 D-form 2 , 4-dihydroxybutyr i c acid 생산을 위한 EcW20(pDHB-D) 균주의 유가식 생물반웅기 실험결과를 나타낸다.  24B shows the results of a flow-through biotite assay of the EcW20 (pDHB-D) strain for the production of D-form 2, 4-dihydroxybutyric acid.
도 25는 2,4— dihydroxybutyr i c acid를 HGBL로 전환하는데 필요한 락토네이즈를 발현하는 pACYC_Ponl 플라스미드 맵을 보여준다. 과 (G3C9) 유전자는 tac promoter 에 의해 발현되고 생산된 단백질이 세포막으로 이동하는데 필요한 lead sequence를 갖도록 제작되었다.  Figure 25 shows the pACYC_Ponl plasmid map expressing lactonase necessary to convert 2,4-dihydroxybutyr ic acid to HGBL. And (G3C9) genes were expressed by the tac promoter, and the produced proteins were designed to have the necessary lead sequence to move to the cell membrane.
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. FIG. 26A shows a two-stage bioreactor culture for the production of L-DHB and HGBL As a result, the amount of 2, 4-DHB and HGBL produced and biomass of L-form, which are produced with time in the case of using glucose as a substrate, are shown.
도 26b는 D-DHB 와 HGBL 생산을 위하여 생물반웅기 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 Ponl 효소의 활성이 2 , 4-DHB에 대하여 매우 낮았기 때문이다.  FIG. 26B shows the result of D-form 2, 4-DHB and HGBL production with time as a result of the biotransformant culture for the production of D-DHB and HGBL, . The yield of D-HGBL was very low, below 0.1 g / L, because the activity of Ponl enzyme was very low for 2,4-DHB.
【발명의 실시를 위한 형태】  DETAILED DESCRIPTION OF THE INVENTION
호모세린 (homoser ine)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 반웅  Step 1: Removal of alpha position amine from homoserine
Step 2 : 환원 반응 (2번 탄소에 결합된 산소 환원)  Step 2: Reduction reaction (oxygen reduction bound to carbon # 2)
Step 3: 락톤화 반웅 (Lactoni zat ion)  Step 3: Lactoni zat ion
상기 첫번째 반응은 호모세린 디아미네이즈 (homoser ine deaminase) 또는 호모세린 트랜스아미네이즈 (homoser ine transaminase) 효소에 의해 촉매될 수 있으며, 호모세린 디아미네이즈에 의한 반웅은 도 2에  The first reaction may be catalyzed by a homoserine ine deaminase or a homoserine transaminase enzyme, and the homoserine diamine-catalyzed reaction is shown in FIG. 2
나타냈으며, 호모세린 트랜스아미네이즈에 의한 반웅은 도 3에 각각 나타냈다. And the reaction by homoserine transaminase was shown in FIG. 3, respectively.
도 2에 제시한 아미노산의 알파 위치 아민기를 제거하는 효소로는 글루타메이트 (glutamate)에 높은 활성을 보이는 글루타메이트  As an enzyme that removes the alpha-position amine group of the amino acid shown in FIG. 2, glutamate having high activity on glutamate
디아미네이즈 (glutamate deaminase ; dehydrogenase) , 그리고 Glutamate deaminase (dehydrogenase), and
세린 (ser ine)에 높은 활성을 보이는 세린 디아미네이즈 (ser ine deaminase) , 그리고 활성이 매우 낮지만 다른 많은 아미노산들에 조금씩 활성을 보이는 아미노산 산화효소 (amino acid oxidase)를 사용할 수 있으며 도 2의 반웅에는 이 효소들을 직접 사용하거나 또는 높은 활성을 갖도록 변이시킨 후 사용할 수 있다. Serine deaminase, which shows high activity in serine, and amino acid oxidase, which is very active but slightly active in many other amino acids, can be used. These enzymes can be used directly or after being mutated to have high activity.
도 3에 제시한 transaminat ion 반응에는 변이를 통해 homoser ine에 대해 활성을 갖도록 제작된 변이 alanine aminotransferase를 비롯하여 이와 유사한 amino transferase를 사용할 수 있다. 또한 In the transaminat ion reaction shown in Fig. 3, the mutant alanine aminotransferase prepared to have activity against homoserine ine Similar amino transferases can be used. Also
homoser ine으로부터 amino기를 받는 amino acceptor로는 a-ketoglutarateAmino acceptors that receive an amino group from homoser ine include a-ketoglutarate
(α-KG)를 사용할 수 있으며 (transaminase 반웅에서 α-KG/glutamic acid pair가 이 목적으로 가장 널리 사용됨), α-KG로부터 생산되는 glutamic acid로부터 amino acceptor인 oHCG를 재생함과 동시에 homoser ine 생합성 전구체인 aspart ic acid의 생산을 위해 aspartate transaminase를 동入 1에 사용하는 것이 바람직하다. (α-KG / glutamic acid pair is most widely used for this purpose in the transaminase reaction), oHCG, which is an amino acceptor from glutamic acid produced from α-KG, is regenerated and homoserine ine biosynthesis It is desirable to use aspartate transaminase for the production of the precursor aspartic acid.
도 1에 제시한 두 번째 단계 반응의 효소로는 L— lactate  As the enzyme of the second step reaction shown in Fig. 1, 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 butyro lactone의 구조도dehydrogenase and D-lactate dehydrogenase can be used. Depending on the enzyme used, the hydroxy group is bound to the 2-position carbon in the (R) or (S) form and the structure of the 2-hydroxy gamma butyrolactone
(R) 혹은 (S) 형태의 isomer가 얻어지게 된다. (R) or (S) -form isomer is obtained.
도 1에 제시한 paraoxonase는 인간유래로 lactonase 활성을 갖는 유전자 (P0N1)를 변이시켜 사용할 수 있으며 P0N1의 반웅이 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 (P0N1) having a lactonase activity from a human, and it is preferable to express it in a non-cytoplasmic periplasm since P0N1 is reversible and lactone can be produced only at an acidic pH.
도 1에 보여주는 경로를 통해 homoser ine 으로부터 2-hydroxy gamma butyrolactone 및 이의 유기산 전구체를 효율적으로 생산하려면 도면 4에 제시한 것과 같이 당으로부터 homoser ine올 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 in the pathway shown in FIG. 1, it is necessary to develop a gene-mutant microorganism that efficiently produces homoserine ine from glucose 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. phosphoenol pyruvate (PEP)를 oxaloacetic acid (0AA)로 효율적으로 전환하는 phosphoenol pyruvate carboxylase ippc) 유전자의 과발현;  1. overexpression of the phosphoenol pyruvate carboxylase ippc gene, which efficiently converts phosphoenol pyruvate (PEP) to oxaloacetic acid (0AA);
2. 발효 중 생성되는 acetate를 acetyl CoA로 전환하여 미생물이 다시 사용할 수 있도록 도와주는 acetyl-CoA synthase iacc) 유전자의 과발현;  2. overexpression of acetyl-CoA synthase iacc gene which helps convert microorganisms into acetyl-CoA during fermentation;
3. glyoxylate shunt의 활성을 높이기 위해 glyoxylate shunt 유전자 발현을 억재하는 /c/ 유전자의 제거 ; 3. To increase the activity of glyoxylate shunt, glyoxylate shunt gene Removal of the / c / gene to suppress expression;
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. Apspart ic aci d의 aspartyl phosphate 전환 효율을 높이기 위해 apspartate kinase의 활성을 향상시킴. apspartate kinase를 coding 하는 iAr ¾:유전자의 발현을 향상시키기 위해 isoleucine에 의해 작동하는  5. Improve the activity of apspartate kinase to increase aspartyl phosphate conversion efficiency of Apspart ic acid. iAr ¾ coding for apspartate kinase: functioning by isoleucine to improve gene expression
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;  Removes operon riboswitches and boosts promoter strength. 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 /ya4을 제거 ;  6. coding for diaminopimelate dcarboxylase to remove lysine production / remove ya4;
7. methionine 생산을 제거하기 위해 homoserine  7. Homoserine to eliminate methionine production
succinyl trans ferase-9- coding 하는 met A유전자를.제거; succinyl trans ferase-9-coding the met A gene;
8. 생산된 homoserine이 homoserine phosphate로 전환되지 않도록 homoserine kinase를 coding하는 유전자를 제거 .  8. Remove the gene encoding homoserine kinase so that homoserine produced is not converted to homoserine phosphate.
Homoserine≤ 생산이 활성화 되면 homoserine을 4一 hydroxy— 2— oxo_ butanoic acid (HOB)로 전환하기 위하여 도면 2에 제시된 deaminase 효소 또는 도면 3에 제시된 transaminase 효소를 coding 하는 유전자를  In order to convert homoserine to 4-hydroxy-2-oxo-butanoic acid (HOB) when the homoserine production is activated, the deaminase enzyme shown in Fig. 2 or the gene encoding the transaminase enzyme shown in Fig. 3
발현人!킨다. 더 나아가 transaminase의 cofactor인 pyr i doxa 1 -5 ' -phosphat e (vitamin B6의 일종임)의 생합성을 촉진하기 위해 vitamine B6 생합성 경로를 강화시킨 균주를 사용하는 것이 좋다. Expressionist! Furthermore, it is recommended to use a strain that has enhanced the vitamine B6 biosynthetic pathway to promote the biosynthesis of pyridoxal 1 -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 P0N1을 발현시켜 2,4— di hydroxy— butanoic acid (dHBA)와 2-hydroxy gamma butyro lactone (HGBL)을 생산할 수 있다. 일반적으로 P0N1올 사용하여 dHBA를 HGBL로 전환하는 생물학적 반웅은 효율이 낮으므로 dHBA를  Furthermore, strains engineered to efficiently biosynthesize HOB from homoserine can produce 2,4-dihydroxy-butanoic acid (dHBA) and 2-hydroxy gamma butyrolactone (HGBL) by additionally expressing homoserine dehydrogenase and / or P0N1. In general, biologic reactions that convert dHBA to HGBL using P0N1 all are less efficient, so dHBA
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. Biologically produced, purified and chemically converted 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 Thus, the homoserine biosynthetic pathway suggested in Fig. When the homozygous transformation of the proposed homoserine in FIG. 1 is performed, a microorganism that has been engineered to produce dHBA or HGBL can be cultured as a main carbon source to produce dHBA and HGBL as target products. In order to efficiently cultivate the microorganism and efficiently produce the desired product, yeast extract and ammonium salt are added to the culture medium as a nitrogen source, and the amino acid,
methionine, lysine, threonine, 그리고 isoleucine을 적절히 첨가할 필요가 있다. 또한 생물 반웅기를 이용한 고농도 생산에서는 유가식으로 발효 중간 중간에 당과 아미노산 흔합물을 적절히 첨가해 주는 것이 methionine, lysine, threonine, and isoleucine. In addition, in the high-concentration production using the biological repulse, the sugar and the amino acid mixture are appropriately added in the middle of fermentation
바람직하다. desirable.
도 4는 포도당을 이용하여 homoserine을 대량으로 생산하기 위한 균주 개량 전략, 그리고 이로부터 2-hydroxy gamma butyro lactone 및 이의 전구체인 2,4-dihydroxybutanoic acid를 생합성하는 전략을 모식적으로 보여준다. 도 4에서, 녹색 화살표는 강화해야하는 반웅, 붉은색 화살표는 효소의 생산이나 효소 활성을 억제하는 기작, 살구색 X표는 억제 반웅의 제거 혹은 유전자 제거의 표시, 이탤릭체 작은 글씨는 조작 대상 유전자를 각각 나타낸다.  FIG. 4 schematically shows a strategy for producing a large amount 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 antagonism to be strengthened, the red arrow indicates the mechanism of inhibiting enzyme production or enzymatic activity, the apricot X mark indicates the elimination or elimination of the repelling antagonist, .
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 제작  Example 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 thrB( 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 thrB (threonine synthase encoding) (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. (3) 다음으로, 아세트산의 생산을 최소화하기 위하여 acs 유전자의 발현을 증대시켰다. Acs의 발현을 증대시키기 위해, acs 발현 유전자 프로모터를 trc 프로모터로 바꾸었다. (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) 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 유전자를 pop- in pop-out 방법으로 제거하였다.  (4) As a method for promoting acetyl reuse, a negative regulator gene was removed by pop-in pop-out method to enhance the activity of glyoxylate shunt.
(5) acetate 생성을 막고 homoser ine으로의 탄소 flux를 향상시키기 위하여 anapl erotic 경로의 핵심 효소인 phosphoenol pyruvate carboxylase를 coding 하는 ppc유전자의 발현을 합성프로모터 8(Synthetic promoter 8: SP8) (서열번호 23: mCMmMTCATCCG( TCGTATMTGTGTGGA)를 사용하여 증가시켰다. 이를 위해 ppc 유전자의 promoter를 pop-in pop-out 방법으로 synthetic promoter 8로 치환하였다.  (5) Expression of the ppc gene encoding phosphoenol pyruvate carboxylase, which is the key enzyme of the anapl erotic pathway, to inhibit acetate formation and improve the carbon flux to homoserine in the synthetic promoter 8 (SP8) (SEQ ID NO: 23) mcMmMTCATCCG (TCGTATMTGTGTGGA). For this purpose, the promoter of the ppc gene was replaced with the synthetic promoter 8 by the 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 Cat abolite 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 transport proteins such as GalP, and prevention of Carbon Cat abolite 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, ΑΚΠΙ 두개의 효소만이 높은 호모세린 농도에서 우수한 활성을 보인다. 또한, ΑΚΠ 및 ΑΚΙΠ 중에서 ΑΚΠ는 aspartate kinase 와 serine dehydrogenase 활성을 동시에 가지고 있어 ΑΚΙΠ 보다는 좀더 유리하다고 알려져 있다. 따라서 이번 균주 개발에서는 ΑΚΠ 효소를 coding하는 metL 유전 ]·를 medium copy plasmid인 pUCPK plasmid를 이용하여 과발현시켰다. 이 때 metL^\ 발현 크기를 조절하기 위해 lac promoter 와 trc promoter 두 가지를 사용하였다. 아래 표 1에 호모세린을 과발현시키기 위해 제거 (deletion), 치환 (substitution) 또는 과발현 (overexpression)한 경쟁경로 유전자와 제거 목적, 제거 방법올 나타냈다. (8) Finally, we optimized the pathway after aspartic acid, known as the bottleneck of the homoserine production pathway. Three homologous aspartate kinases AKI, AKII and AKIII are known to play an important role in homoserine production. Only two enzymes, AKII and ΑΚΠΙ, exhibit superior activity at high homoserine concentrations. It is also known that ΑΚΠ among ΑΚΠ and ΑΚΙΠ have aspartate kinase and serine dehydrogenase activity, which is more advantageous than ΑΚΙΠ. Therefore, in this strain development, metL gene coding for ΑΚΠ enzyme was overexpressed using pUCPK plasmid, a medium copy plasmid. Two types of lac promoter and trc promoter were used to control metL ^ \ expression size. In Table 1 below, competing pathway genes for deletion, substitution or overexpression, and methods for elimination and removal of homoserine were shown.
[표 1]  [Table 1]
Figure imgf000031_0001
대장균인 W3110 균주 및 BL2KDE3)은 Korean Collection for Type Cultures (KCTC)에서 구입하였다. 대장균 TOPIO 균주는 플라스미드의 cloning 과 유지에 사용하였다. 유전자 제거에는 multiplex automated genome engineering (MAGE) 기법과 pop- in pop-out 방법을 사용하였다. 본 발명자들이 개발한 Homoserine 과생산 변이 대장균주의 종류 아래 표 2에 나타냈다.
Figure imgf000031_0001
Escherichia coli W3110 strain and BL2KDE3) were purchased from Korean Collection for Type Cultures (KCTC). Escherichia coli TOPIO strain is a plasmid cloning and maintenance. Multiplex automated genome engineering (MAGE) and pop-in pop-out methods were used for gene deletion. The present inventors developed a mutant E. coli Homoserine and production Note the type shown in Table 2 below.
[표 2]  [Table 2]
Figure imgf000032_0001
유전체 분리 kit는 Promega (Madison, WI, USA)에서 구매하였다. High-f idel ity 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 (Bee ton-Dickinson, NJ, USA)사에서 구입하였다. 그 외 시약과 효소는 모두 Sigma-Aldrich (St. Louis, MO, USA)사에서 구입하였다.
Figure imgf000032_0001
Dielectric isolation kit was purchased from Promega (Madison, WI, USA). High-f idelity pfu-a polymerase was purchased from Invitrogen (Seoul, Korea). DNA cleavage enzymes and DNA enhancers were obtained from New England Bio-LAb (Beverly, Mass., 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 (Bee ton-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 치환  1-1 Gene deletion and substitution using MAGE method
MAGE (Multiplex Automated Genome Engineering) 방법은 in vivo genome editing에 매우 강력한 to 이다. 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 도밉할 수도 있다.  MAGE (Multiplex Automated Genome Engineering) method is very powerful to 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 may also be able to mock different kinds 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 recombinat ion을 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 oligos were constructed so that a homology arm, 5 '-terminal homolgy arm and 3' terminal homology arm, overlap with the target gene sequence 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 mutation in the target gene through homologous recombinat ion.
MAGE 실험을 위해 한천 고체 배지에서 자라는 single colony를 따서 섭씨 30도, LB+Qn 배지에서 12시간 배양하였다. 이후 이 배양액 100 microliter를 새로운 LB+Cm 배지에 옮기고 섭씨 30도에서 0.6 0D (600 nm)가 될 때까지 배양하였다. 이후 culture tube를 15분간 섭씨 42도에서 방치하였다가 30 분간 얼음물 속에 넣었다. 이 후 준비된 competent cell을 4°C, 5000 rpm에서 10분간 원심분리하고 차가운 증류수로 3차례 세척하였다. 그리고 세포 pellet을 10% glycerol 용액 100 mL에 현탁시켰다. 이후 ssDNA oligo 50 uM을 세포현탁액과 흔합하고 이를 elctro cuvette에 넣은 후 electroporation 시켰다. 이 후 이 흔합액을 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 pl ate에 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 in LB + Qn medium for 12 hours at 30 ° C. Then, 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 4 ° C and 5000 rpm for 10 min and washed three times with cold distilled water. The cell pellet was suspended in 100 mL of 10% glycerol solution. Then, 50 μM of ssDNA oligo was mixed with the cell suspension, placed in an elctro cuvette and electroporated. Then, the resulting suspension was transferred to a culture tube, and 5 mL of LB was added thereto and cultured at 30 ° C. for 3 hours (first cycle). Such The procedure was repeated 6-10 times (6-10 cycles) and 100 uL of cell culture was plated on agar platel and incubated 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.
[표 3]  [Table 3]
Figure imgf000034_0001
Apta_FP gacca cgtcagccttggcgtgatccgtgcaatggaacgcaaaTAActcagccgcgtaccggtggcg
Figure imgf000034_0001
Apta_FP gacca cgtcagccttggcgtgatccgtgcaatggaacgcaaaTAActcagccgcgtaccggtggcg
34 atgcgcccgatcagactacg  34 atgcgcccgatcagactacg
Apta_RP cgtagtctgatcgggcgcatcgccaccggtacgcggctgagTTAtttgcgttccattgcacggatca  Apta_RP cgtagtctgatcgggcgcatcgccaccggtacgcggctgagTTAtttgcgttccattgcacggatca
35 cgccaaggctgacgctggtc  35 cgccaaggctgacgctggtc
ᅀ pta— seq Ggcgt t cgtctgagcgt tttc 36 _FP ᅀ pta-seq Ggcgt t cgtctgagcgt tttc 36 _FP
Apta_seq Ggattcttgcagcttcgcc  Apta_seq Ggattcttgcagcttcgcc
37 _RP  37 _RP
AackA_FP gaaatttgccatcatcgatgcagtaaatggtgaagagtaccttTGAaagcacgtatcaaatggaaaa  AackA_FP gaaatttgccatcatcgatgcagtaaatggtgaagagtaccttTGAaagcacgtatcaaatggaaaa
38 t gacggcaat aaacaggaagc  38 t gacggcaat aaacaggaagc
ᅀ ackA一 RP gcttcctgtttattgccgtccattttccatttgatacgtgcttTCAaaggtactcttcaccatttac ᅀ ackA 1 RP gcttcctgtttattgccgtccattttccatttgatacgtgcttTCAaaggtactcttcaccatttac
39 tgcatcgatgatggcaaatttc  39 tgcatcgatgatggcaaatttc
AackA_se Tctggtttagccgaatgtttcc  AackA_se Tctggtttagccgaatgtttcc
40 q_FP  40 q_FP
AackA_se Atgt cagttcttctaccgg  AackA_se Atgt cagttcttctaccgg
41 q_RP  41 q_RP
AldhA_RP gcaacaggtgaacgagt cctt tggctttgagct gaat tttt tTAActgccaatggctgcgaagcgg  AldhA_RP gcaacaggtgaacgagt cctt tggctttgagct gaat tttt tTAActgccaatggctgcgaagcgg
42 tatgtattttcgtaaacgatg  42 tatgattattcgtaaacgatg
AldhA_se Tt t tccggtgtcagcggg  AldhA_se Tt t tccggtgtcagcggg
43 q_FP  43 q_FP
AldhA— se Gactttctgctgacgg  AldhA-se Gactttctgctgacgg
44 q_RP  44 q_RP
AadhE_RP gtaaaaaaagcccagcgtgaatatgccagtttcactcaagagcaaTAAct ctgcagatgctcgaat  AadhE_RP gtaaaaaaagcccagcgtgaatatgccagtttcactcaagagcaaTAAct ctgcagatgctcgaat
45 cccactcgcgaaaatggccgttg  45 cccactcgcgaaaatggccgttg
ᅀ adhE_se Ggagc tgtat gcggc ttt aac ᅀ adhE_se Ggagc tgtat gcggc ttt aac
46 q_FP  46 q_FP
AadhE_se Gtagacaaaatcttccgcgccg  AadhE_se Gtagacaaaatcttccgcgccg
47 q_RP  47 q_RP
ApflB_RP ccaaaggtgactggcagaatgaagtaaacgtccgtgacttcattTAAagtccttcctggctggcgct  ApflB_RP ccaaaggtgactggcagaatgaagtaaacgtccgtgacttcattTAAagtccttcctggctggcgct
48 actgaagcgaccaccaccctg ApflB_se Tgcagagaagtgaactgtgcc 48 actgaagcgaccaccaccctg ApflB_se Tgcagagaagtgaactgtgcc
49 q_FP  49 q_FP
ApflB_se Cagaaaaactacactccgtacg  ApflB_se Cagaaaaactacactccgtacg
50 q_RP  50 q_RP
ᅀ iclR— FP Cccggcttgttgcgccagttccgtgagtgccacactgccattcagccacgcgttaaagactgaacct ᅀ iclR- FP Cccggcttgttgcgccagttccgtgagtgccacactgccattcagccacgcgttaaagactgaacct
51 gtccagtcgctggtgcggtggc  51 gtccagtcgctggtgcggtggc
ᅀ iclR_RP Gccaccgcaccagcgactggacaggttcagtctttaacgcgtggctgaatggcagtgtggcactcac ᅀ iclR_RP Gccaccgcaccagcgactggacaggttcagtctttaacgcgtggctgaatggcagtgtggcactcac
52 ggaactggcgcaacaagccggg  52 ggaactggcgcaacaagccggg
AiclR_se Gcgcgtttgggcgagatc  AiclR_se Gcgcgtttgggcgagatc
53 q_FP  53 q_FP
ᅀ iclR_se Ctgaaattactggagtgg ᅀ iclR_se Ctgaaattactggagtgg
54 q_RP  54 q_RP
AlysA_FP ggctttctgtgcaaagcgcaccacatcaaactgtttcagcgctgTTAgacccacaccgggcagccaa  Alysa_FP ggctttctgtgcaaagcgcaccacatcaaactgtttcagcgctgTTAgacccacaccgggcagccaa
55 attcagcgggcaaacgcagcag  55 attcagcgggcaaacgcagcag
AlysA_RP ctgctgcgtttgcccgctgaatttggctgcccggtgtgggtcTAAcagcgctgaaacagtttgatgt  Alysa_RP ctgctgcgtttgcccgctgaatttggctgcccggtgtgggtcTAAcagcgctgaaacagtttgatgt
56 ggtgcgct t tgcacagaaagcc  56 ggtgcgct t tgcacagaaagcc
ᅀ lysA_se Cctgtt ccagatgggcat aat c ᅀ lysA_se Cctgtt ccagatgggcat aat c
57 q_FP  57 q_FP
MysA— se . Gggtctacgat cgcaaat tattcg  Mace-se. Gggtctacgat cgcaaat tattcg
58 q_RP  58 q_RP
AthrB_RP cttatcggcaaagcgtccgaggttgttgagactgaatgtctctgTTAtgcaccatcaacaggtgtca  AthrB_RP cttatcggcaaagcgtccgaggttgttgagactgaatgtctctgTTAtgcaccatcaacaggtgtca
59 ccgccgccccgagcacatcaaac  59 ccgccgccccgagcacatcaaac
AthrB_se Tt get cggagatgt agtcacgg  AthrB_se Tt get cggagatgt agtcacgg
60 q_FP  60 q_FP
ᅀ thrB— se Gcgat actgcgccggtaaaat ag ᅀ thrB-se Gcgat actgcgccggtaaaat ag
61 q_RP  61 q_RP
AmetA_FP Gaagaaaacgtctttgtgatgacaacttctcgtgcgtctggttaattaacctgatgccgaagaagat  AmetA_FP Gaagaaaacgtctttgtgatgacaacttctcgtgcgtctggttaattaacctgatgccgaagaagat
62 tgaaactgaaaatcagtttctg  62 tgaaactgaaaatcagtttctg
AmetA_RP Cagaaactgattttcagtttcaatcttcttcggcatcaggttaattaaccagacgcacgagaagttg  AmetA_RP Cagaaactgattttcagtttcaatcttcttcggcatcaggttaattaaccagacgcacgagaagttg
63 tcatcacaaagacgttttcttc AmetA_se Ctggt caggaaat t cgt ccac 63 tcatcacaaagacgttttcttc AmetA_se Ctggt caggaaat t cgt ccac
64 q_FP  64 q_FP
AmetA_se Cgaatcaacgctgccggaaag  AmetA_se Cgaatcaacgctgccggaaag
65 q_RP  65 q_RP
AlacI_RP cttatcagaccgtt tcccgcgtggtgaaccaggccagccacgttTGAcgatggcggagctgaattac 66 attcccaaccgcgtggcacaac  AlacI_RP cttatcagaccgtt tcccgcgtggtgaaccaggccagccacgttTGAcgatggcggagctgaattac 66 attcccaaccgcgtggcacaac
Alacl_se Gatatttatgccagccagcc 67 q_FP  Alacl_se Gatatttatgccagccagcc 67 q_FP
ᅀ lacl_se Ccacgt 11 c t gcgaaaacgcggg 68 q_RP ᅀ lacl_se Ccacgt 11 c t gcgaaaacgcggg 68 q_RP
Primer for gene deletion using Pop- in Pop-out method  Primer for gene deletion using Pop-in Pop-out method
US-Ptrc-acs- gtttaatcggtacccggggatcgcggccgccgcgcccttcctgccagtcaattttc 69 FP US-Ptrc-acs- gtttaatcggtacccggggatcgcggccgccgcgcccttcctgccagtcaattttc 69 FP
US-Ptrc-acs- Cttttaagaaggagatatacatatgagccaaattcacaaacacacc US-Ptrc-acs- Cttttaagaaggagatatacatatgagccaaattcacaaacacacc
70 RP  70 RP
Ptrc-acs-FP Ggtgtgtttgtgaatttggctcatatgtatatctccttcttaaaag 71 Ptrc-acs-FP Ggtgtgtttgtgaatttggctcatatgtatatctccttcttaaaag 71
Ptrc-acs-RP Cggcgtgcgtttatttttatccttgtcatcgactgcacggtgcaccaatgc 72Ptrc-acs-RP Cggcgtgcgtttatttttatccttgtcatcgactgcacggtgcaccaatgc 72
DS-Ptrc-acs- Gcattggtgcaccgtgcagtcgatgacaaggataaaaataaacgcacgccg DS-Ptrc-acs- Gcattggtgcaccgtgcagtcgatgacaaggataaaaataaacgcacgccg
73 FP 73 FP
DS-Ptrc-acs- Gggcgcgcgccat tct ccggt cgactctagat catgccgt catcccac DS-Ptrc-acs- Gggcgcgcgccat tct ccggt cgactctagat catgccgt catcccac
74 RP 74 RP
US-ldhA-FP At cgcggccgc tgtctgtttt cggt c 75US-ldhA-FP At cgcggccgc tgtctgtttt cggt c 75
US-ldhA-RP Ctggagaaagtcttatgtaatcttgccgctcccc 76US-ldhA-RP Ctggagaaagtcttatgtaatcttgccgctcccc 76
DS-ldhA-FP Ggggagcggcaagattacataagactttctccag 77DS-ldhA-FP Ggggagcggcaagattacataagactttctccag 77
DS-ldhA-RP Ct agaggatcccaagcagaat caagt tctaccg 78DS-ldhA-RP Ct agaggatcccaagcagaat caagt tctaccg 78
Gn-ldhA-FP Gatggtacggcgattgggatg 79Gn-ldhA-FP Gatggtacggcgattgggatg 79
Gn-ldhA-RP Gccagggagaaaaaatcag 80Gn-ldhA-RP Gccagggagaaaaaaatcag 80
US-iclR-FP Ctgcgcggacgctgaggatcccggtgatcccgtcctctcacg 81US-iclR-FP Ctgcgcggacgctgaggatcccggtgatcccgtcctctcacg 81
US-iclR-RP Ttcgaaccccagagtcccgcctgccgctcgtaggtcctg 82US-iclR-RP Ttcgaaccccagagtcccgcctgccgctcgtaggtcctg 82
DS-iclR-FP Gcaggacct acgagcggcaggcgggac t ctggggt t cgaaatg 83 DS-iclR-RP Tgcctgcaggtcgactctagattatttgttaactgttaattgtccttgttc 84DS-iclR-FP Gcaggacct acgagcggcaggcgggac t ctggggt t cgaaatg 83 DS-iclR-RP Tgcctgcaggtcgactctagattatttgttaactgttaattgtccttgttc 84
Gn-iclR-FP Ctgaacagcaggtcgtcc 85Gn-iclR-FP Ctgaacagcaggtcgtcc 85
Gn-iclR-RP Gcgtcgaaaccttcgatg 86 Gn-iclR-RP Gcgtcgaaaccttcgatg 86
1-2. Pop-in pop-out 방법에 의한 유전자 결실 또는 유전자 치환 ldhA 및 iclR유전자를 제거하기 위해 sacB-Km 카세트를 갖는 pKOV 플라스미드를 사용하였다 (표 4). 구체적으로, target 유전자의 upstream과 downstream region 600-700 bp 를 갖는 fragment를 E. col i W3110 염색체로부터 해당 primer를 사용하여 PCR 증폭하였다 (표 3). 이 fragment의 sequence를 확인한 早 Gene Art Seamless Cloning and Assembly Kit (Invitrogen, USA)를 이용하여 pKOV plasmid에 삽입하였다. 이 후 E. col i 3110 에 recombinant pKOV lasmid 를 도입하고 homology recombination을 통해 해당 유전자를 결실시켰으며, 설탕 배지 배양을 통해 pKOV plasmid를 제거하였다. 최종적으로 PCR 방법으로 스크리닝하고 유전자가 결실된 변이주들을 확인, 확보하였다. 1-2. To remove the gene deletion or gene replacement ldhA and iclR genes by the pop-in pop-out method, the pKOV plasmid with sacB-Km cassette was used (Table 4). Specifically, a fragment with an upstream and downstream region of 600-700 bp of the target gene was amplified by PCR using the corresponding primers from the E. coli W3110 chromosome (Table 3). The sequence of this fragment was inserted into the pKOV plasmid using the early Gene Art Seamless Cloning and Assembly Kit (Invitrogen, USA). Then, recombinant pKOV lasmid 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 제작  1-3. 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 (0RF)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, pBbAlk, 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 (0RF) of the target gene was amplified by PCR using the appropriate primer (Table 3). Thus cutting the resulting DNA fragment with a restriction enzyme cloning was the plasmid, or the like that is pUCPK, P ET-T7p, pBbAlk, P Trc-99a. This plasmid was then introduced into the corresponding host strain.
[표 4]  [Table 4]
Plasmid Description Source  Plasmid Description Source
pSIM5 MAGE, β recombinant protein, Cm25 Addgene, USA pUCPK Overexpression, lac promoter , medium copy, K50 Addgene, USA ET Over express km, T7 promoter , high copy, K50 Addgene, USA pBbAlk■ Overexpression, trc promoter , medium copy, K50 Addgene, USA pTrc一 99a Overexpression, trc promoter , low copy, K50 Addgene, USA pKOV Pop" in-Pop out , Cm25 Addgene , USA p iCPK-wetl Ptrc-metL Thi s study Overexpression, trc promoter, medium copy, K50, T50 promoter, high copy, T50 promoter, high copy, pSIM5 MAGE, β recombinant protein, Cm25 Addgene, USA pUCPK Overexpression, lac promoter, Addgene, USA pTrc-99a Overexpression, trc promoter, low copy, K50 Addgene, USA pKOV Pop " in-Pop out, Cm25 Addgene, USA p iCPK-wetl Ptrc-metL Thi s 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 갖는프로모터의 경우 유전자 불안정성을 크게 증가시키므 서로 다른 서열의 합성 프로모터 (synthet i c promoters) 라이브러리를 구축하였다 (표 5) . 또한 이들의 상대 활성올 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 promoter having the same sequence, the gene instability was greatly increased, and a library of synthetically promoters of different sequences was constructed (Table 5). And their relative activities were measured using GFP (Fig. 5). A total of 9 promoters were synthesized and selected, and their activities ranged from about 4 times.
[표 5]  [Table 5]
Figure imgf000039_0001
실시예 2: 호모세린의 아미노기 제거를 위한 트랜스아미네이즈 (TA) 효소의 스크리닝 및 변이효소 제작
Figure imgf000039_0001
Example 2: Screening of transaminate (TA) enzyme for the removal of amino group of homoserine and production of mutant enzyme
2-1. 트랜스아미네이즈 (TA) 효소의 스크리닝  2-1. Screening of transaminate (TA) enzymes
효소의 확보  Securing enzyme
트랜스아미네이즈 (TA) 효소는 생명체 내에서 중요한 역할을 담당하고 오랫동안 다양하게 연구되어 왔지만 homoser ine에 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., Baci 1 lus subti lis, Mesorhizobium lot i , Agrobacterium tumef aciens, Pseudomonas denitri f icans , P. put i da and P. //i/oresce2s'등으로부터 확보하였다: Transaminate (TA) enzymes play an important role in life and have been studied extensively for a long time, but the TA enzyme specific for homoserine ine has not yet been revealed in vivo. In general, many enzymes 17 of the known TA enzymes, aspartate transaminase, alanine transaminase, branched chain transaminase, and aromatic transaminase, were selected for activity against homoserine (Table 6). These enzymes were obtained from E. coli, Enterobacter spp., Baci 1 lus subtilis, Mesorhizobium loti, Agrobacterium tumefaciens, Pseudomonas denitri ficans, P. puti da and P. // i / oresce2s '
Figure imgf000040_0001
Figure imgf000041_0001
asp aspatate P. AGI228 F:
Figure imgf000040_0001
Figure imgf000041_0001
asp aspatate P. AGI228 F:
AT transaminase denitrific 91.1 t tcagaat t caaaagat ctttt aagaaggagat at 124 AT transaminase denitrific 91.1 t tcagaat t caaaagat ctttt aagaaggagat at 124
.ans acatatgctcggacccggcg  .ans acatatgctcggacccggcg
tcgagtttggatcctcagcgattctggtgcgcg 125 aro acromat ic P. AGI229 F:  tcgagtttggatcctcagcgattctggtgcgcg 125 aro acromat ic P. AGI229 F:
AT transaminase denitrific 71.1 gtggtggtggtggtggtgctcgagcagtttcaggc 126 ans gcagcgc AT transaminase denitrific 71.1 gtggtggtggtggtggtgctcgagcagtttcaggc 126 ans gcagcgc
R:  R:
aacttt aagaaggagat at acatatgatgagcaag 127 ttctggagtccc  aacttt aagaaggagat at acatatgatgagcaag 127 ttctggagtccc
aro acromat ic P. AUM683 F: aro acromat ic P. AUM683 F:
AT transaminase fluorescen 13.1 gtggtggtggtggtggtgctcgaggagttcgccga 128 s gggc AT transaminase fluorescen 13.1 gtggtggtggtggtggtgctcgaggagttcgccga 128 s gggc
R:  R:
act tt aagaaggagat at acat at gat gagtaaat 129 tctggagcccg 효소의 생산  act tt aagaaggagat at acat at gat gagtaaat 129 tctggagcccg production of enzymes
E. coli BL2KDE3) 숙주에서 표 6의 효소를 발현하였다. LB medium을 사용하였고 plasmid의 유지와 보존을 위해 kanamycin 50 mg/L 를 사용하였다. 발현 백터로는 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 Top 10 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.  E. coli BL2KDE3) expresses the enzyme of Table 6 in the host. LB medium was used and kanamycin 50 mg / L was used for the maintenance and preservation of plasmids. PET as an expression vector and T7 as a promoter. For the TA gene cloning, the coding sequence was amplified from the genomic DNA of the microorganism, cloned into the E. coli Top 10 strain using the seamless cloning and assembly kit (Invitrogen) with the pET vector containing His-tag Respectively. 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.
[표 τ_  [Table τ_
Gene Enzyme Source Acc. No. pET/TAi aspAT Aspatate transaminase M. loti ANN59010.1 ΓΑ1 aspAT Aspatate transaminase M. loti ANN57047.1 ΓΑ2 asp AT Aspatate transaminase A. tumefaciens ACM26415.1 ΓΑ3 aspAT Aspatate transaminase B. subtil is AQR85543.1 ΓΑ4 aspAT Aspatate transaminase E. col i AVI 55449.1 ΓΑ5 bcAT branched chain D. mccartyi AQY72500.1 ΓΑ6 Gene Enzyme Source Acc. No. pET / TAi aspAT Aspatate transaminase M. loti ANN59010.1 ΓΑ1 aspAT Aspatate transaminase M. loti ANN57047.1 ΓΑ2 asp AT Aspatate transaminase A. tumefaciens ACM26415.1 ΓΑ3 aspAT Aspatate transaminase B. subtilis AQR85543.1 ΓΑ4 aspAT Aspatate transaminase E. coli AVI 55449.1 ΓΑ5 bcAT branched chain D. McCarty AQY72500. 1 ΓΑ6
transaminase  transaminase
bcAT branched chain E. col i AVI53929.1 ΓΑ7 bcAT branched chain E. coli AVI53929.1 ΓΑ7
transaminase  transaminase
bcAT branched chain E. spp A0L13801.1 ΓΑ8 bcAT branched chain E. spp A0L13801.1 ΓΑ8
transaminase  transaminase
aroAT acromat ic transaminase E. col i AUV21492.1 ΓΑ9 aroAT acromat ic transaminase E. col i AVI54196.1 TA10 alaAT alanine transaminase P. den i tr ifi cans AGI 22743.1 TA11 alaAT alanine transaminase P. put i da AE015451.2 TA12 alaC alanine transaminase E. col i AAN66442.1 TA13 alaC alanine transaminase E. col i AAN66442.1 TA14 aroAT acromat ic transaminase E. coli AUV21492.1 ΓΑ9 aroAT acromat ic transaminase E. coli AVI54196.1 TA10 alaAT alanine transaminase P. den i tr i cans AGI 22743.1 TA11 alaAT alanine transaminase P. puti da AE015451.2 TA12 alaC alanine transaminase E. coli AAN66442.1 TA13 alanine transaminase E. coli AAN66442.1 TA14
A140P-Y275D  A140P-Y275D
aspAT ' aspatate transaminase P. denitr i ficans AGI22891.1 TA15 aroAT acromat ic transaminase P. den i tr ifi cans AGI 22971.1 TA16 aroAT acromat ic transaminase P. fluorescens AUM68313.1 TA17 효소생산을 위해 TA를 생산하는 재조합 균주는 50 mg/L kanamycin을 포함하는 LB 배지에서 배양하였다. 액체부피 20 mL, 250 mL 플라스크를 사용하였고 20°C, 200 rpm 조건에서 배양하였다. 세포농도가 0.6 0D (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 완층용액 (pH 7.0)으로 세척하고 binding 완층용액 (20 mM 인산 완층용액, 0.5 M NaCl, 20 mM imidazole)에 현탁하였다. 이 후 세포 » Sonicator로 파쇄하고 원심분리하여 미파쇄부분과 고형분을 제거하고 용액부분만 모아서 세포활성과 SDS-PAGE 를 이용한 단백질 분석에 이용하였다. aspAT 'aspatate transaminase P. denitr ficans AGI22891.1 TA15 aroAT acromat ic transaminase P. den i tr ifi cans AGI 22971.1 TA16 aroAT acromat ic transaminase P. fluorescens AUM68313.1 TA17 The recombinant strain producing TA for enzyme production is 50 and cultured in LB medium containing 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 solution (pH 7.0) and the binding complete solution (20 mM Phosphate buffer solution, 0.5 M NaCl, 20 mM imidazole). Then, 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 The pure separation of the enzyme was carried out on a Ni-NTA-HP resin packing column (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 흑은 non—naturing 조건에서 전기 영동하였다 (도 6a 및 6b). 그리고 glycerol 이 20% 되도록 첨가한 후 - 80°C에 보관하였다. 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 black non-naturing conditions (FIGS. 6A and 6B). Then, glycerol was added at 20% and stored at -80 ° C.
효소의 활성측정  Enzyme 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-hydroxybutyr i c acid + alanine  (i) Homoserine + pyruvate 2-oxo-4-hydroxybutyrate acid + domain
(ii)Homoserine + 2-oxoglutarate <→ 2_oxo_4ᅳ hydroxybutyr ic acid + glutamate  (ii) Homoserine + 2-oxoglutarate <- 2_oxo_4 ᅳ hydroxybutyr ic 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 반웅, 즉 pyruvate를 받게로 하는 반웅을 이용해 TA 활성을 조사하였다. 생성물인 alanine은 0PA로 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, the enzyme activity was not observed. Therefore, the TA activity was investigated using the reaction of (i), pyruvate. The product, alanine, was determined by HPLC after being modified with 0PA.
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 반웅 용액을 제조하였다. 이 용액을 약 5분간 37°C에서 incubation 한 후 10 mM의 pyruvate를 첨가하여 반응을 시작하였다. 반응 10분 후 12 mM의 과염소산 (per chloric acid)을 첨가하여 반웅을 중단시키고 10,000g에서 5분간 원심분리하였다. 이후 5 uL sample을 취하여 12.5 μ!의 OPA 용액 (25 mg OPA, 50 yL 2-mercaptoethanol , pH 9.5 0.5 mM 포화 sodium borate 용액, 4.5 mL methanol)과 흔합하고 상온에서 incubation 한 후 filter 정제하였다. 0PA 에 의해 변형된 alanine은 Zorbax eclipse XBD- C18 column을 장착한 HPLC로 정량 분석하였다. 0PA로 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. TA activity was measured using a 50 mM phosphate buffer solution (pH 7.0), 0.1 mM cofactor pyridoxal phosphate (PLP), 10 mM homoserine, and an appropriate amount of TA enzyme. The solution was incubated at 37 ° C for about 5 minutes and then added with 10 mM pyruvate. After 10 minutes of reaction, 12 mM perchloric acid was added to stop the reaction and centrifuged at 10,000 g for 5 minutes. A 5 μL sample was then taken to obtain 12.5 μ! OPA solution (25 mg OPA, 50 yL 2-mercaptoethanol, pH 9.5 0.5 mM saturated sodium borate solution, 4.5 mL methanol) was incubated at room temperature and filtered. The alanine modified by 0PA was quantitatively analyzed by HPLC on a Zorbax eclipse XBD-C18 column. The alanine derivative modified with 0PA 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 TAl, 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, four types of TAl, TA2, TA3, and TA6 were produced in 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 엔지니어링  2-2 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 조사  Development 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. M9 minimal medium with no nitrogen source 10 mM homoserine as a nitrogen source, and a small amount (0.5 mM) of threonine and methionine were added to induce microbial growth, respectively. 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.
TA4효소 라이브러리 제작  TA4 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/lASM을 참고하였다. 1ASM 구조는 조효소로 인산피리독살 (pyridoxal-5' -phosphate : PLP)을, 기질의 유사체로 말레산 (maleic acid)를 갖고 있다. 이 모델은 M0E(Molecular Operat ing Environment )를 사용해 제작했으며, PROCHECK 및 ProSA 온라인 구조 분석을 통해 평가하였다. PYM0L뷰어 (http : //www.pymo 1 . 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 structure and sequence of the 1ASM can be found at https: // www. Refer to rcsb.org/structure/lASM. 1ASM structure has pyruvate phosphodiester (p yr idoxal-5 '-phosphate (PLP) as coenzyme and maleic acid as substrate analogue. This model was created using MOE (Molecular Operating Environment) and evaluated through PROCHECK and ProSA online structure analysis. The protein structure was confirmed using a PYM0L viewer (http://www.pymo 1. org) (FIG. 10).
1ASM 구조로부터 말레산의 카르복실산 (아스파르트산 잔기의 카르복실산)과 상호작용을 하고 있는 4개의 아미노산 ( I lel7 , Gly38 , Asnl94, Arg386)을 확인하였고ᅳ TA4 모델 구조를 통해서 아스파르트산 잔기의 카르복실산과 상호작용을 할 것으로 예상되는 4개의 아미노산 (Lysl4, Gly40, Asnl78 , Try364)를 선정하였다 (도 11) . Was from 1ASM structure confirmed acid (aspartic acid residue of a carboxylic acid) and the cross-four amino acids (I lel7, Gly38, Asnl94, Ar g 386) that the action of maleic acid, aspartic acid via the eu TA4 model structure Four amino acids (Lysl4, Gly40, Asnl78, Try364) expected to interact with the carboxylic acid of the residue were selected (Fig. 11).
도 10과 도 11의 정보를 이용하여 TA4 효소 mutant l ibrary를 구축하였다. 아스파르트산 잔기의 카르복실산과 상호작용을 할 것으로 예상되는 TA4의 4개의 아미노산 (Lysl4, Gly40 , Asnl78 , Try364)을 에셈블리 PCR 방법으로 무작위로 변형시켜 TA4 라이브러리를 제작하였다. 이 때, PET30b/TA4 플라스미드를 주형으로 사용하였으며,ᅳ 사용한 프라이머를 표 8에 나타냈다. 제한효소 Xbal 및 Xhol 부위를 이용하여, 프라이머 1 및 2 로부터의 PCR 산물을 pET30b 플라스미드에 클로닝하였다. 생성된 TA4 라이브러리를 대장균 Ε)Η10 로 형질전환시켰고, 플라스미드를 분리정제하였다. 아래 표 8은 TA4 변이효소 라이브러리 제작에 사용한 프라이머 서열을 나타낸다. Using the information in FIG. 10 and FIG. 11, the TA4 enzyme mutant libary was constructed. TA4 library was constructed by randomly modifying four amino acids of TA4 (Lysl4, Gly40, Asnl78, Try364) expected to interact with the carboxylic acid of aspartic acid residues by means of the assemble PCR method. In this case, we used the P ET30b / TA4 plasmid as a template, the primers used are shown in Table 8. eu. Using the restriction enzyme Xbal and Xhol sites, the PCR products from primers 1 and 2 were cloned into a pET30b plasmid. The resulting TA4 library was transformed with E. coli E ) 10 and the plasmid 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)
[표 8]  [Table 8]
Figure imgf000047_0001
TA4 효소 라이브러리 스크리닝 및 우수 효소 개발
Figure imgf000047_0001
TA4 enzyme library screening and excellent enzyme development
제작된 라이브러리를 대장균 BL2 DE3)로 형질전환시킨 후, 50 mL LB 배지에서 밤새 배양하였다. 이후 cell pellet을 원심분리하여 얻고 이를 100 mM 인산 완층용액으로 3번 세척한 후 20 mM homoserine을 질소원으로 갖는 M9 최소배지 (50 um/mL kanamycin 포함)에 0D 0.05가 되도록 접종하였다. 이 후. 37Ϊ:, 200 rpm에서 3시간 배양하고 0.05 mM IPTG를 첨가하여 TA4 효소를 induction 시켰다. 0D 값이 0.5가 되었을 때 배양액을 100 배 희석하여 다시 배양하였다. 이와 같은 배양 -희석 cycle을 10회 반복한 후 배양액을 50 ug/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 BL2 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 solution, and then inoculated to 0 M 0.05 minimal medium (including 50 um / mL kanamycin) with 20 mM homoserine as a nitrogen source. after. After culturing at 37 rpm for 3 hours at 200 rpm, TA4 enzyme was induced by adding 0.05 mM IPTG. When the 0D value reached 0.5, the culture was diluted 100-fold and then re-cultured. Such a culture-dilution cycle was repeated 10 times After repetition, the culture was spread on an LB agar medium containing 50 ug / mL kanamycin, and 50 colonies were grown on a well grown M9 medium. A total of five mutants were obtained from these gene sequences and named 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 model ling을 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). These results demonstrate the importance of sites that interact with homoserine, with the results obtained through model ling, that Y364 and N178 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, 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. 실시예 3: 2-Qxo-4-hydr oxy butyr i c acid (0HB)의 (2S)-L-reductase 효소의 스크리닝 및 변이효소 제작  In the subsequent experiments, the most active TA4-1 and TA4-6 variants were used. Example 3: Screening and mutagenesis of (2S) -L-reductase enzyme of 2-Qxo-4-hydoxybutyric acid (0HB)
3-1. 0HB L-reductase 효소의 스크리닝  3-1. Screening of 0HB L-reductase enzyme
0HB L-reductase 효소의 확보  0HB L-reductase enzyme assurance
0HB는 prochiral 화합물로 환원되면 2번 위치에 L (2S) 또는 D (2R) 히드록시기를 갖는 2,4-dihydroxy butyric acid (DHB)로 전환된다. 0HB reductase는 2— hydroxy acid dehydrogenase에 속하며 여기에는 lactate dehydrogenase (ECl.1.1.27, ECl.1.1.28), ma late dehydrogenase (ECl.1.1.37, ECl.1.1.82, ECl.1.1.299) 그리고 branched chain (D), (L)- 2-hydroxyacid dehydrogenase (ECl.l.l. 272, ECl.1.1.345) 등이 잘 알려져 있다. 먼저 L— form isomer를 생산하기 위하여 Alcal igeneseutrophus H16, Cupriavidus basi lensis , Achromobacter xylosoxidans , Burkholderiaglumae , Escherichia fergusoni i , Escherichia col i , Lactobaci 1 lusmal i 및 Escherichia coli K-12 등으로부터 0HB에 대해 활성이 높을 것으로 예상되는 (L)-lactate dehydrogenase 효소들을 선정하였다 (표 9). OHB 2S— reductase 효소 screening을 위한 (L)— l actate dehydrogenase 후보 효소들을 표 9에 나타냈다. OH 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. The 0HB reductase belongs to 2-hydroxy acid dehydrogenase, including lactate dehydrogenase (EC1.1.1.27, EC1.1.1.28), ma late dehydrogenase (EC1.1.37, EC1.1.82, EC1.1.299) And branched chain (D), (L) -2-hydroxyacid dehydrogenase (ECl.ll 272, ECl.1.1.345) are well known. First, the activity of 0HB is expected to be high from Alcaligenes eutrophus H16, Cupriavidus basi lensis, Achromobacter xylosoxidans, Burkholderiaglumae, Escherichia fergusonii, Escherichia coli, Lactobaci 1 lusmali and Escherichia coli K-12 to produce L-form isomer (L) -lactate dehydrogenase enzymes were selected (Table 9). (L) -l actate dehydrogenase candidate enzymes for OHB 2S reductase enzyme screening are shown in Table 9.
[표 9]  [Table 9]
Gene Source Acc . n Pr imer sequence (FP/RP) 서열  Gene Source Acc. Pr primer sequence (FP / RP) sequence
0. 번호 l act ate dehdydr Alcal ige YP 725 ATAACATATGAAGATCTCCCTCACCAGCGC 140 ogemses i IdhO) nes 182. 1 TAATAGGATCCTCAGTGATGGTGATGGTGATGGGCCG 141 eutrophu TGGGGACGGCCACGTTG  0. Number l act ate dehdydr Alcal ige YP 725 ATAACATATGAAGATCTCCCTCACCAGCGC 140 ogemses i IdhO) nes 182. 1 TAATAGGATCCTCAGTGATGGTGATGGTGATGGGCCG 141 eutrophu TGGGGACGGCCACGTTG
s (Ae)  s (Ae)
ma l ate/L- Cupriavi WP 043 ATAACATATGAAGATAACACTGCAATC 142 l actate dehydro dus 344208. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGGCCCG 143 genase ( 1 dhᅳ 2) basi lens CCGGCAGTGCCACGCCAAGTTC ma l ate / L- Cupriavi WP 043 ATAACATATGAAGATAACACTGCAATC 142 l actate dehydro dus 344208. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGGCCCG 143 genase (1 dh ᅳ 2) basil lens CCGGCAGTGCCACGCCAAGTTC
is(Cb)  is (Cb)
mal ate/L- Achromob WP 006 ATAACATATGAAGATCTCCATTACCCAAG . 144 l actate dehydro acter 389860. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGAGGCA genase ( ldh~2) xylosoxi ACGCGTCAGC 145 dans (Ax) mal ate / L- Achromob WP 006 ATAACATATGAAGATCTCCATTACCCAAG. 144 l actate dehydroacter 389860. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGAGGCA genase (ldH ~ 2) xylosoxi ACGCGTCAGC 145 dans (Ax)
ma l ate/L- Burkhold YP 002 ATAACATATGCAGATATCCCTCGACGATG 146 l actate dehydro eria 909484. 1 TAATAGGATCCCTAGTGGTGATGATGGTGATGGGCCC genase ( ldh-2) glumae(B GTGCGGCCGGCGGCACCACGCCGAGTTCGTC 147 g) (ldh-2) glumae (B GTGCGGCCGGCGGCACCACGCCGAGTTCGTC 147 g) (SEQ ID NO: 2) ATAACATATGCAGATATCCCTCGACGATG 146 l actate dehydro eria 909484. 1 TAATAGGATCCCTAGTGGTGATGATGGTGATGGGCCC genase
mal ate/L- Escheric WP 002 ATAACATATGTATGGGTACAGATACCTTC . 148 l actate dehydro hi a 431747. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGATGCT genase ( ldhᅳ 2) ferguson GATTCCTGAGGATGTAAC 149 ii (Ef) mal ate / L- Escheric WP 002 ATAACATATGTATGGGTACAGATACCTTC. 148 l actate dehydro hi a 431747. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGATGCT genase (ldH 2) ferguson GATTCCTGAGGATGTAAC 149 ii (Ef)
D-2- Escheric WP024240 ATAACATATGATGTCATTACAAATTGCTG 150 hydroxyac id deh hi a 865. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGTCCAG ydrogenase ( ldh一 coli (Ec) CTAATGCTGATTCCTG 151 2) D-2- Lactobac WP 003 ATAACATATGACAAGAATAATTGCTTATCATG 152 hydroxyac id deh illus 689565. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGTTCTC D-2-Escheric WP024240 ATAACATATGATGTCATTACAAATTGCTG 150 hydroxyac id deh hi a 865. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGTCCAG ydrogenase (ldh 1 coli (Ec) CTAATGCTGATTCCTG 151 2) D-2-Lactobac WP 003 ATAACATATGACAAGAATAATTGCTTATCATG 152 hydroxyac id deh illus 689565. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGTTCTC
153 ydrogenase ( ldM) mali(Lm) CCTTGAAACTTATTTCATGTG  153 ydrogenase (ldM) mali (Lm) CCTTGAAACTTATTTCATGTG
D- Escheric NP 415 ATAACATATGA CTCGCCGTTTATAG 154 l act at e dehydro hi a col i 898. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGAACCA genase( IdhA) K- GTTCGTTCGGGCAG 155  D-escheric NP 415 ATAACATATGA CTCGCCGTTTATAG 154 l act ate dehydroh i a col i 898. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGAACCA genase (IdhA) K- GTTCGTTCGGGCAG 155
12(Eck) 선정된 효소들의 아미노산서열을 비교한 결과, 대개 44-66%의 높은 유사성을 보였다. 그러나 eutrophus, L.mali그리고 E. coli유래 효소는 낮은 유사성을 보였다.  12 (Eck) The amino acid sequences of the selected enzymes were compared, showing a high similarity of 44-66%. However, eutrophus, L.mali and E. coli-derived enzymes showed low affinity.
0HB L-reductase 효소의 생산 및 활성 측정  Production and activity measurement of 0HB L-reductase enzyme
효소발현을 위해 E. coli BL2KDE3) star 균주를 숙주균주로 이용하였으며, 클로닝과 폴라스미드 보존을 위해서는 E. coli DH5a 균주를 숙주로 이용하였다. kanamycin 50 pg/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 Hi s-tag을 붙여 주었다. PCR fragment는 pET vector (표 10)에 넣은 후 E. coli DH5a 균주에 클로닝하고 유전자 서열을 확인한 후 E. coli BL2KDE3) star에 도입하였다. Ldh 유전자의 soluble expression을 촉진하기 위해 Chaperon 을 발현하는 pG-Tf2 플라스미드를 추가로 도입해 주었다.  E. coli BL2KDE3 star strain was used as a host strain for E. coli expression and E. coli DH5a strain was used as a host for cloning and preservation of polar powder. LB medium containing kanamycin 50 pg / 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 attached Hi s-tag to the C-terminus. The PCR fragment was inserted into a pET vector (Table 10), cloned into the E. coli strain DH5a, and sequenced into E. coli BL2KDE3 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 ug/mL kanamycin을 포함하는 LB 배지에서 호기적으로 배양하였다. 배양온도는 30°C , 교반속도는 100 rpm 이었고 1 L 플라스크에 250 mL 배지를 넣어 주었다. 세포농도가 0.5 0D에 도달했을 때, 0. 1 mM의 IPTG를 첨가하고 추가적으로 10시간 배양하였다. 이 후 세포를 원심분리하고 25 mM 인산 완층용액 (pH 7.0)으로 3번 세척한 후 binding buf fer (20 mM pH 7.0 인산 완층용액, 0.5 M NaCl , 10 mM imidazol e)에 현탁한 후 soni cator를 이용하여 파쇄하였다. 세포 파쇄액은 4°C, 25,000 g 에서 30분 원심분리하여 비용해성 부분을 제거하고 용해성 부분을 모아 효소 활성 분석이나 추가적인 효소분리에 이용하였다. For LDH enzyme production, the 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 ug / 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 0D, 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 solution (pH 7.0), suspended in binding buffer (20 mM pH 7.0 phosphate buffer solution, 0.5 M NaCl, 10 mM imidazole) 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 0HB를 기질로 사용하여 분석하였다. 0HB는 상업적으로 구입할 수 없으므로 homoserine으로부터 합성하였다. 즉, 125 mM homoserine 용액을 pH 7.8, 100 mL Tris 완층용액으로 제조하고 1.25 U/raL의 snake venom (L)— amino acid oxidase 효소와 4400 U/mL의 catalase 효소와 흔합한 후 37°C에서 90분간 반웅시켰다. 이 후 반웅액을 Ultracentrifugal filter (10 kDa, Amicon)필터로 여과하여 0HB를 얻었다. 효소 활성 측정을 위해 효소 반웅액은 60 mM Hepes buffer (pH 7.0), 50 mM NaCl, 5 mM MgCl2, 5 mM f ructose-1 , 6-bi sphosphate , 적당량의 효소 등을 흔합하여 제조하고 37°C에서 2분간 incubation 하였다. 이 후 0.1 mM의 NAD(P)H 와 적당량의 0HB 흑은 pyruvate를 첨가하여 반웅을 개시하였다. 효소 활성은 NAD(P)H의 감소 속도와 extinction coefficient를 이용하여 계산하였다. LDH enzyme activity was analyzed using pyruvate and 0HB as substrates. 0HB was synthesized from homoserine since it was not commercially available. That is, 125 mM homoserine solution to pH 7.8, 100 mL Tris prepared by wancheung solution of 1.25 U / raL snake venom (L ) - from the amino acid oxidase enzyme with 4400 U / mL of 37 ° C and then combined common and catalase enzyme 90 For a while. Then, the solution was filtered with an Ultracentrifugal filter (10 kDa, Amicon) filter to obtain 0HB. For enzyme activity, the enzyme solution was prepared by mixing 60 mM Hepes buffer (pH 7.0), 50 mM NaCl, 5 mM MgCl 2 , 5 mM fructose-1, 6-bi sphosphate and an appropriate amount of enzyme at 37 ° C for 2 min. After that, 0.1 mM NAD (P) H and an appropriate amount of 0HB black were added pyruvate 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.
[표 10] [Table 10]
Figure imgf000051_0001
pET-Ef-LDH pET containing LDH from Escherichia fergusoni i Thi s study pET-Ec-LDH pET containing LDH from Escherichia col i Thi s study pET-Lm-LDH pET containing LDH from Lactobacillus mali Thi s study pET-LDHAl pET containing mutant enzyme LDH I48S from Thi s study
Figure imgf000051_0001
pET-Ef-LDH pET containing mutant LDH from Escherichia fergusoni i Thi s study containing pET-Ec-LDH pET containing Escherichia coli Thi s study enzyme LDH I48S from Thi s study
Al cali genes eutrophus  Al cali genes eutrophus
PET-LDHA2 pET containing mutant enzyme LDH I48T from Thi s study  PET-LDHA2 containing pET containing mutant enzyme LDH I48T from Thi s study
Al cali genes eutrophus  Al cali genes eutrophus
PET-LDHA3 pET containing mutant enzyme LDH I48N from Thi s study  PET-LDHA3 pET containing mutant enzyme LDH I48N from Thi s study
Al cali genes eutrophus  Al cali genes eutrophus
PET-LDHA4 pET containing mutant enzyme LDH I48D from Thi s study  PET-LDHA4 containing pET containing mutant enzyme LDH I48D from Thi s study
Alcal i genes eutrophus  Alcaligenes eutrophus
PET-LDHA5 pET containing mutant enzyme LDH I48 from Thi s study  PET-LDHA5 containing pET containing mutant enzyme LDH I48 from Thi s study
Alcal i genes eutrophus 대부분의 효소들은 pyruvate에 높은 활성을 가지고 있었고 그에 비해 0HB에는 낮은 활성을 보였다 (도 13) . pyruvate를 기질로 할 경우 Ec/Eck— LDH 가 6 umol /mg protein/min 로 가장 높은 활성을 나타내었다. 0HB에 대해서는 Ae-LDH가 1.8 umol/mg protein/min의 가장 높은 활성을 보였다. 따라서 Ae-LDH를 선택하여 0HB에 높은 활성을 갖는 변이 효소를 제작하였다.  Alcaligenes eutrophus Most enzymes had high activity on pyruvate and low activity on 0HB (Fig. 13). When pyruvate was used as a substrate, Ec / Eck-LDH showed the highest activity at 6 μmol / mg protein / min. For 0HB, Ae-LDH showed the highest activity of 1.8 μmol / mg protein / min. Therefore, Ae-LDH was selected and a mutant enzyme having high activity at 0HB was prepared.
OHB L-reductase 효소의 생성물의 광학활성 측정  Measurement 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 color imetr i c 분석 ki t을 사용하여 분석하였다. 먼저 생성된 l actate에 대하여 HPIX 분석을 실시하였다. Agi lent 사의 chi ral column (EC 250/4 NUCLEOSIL Chiral-1 , Germany)을 사용하였고 분석온도은 35 °C , 이동상으로는 0.2 mM CuS04 그리고 f low rate는 0.5 ml mirf1 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), it was analyzed using optically active HPLC and colorimetric analysis kit. HPox analysis was performed on the l actate generated first. The analytical temperature was 35 ° C, 0.2 mM CuSO 4 for the mobile phase, and 0.5 ml mirf 1 for the f low rate (Ag 250/4 NUCLEOSIL Chiral-1, Germany). 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 commercially sold lactate assay kit (BioVision, CA, USA), and the lactate produced was also identified as L-form.
0HB의 환원반웅에 의해 생성되는 DHB에 대해서도 2번 위치 0H기의 chirality를 조사하였다. 그러나 DHB의 경우 상업제품이 없고 더 나아가 광학적으로 순수한 화합물은 구할 수 없었다. 그리하여 2번 위치 0H 기가 R-form 및 S_form으로 섞여있는 racemic 2ᅳ hydroxy gamma butyrolactone (HGBL)를 구입 (Sigma-Aldrich, MO, USA)하여 화학적으로 분해시켜 racemic DHB를 합성하였다. 합성 방법은 다음과 같았다.  The chirality of position 0H in position 2 was also investigated for the DHB produced by the reduction reaction of 0HB. However, for DHB, there are no commercial products and further optically pure compounds were not available. Thus racemic DHB was synthesized by chemically degrading racemic 2-hydroxy gamma butyrolactone (HGBL) (Sigma-Aldrich, MO, USA) in position 2 OH at R-form and S_form. The synthesis method was as follows.
Racemic HGBL을 metahn 에 녹인 후 동일 당량의 NaOH를 첨가하고 상온에서 18시간 반응시켰다. 그리고 진공여과 후 건조시켰다. NMR로 확인한 결과 100% 전환이 확인되었다.  Racemic HGBL was dissolved in metahn, 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 colorimetr ic 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 0HB 생성물에 대해 DHB 분석을 chiral column과 colorimetric 분석 kit을 이용하여 동시에 실시하였다. HPIX 분석의 경우 하나의 peak만 얻어졌고 retention time은 L-form으로 추정되는 9.9분 시간대에 나타났다. 또한 colorimetric 분석에서도 L-form 만 얻어졌다. 따라서 Ae-LDH와의 반웅으로 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 colorimetr ic 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 0HB products reacted with Ae-LDH was performed simultaneously using a chiral column and a colorimetric assay kit. For HPIX analysis, only one peak was obtained and retention time appeared at 9.9 min, which is estimated to be L-form. L-form was obtained in colorimetric analysis. Thus, the DHB obtained from the reaction with Ae-LDH was identified as optically pure L-form or (S) -form isomer.
3-2. site-directed mutagenesis를 이용한 0HB Lᅳ reductase 효소의 변이체 개발  3-2. Site-directed mutagenesis was used to develop a mutant of 0HB L reductase enzyme
먼저 대장균 락트산탈수소효소의 크리스탈 구조 (PDB ID: 2G8Y)를 주형으로 사용하여, 상동성 모델링을 통해 Ae_ldhA의 3차원 구조를  First, using the crystal structure of E. coli lactate dehydrogenase (PDB ID: 2G8Y) as a template, the three-dimensional structure of Ae_ldhA was identified through homology modeling
구축하였다. Ae_ldhA모델은 M0E(Molecular Operating Environment )를 사용하여 제작했으며, PR0CHECK 및 ProSA 온라인 구조 분석을 통해 Respectively. Ae_ldhA model uses Molecular Operating Environment (M0E) Through PR0CHECK and ProSA online structure analysis,
평가하였다. PYM0L뷰어 (http://www.pymol.org)를 사용하여 단백질 구조를 확인하였다 (도 14).  Respectively. The protein structure was confirmed using the PYM0L viewer (http://www.pymol.org) (Fig. 14).
Ae_ldhA 변이체 디자인을 위해 피부르산과 HOB 구조 (Pubchem Database)의 구조를 Ae_ldhAl의 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, the structure of the skin leucine and HOB structure (Pubchem Database) was docked simulated using the triangular matching method at the enzyme active site of Ae_ldhAl, 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.. H0B가 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 H0B is larger than phosonic acid, substituted amino acids are limited to amino acids having small residues in the same type.
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-ldh0, 참고문헌 : Zhang et al . "NADH-dependent lactate dehydrogenase from A leal i genes eutrophus H16 reduces 2-oxoadi ate 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 Mi 11 ipore Co. , Darmstadt , Germany)로 다시 한 번 여과한 후 -80°C에 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 the mutated Ae_ldhA (pET24ma_Ae-ldh0, refer to Zhang et al., "NADH-dependent lactate dehydrogenase from lei genes eutrophus H16 reduces 2-oxoadi ate to 2-hydroxyadipate" Biotechnology and Bioprocess Engineering, 19: 1048-1057 (2014)) 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 Mi 11 ipore Co., Darmstadt, Germany), it was stored at -80 ° C.
[표 11]
Figure imgf000054_0001
LDH1_RP cgagggcggtgcggtagt tgggcagactcgacaggccgtggctgatatag 157
[Table 11]
Figure imgf000054_0001
LDH1_RP cgagggcggtgcggtagt tgggcagactcgacaggccgtggctgatatag 157
LDH2_FP ctatatcagccacggcctgtcgactctgcccaactaccgcaccgccctcg 158LDH2_FP ctatatcagccacggcctgtcgactctgcccaactaccgcaccgccctcg 158
LDH2— RP cgagggcggtgcggtagt tgggcagagtcgacaggccgtggctgatatag 159LDH2- RP cgagggcggtgcggtagt tgggcagagtcgacaggccgtggctgatatag 159
LDH3_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 160LDH3_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 160
LDH3_RP cgagggcggtgcggtagt tgggcagat tcgacaggccgtggctgatatag 161LDH3_RP cgagggcggtgcggtagt tgggcagat tcgacaggccgtggctgatatag 161
LDH4_FP ctatatcagccacggcctgtcggacctgcccaactaccgcaccgccctcg 162 ' LDH4_FP ctatatcagccacggcctgtcggacctgcccaactaccgcaccgccctcg 162 '
LDH4_RP cgagggcggtgcggtagt tgggcaggtccgacaggccgtggctgatatag 163LDH4_RP cgagggcggtgcggtagt tgggcaggtccgacaggccgtggctgatatag 163
LDH5_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 164LDH5_FP ctatatcagccacggcctgtcgaatctgcccaactaccgcaccgccctcg 164
LDH5_RP cgagggcggtgcggtagttgggcagat tcgacaggccgtggctgatatag 165 효소의 활성은 NADH의 산화 정도를 340nm의 흡광도를 관찰함으로 측정하였고, 효소의 활성 (speci f i c act ivi ty; 1U)은 1분 동안 1 ymol의 NADH를 NAD로 산화시키는데 필요한 효소의 양으로 정의하였다. 변이체 효소는 모두 pyruvate 와 0HB에 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) . 0HB에 대한 활성도 대부분 감소하였다. 그러나 I48T (LDH2)의 경우 pyruvate에 대해서는 약 50% 정도의 활성이 감소하였으나 ΟΗΒί 대하여는 미미하지만 약간 증가된 활성을 보였다. 즉 pyruvate 대비 0HB에 대한 선택성이 2배 이상 증가한 것으로 나타났다. 향후 실험에서는 LDH2를 선택하였다. LDH5_RP cgagggcggtgcggtagggggcagat tcgacaggccgtggctgatatag 165 The activity of the enzyme was measured by observing the degree of oxidation of NADH at 340 nm and the activity of the enzyme (1 U) was determined by measuring the activity of the enzyme required to oxidize 1 ymol of NADH to NAD Respectively. All of the mutant enzymes showed reduced activity against pyruvate and 0HB. 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 the activity against 0HB was decreased. However, in the case of I48T (LDH2), about 50% of the activity of pyruvate was decreased, but the activity of ΟΗΒί was slight but slightly increased. That is, the selectivity to 0HB over pyruvate was more than doubled. LDH2 was selected in future experiments.
실시예 4: 2-0xo-4-hydroxy butyric acid (OHB)의 (2R)-D-reductase 효소의 스크리닝 및 변이효소 제작  Example 4: Screening of 2R-D-reductase enzyme of 2-0xo-4-hydroxy butyric acid (OHB) and preparation of mutant enzyme
4-1. OHB D-reductase 효소의 스크리닝  4-1. Screening of OHB D-reductase enzyme
OHB D-reductase 효소의 확보  Obtaining of OHB D-reductase enzyme
L-reductase의 경우를 참고하여 D- lactate dehydrogenase 를 대상으로 OHB 2R-reductase 효소를 스크리닝 하였다. E. coli, Pediococcus acidi lact ici , Pseudomonas aeruginosa , Leuconostoc mesenteroides cremoris, Lactobaci 1 lus bulgaricus, L. jensenii , Oenococcus oenii , L. i ant arum, L. reuteri and L. casei 등으로부터 OHB에 대해 활성이 높을 것으로 예상되는 (D)-lactate dehydrogenase 효소들을 선정하였다 (표 12) . 표 12에는 OHB D-reductase 효소 screening dehydrogenase후보 효소들을 나타냈다. In the case of L-reductase, OHB 2R-reductase enzyme was screened for D-lactate dehydrogenase. Lactobacillus listeria, L. jensenii, Oenococcus oenii, L. iantum, L. reuteri and L. casei, which are highly active against OHB, have been identified as E. coli, Pediococcus acidi lacti, Pseudomonas aeruginosa, Leuconostoc mesenteroides cremoris, The expected (D) -lactate dehydrogenase enzymes were selected (Table 12). Table 12 shows candidate enzymes for screening dehydrogenase of the OHB D-reductase enzyme.
[표 12]  [Table 12]
Figure imgf000056_0001
enases
Figure imgf000056_0001
enases
( IdhD) (IdhD)
D- Lactobaci 1 lus ZP_03974 ATAACATATGAAAGGAATGGGAAAAC 176 ' l actate reuteri 385. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGCATTCT dehydrog TATTTCATTTCGTG D- Lactobaci 1 lus ZP_03974 ATAACATATGAAAGGAATGGGAAAAC 176 ' actate reuteri 385. 1 TAATAGGATCCTCAGTGGTGATGATGGTGATGCATTCT dehydrog TATTTCATTTCGTG
177 enases  177 enases
( IdhD) (IdhD)
D- Leuconostoc me ZP_03913 ATAACATATGAAGATTTTTGCTTACG 178 l actate sent ero ides 173. 1  D- Leuconostoc me ZP_03913 ATAACATATGAAGATTTTTGCTTACG 178 l actate sent ero ides 173. 1
TAATAGGATCCTTAGTGGTGATGATGGTGATGATATTC  TAATAGGATCCTTAGTGGTGATGATGGTGATGATATTC
dehydrog cremoris dehydrog cremoris
AACAGCAATAGCTGG  AACAGCAATAGCTGG
179 enases  179 enases
{ IdhD) {IdhD}
D一 Oenococcus oen ZP_01544 ATAACATATGAAAATTTATGCTTATG 180 l actate i 962. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGGAATTT dehydrog AACGAGATTCTTGTCTC  D 1 Oenococcus oen ZP_01544 ATAACATATGAAAATTTATGCTTATG 180 l actate i 962. 1 TAATAGGATCCTTAGTGGTGATGATGGTGATGGAATTT dehydrog AACGAGATTCTTGTCTC
181 enases  181 enases
{ IdhD) {IdhD}
D- Lactobaci 1 lus CAI 96942 AT CATATGACTAAMTTTTTGCTTAC 182 l actate delbruecki i . 1  D- Lactobaci 1 lus CAI 96942 AT CATATGACTAAMTTTTTGCTTAC 182 l actate delbruecki i. One
TAATAGGATCCTTAGTGGTGATGATGGTGATGGAAACT  TAATAGGATCCTTAGTGGTGATGATGGTGATGGAAACT
dehydrog subsp. dehydrog subsp.
CCAGTTAAGGTTGGC  CCAGTTAAGGTTGGC
183 enases bulgaricus  183 enases bulgaricus
{ IdhD) {IdhD}
OHB D-reductase 효소의 생산 및 활성 측정 Production and activity measurement of OHB D-reductase enzyme
L-LDH의 경우와 마찬가지로 효소발현을 위해 E. coli BL2KDE3) star 균주를 숙주균주로 이용하였고 클로닝과 플라스미드 보존을 위해서는 E. coli DH5a 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 pr imer를 사용하여 PCR 증폭하였고 이 때 C 말단에 Hi s-tag을 붙여 주었다. PCR fragment는 pET vector에 넣은 후 E. coli DH5a 균주에 클로닝하고 유전자 서열을 확인한 후 E. coli BL2KDE3) star에 도입하였다. Ldh 유전자의 solubl e express ion을 촉진하기 위해 Chaperon 을 발현하는 pG-Tf2 플라스미드를 추가로 도입해 주었다. As in the case of L-LDH, E. coli BL2KDE3) star strain was used as a host strain for the expression of the enzyme, and E. coli DH5a 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 primer shown in Table 10 below And then Hi s-tag was attached to the C-terminal. The PCR fragment was inserted into a pET vector, cloned into E. coli strain DH5a, and sequenced into E. coli BL2KDE3 star. To facilitate the solubility of the Ldh gene, we introduced the pG-Tf2 plasmid, which expresses the chaperon.
D-LDH 효소 생산을 위해 유전자 재조합 BL21(pET-Xx-LDH) 균주를 50 Ug/mL kanamycin을 포함하는 LB 배지에서 호기적으로 배양하였다. 배양온도는 30 Q C , 교반속도는 100 rpm 이었고 1 L 플라스크에 250 mL 배지를 넣어 주었다. 세포농도가 0.5 0D에 도달했을 때, 0. 1 mM의 IPTG를 첨가하고 추가적으로 10시간쾌양하였다. 이 후 세포를 원심분리하고 25 mM 인산 완층용액 (pH 7.0)으로 3번 세척한 후 binding buf fer (20 mM pH 7.0 인산 완층용액, 0.5 M NaCl , 10 mM imi dazol e)에 현탁한 후 soni cator를 이용하여 파쇄하였다. 세포 파쇄액은 41:, 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 Ug / mL kanamycin. The incubation temperature was 30 Q C, stirring speed was 100 rpm, and 250 mL medium was added to a 1 L flask. When the cell concentration reached 0.5 0D, 0.1 mM IPTG was added and an additional 10 hours of pleasure. The cells were then centrifuged, washed three times with 25 mM phosphate buffer solution (pH 7.0), suspended in binding buffer (20 mM pH 7.0 phosphate buffer solution, 0.5 M NaCl, 10 mM imidazole) . The cell lysate was centrifuged at 41: 25,000 g for 30 minutes 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 0HB를 기질로 사용하여 분석하였다. L-LDH와 마찬가지로 대부분의 효소들은 pyruvate에 3-10배 높은 활성을 가지고 있었고 그에 비해 0HB에는 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 umol/mg protein/min 의 활성을 보였다. 0HB에 대해서는 Lb—LDH가 0.8 umol /mg protein/min의 가장 높은 활성을 보였다. pyruvate 와 0HB에 대한 선택도는 약 1 : 0.2 수준이었다. Lb-LDH를 선택하여 0HB에 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 0HB were used as substrates. Like L-LDH, most of the enzymes had 3-10-fold higher activity on pyruvate than on 0HB (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. For 0HB, Lb-LDH showed the highest activity of 0.8 umol / mg protein / min. The selectivity for pyruvate and 0HB was about 1: 0.2. L-LDH was selected to produce D-reductase mutant enzyme having high activity at 0HB.
OHB D-reductase 효소의 생성물의 광학활성 측정  Measurement 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 color imetr i c 분석 ki t을 사용하여 분석하였다. Lb-LDH와 반응시킨 0HB 생성물의 HPLC 분석의 경우 하나의 peak만 얻어졌고 retent ion t ime은 D— form으로 추정되는 11.3분 시간대에 나타났다. 또한 color imetr i c 분석에서도 D-form 만 얻어졌다. 따라서 Lb-LDH와의 반웅으로 얻어진 DHB는 광학적으로 순수한 D-form 흑은 (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 0HB product reacted with Lb-LDH showed only one peak, and retention ion ime appeared in the D-form at 11.3 minutes. In color imetr ic analysis, only D-form was obtained. Thus, the DHB obtained from the reaction with Lb-LDH was identified as optically pure D-form black as (R) -form isomer.
4-2. site-directed mutagenesis를 이용한 0HB D-reductase 효소의 변이체 개발  4-2. site-directed mutagenesis of 0HB D-reductase enzyme
PDB databank에 나와있는 Lb-LDH 효소의 결정구조를 이용하여 engineering 해야 할 아미노산 잔기를 확인하였다 (도 18). 즉 피루브산과의 docking을 통해 His296, Arg235, Glu264 둥 3개의 잔기가 피루브산과의 반웅 활성부위에 존재하고 추가적으로 두 개의 hydrophobic 아미노산 잔기인 Val78과 TyrlOl이 활성부위 주위에 존재하는 것을 확인하였다. 이러한 hydrophobic 잔기들은 0HB와의 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 of His296, Arg235, and Glu264 were present in the antagonistic activity site with pyruvate, and additionally two hydrophobic amino acid residues Val78 and TyrlOl were present around the active site. Since these hydrophobic residues are likely to interfere with the reaction with 0HB, 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 (Am icon, Merck Millipore Co. , Darmstadt, Germany)로 다시 한 번 여과한 후 -80°C에 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 (Am icon, Merck Millipore Co., Darmstadt, Germany), it 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.
[표 13]  [Table 13]
Pr imers 염기서열 서열번호 Pr imers nucleotide sequence SEQ ID NO:
LDH6_FP cat cactaagatgagcctgcgtaactccggtgt tgacaacatcgacatggct 184LDH6_FP cat cactaagatgagcctgcgtaactccggtgt tgacaacatcgacatggct 184
LDH6_RP tagccatgtcgatgttgtcaacaccggagt tacgcaggctcatcttagtgatg 185LDH6_RP tagccatgtcgatgttgtcaacaccggagt tacgcaggctcatcttagtgatg 185
LDH7_FP cat cactaagatgagcctgcgtaacaacggtgt tgacaacatcgacatggcta 186LDH7_FP cat cactaagatgagcctgcgtaacaacggtgt tgacaacatcgacatggcta 186
LDH7_RP tagccatgtcgatgttgtcaacaccgttgt tacgcaggctcatcttagtgatg 187LDH7_RP tagccatgtcgatgttgtcaacaccgttgt tacgcaggctcatcttagtgatg 187
LDH8_FP cat cactaagatgagcctgcgtaacaccggtgt tgacaacatcgacatggcta 188 LDH8_RP tagccatgtcgatgttgtcaacaccggtgttacgcaggctcatcttagtgatg 189LDH8_FP cat cactaagatgagcctgcgtaacaccggtgt tgacaacatcgacatggcta 188 LDH8_RP tagccatgtcgatgttgtcaacaccggtgttacgcaggctcatcttagtgatg 189
LDH9_FP catcactaagatgagcctgcgtaacgacggtgttgacaacatcgacatggcta 190LDH9_FP catcactaagatgagcctgcgtaacgacggtgttgacaacatcgacatggcta 190
LDH9— RP tagccatgtcgatgttgtcaacaccgtcgttacgcaggctcatcttagtgatg 191LDH9- RP tagccatgtcgatgttgtcaacaccgtcgttacgcaggctcatcttagtgatg 191
LDH10_FP catcactaagatgagcctgcgtaacaagggtgttgacaacatcgacatggcta 192LDH10_FP catcactaagatgagcctgcgtaacaagggtgttgacaacatcgacatggcta 192
LDH10— RP tagccatgtcgatgttgtcaacacccttgttacgcaggctcatcttagtgatg 193 효소의 활성은 NADH의 산화 정도를 340nm의 흡광도를 관찰해 측정하였고, 효소의 활성 (specific activity; 1U)은 1분 동안 1 n 의 NADH를 NAD+로 산화시키는데 필요한 효소의 양으로 정의하였다. 변이체 효소는 모두 pyruvate 와 0HB에 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. LDH10- RP tagccatgtcgatgttgtcaacacccttgttacgcaggctcatcttagtgatg 193 Enzymatic activity was measured by measuring the degree of oxidation of NADH at 340 nm. The specific activity (1 U) of the enzyme was determined by the amount of enzyme required to oxidize 1 n of NADH to NAD + Respectively. All of the mutant enzymes showed reduced activity against pyruvate and 0HB. 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) 생산용균주의 제작  Example 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 반웅의 cofactor인 pyr idoxal-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, the increase of TA activity should increase the expression of pyridoxal-5-phosphate (PLP) which is a cofactor of TA antagonist as well as the expression of TA enzyme itself, that is, the biosynthesis of vitamin B6. 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 증가  5-1. 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. 결과적으로 PLP 생합성이 강화된 3개의 균주를 얻을 수 있었다 (표 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 expression of epd, dxs ' , and pdxJ genes to increase vitamin B6 biosynthesis. As a result, three strains with enhanced PLP biosynthesis were obtained (Table
[표 14] [Table 14]
Primer 염기서열 서열번 s 호 us- atctaagcggccgcggaattatgcaattcgtggtac 194 Primer nucleotide sequence SEQ ID NO: us- atctaagcggccgcggaattatgcaattcgtggtac 194
Psp5一 Psp5
epd-FP epd-FP
us- aaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatgaccgtacgcgtagcgataa 195us- aaaaaatttatttgctttcgcatctttttgtacctataagtgtggaatgaccgtacgcgtagcgataa 195
Psp5- atg Psp5-ATG
epd-RP epd-RP
Psp5- gccagtttagtatcgacc 196 epd-FP  Psp5- gccagtttagtatcgacc 196 epd-FP
Psp5- acaaaagcatgatcctgttgaagatgcg 197 epd-RP  Psp5- acaaaagcatgatcctgttgaagatgcg 197 epd-RP
DS- catttatcgctacgcgtacggtcattccacacttataggtacaaaaagatgcgaaagcaaataaattt 198 DS- catttatcgctacgcgtacggtcattccacacttataggtacaaaaagatgcgaaagcaaataaattt 198
Psp5- ttttc Psp5-TTTTc
epd-FP epd-FP
DS- tagtaatctagaggagttggcatctttctgcgatttc 199 DS- tagtaatctagaggagttggcatctctctgcgatttc 199
Psp5- epd-RP Psp5-EPd-RP
us- atctaagcggccgctcgacatttcattgtcgttgag 200us- atctaagcggccgctcgacatttcattgtcgttgag 200
Psp5- dxs-FP Psp5- dxs-FP
us- caaaaaatttatttgctttcgcatct ttttgtacctataagtgtggaatgagttttgatattgccaaa 201us- caaaaaatttatttgctttcgcatct ttttgtacctataagtgtggaatgagttttgatattgccaaa 201
Psp5- tac Psp5- tac
dxs-RP dxs-RP
Psp5- gtaaagcttaccggaaagcagctgt 202 dsx-FP  Psp5- gtaaagcttaccggaaagcagctgt 202 dsx-FP
Psp5- agcaactcgaagcctgcgttaag 203
Figure imgf000062_0001
[표 15]
Psp5- agcaactcgaagcctgcgttaag 203
Figure imgf000062_0001
[Table 15]
Figure imgf000063_0001
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.
Figure imgf000063_0001
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 조절 5-2. 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.
[표 16] [Table 16]
Protein Names Gene  Protein Names Gene
MFS type transporter YhjX  MFS type transporter YhjX
Probable formate transporter focA  Probable formate transporter focA
Lactate premease lldp  Lactate premease lldp
Inner membrane metabolite transporter yhjE  Inner membrane metabolite transporter yhjE
C4-dicarboxylate transporter dcuA  C4-dicarboxylate transporter dcuA
C4一 dicarboxylate transporter dcuB C4-dicarboxylate transporter dcuC C4-dicarboxylate transporter dcuB C4-dicarboxylate transporter dcuC
[표 17] [Table 17]
Figure imgf000064_0001
이들 중 가장 가능성이 높은 2개의 exporter, 즉 ldp, (fcuA의 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, actP : 제거했다 . Transporter engineering 에 Λ1 "용한 primer 서열을 표 18에 나타내었다.
Figure imgf000064_0001
The expression level of the two most potent exporters, ldp, (fcuA), was elevated using a synthetic promoter. The overexpression of membrane protein interfered with cell growth, so SP5, a mid-level synthetic promoter, was used. In addition, three membrane proteins, kgtP, dsdx, and actP, which are expected to be the most likely importer, were removed: Λ 1 "soluble primer sequences in transporter engineering are shown in Table 18.
[표 18]  [Table 18]
Primer 염기서열 서열번 Primer sequence SEQ ID NO:
(과발현 호 ) (Overexpression)
us- atctaagcggccgcgattggaatgcccatcgcac 212us- atctaagcggccgcgattggaatgcccatcgcac 212
Psp4- lldp-FP Psp4- lldp-FP
US— ttgacaat taat cat ccggctcgtaattt atgtggaatgaat etc tggcaacaaaactacg 213 Psp4- lldp-RP Psp5- ccatctgaccaatctcaaagctgt 214 lldp-FP US- ttgacaat taat cat ccggctcgtaattt atgtggaatgaat etc tggcaacaaaactacg 213 Psp4- lldp-RP Psp5- ccatctgaccaatctcaaagctgt 214 lldp-FP
Psp5- t tgtgaacaaagcacctggtcgcg 215 lldp-RP  Psp5- t tgtgaacaaagcacctggtcgcg 215 lldp-RP
DS- t ccacat aaat t acgagccggat at t aat tgt caattggt agggccaat tcttgtg 216 DS- ccacat aaat t acgagccggat at a tgt caattggt agggccaat tcttgtg 216
Psp4- lldp-FP Psp4- lldp-FP
DS- tagtaatct agagaaagaagt cgaaaaaaacgaaat c 217 DS- tagtaatct agagaaagaagt cgaaaaaaacgaaat c 217
Psp4- lldp-RP Psp4-1LLDP-RP
us- atctaagcggccgcgttgatgattgcatagataacc 218us- atctaagcggccgcgttgatgattgcatagataacc 218
Psp4- ducA-FP Psp4-ducA-FP
us- ttgacaattaatcatccggctcgtaatttatgtggaatgcaggttgctggcggtctggactatctg 219us-ttgacaattaatcatccggctcgtaatttatgtggaatgcaggttgctggcggtctggactatctg 219
Psp4- gttc Psp4-gttc
ducA - RP ducA - RP
Psp4- ccatctgaccaatctcaaagctgt 220 ducA-FP  Psp4- ccatctgaccaatctcaaagctgt 220 ducA-FP
Psp4- t tgtgaacaaagcacctggtcgcg 221 ducA-RP  Psp4- t tgtgaacaaagcacctggtcgcg 221 ducA-RP
DS- t ccacat aaattacgagccggatgattaattgtcaatgttttttaacaagttgatattagattg 222 Psp4- ducA - FP DS- t ccacat aaattacgagccggatgattaattgtcaatgttttttaacaagttgatattagattg 222 Psp4- ducA-FP
DS- tagtaatct agagaaagaagt cgaaaaaaacgaaat c 223 Psp4- ducA - RP Pr imers 염기서열 DS- tagtaatct agagaaagaagt cgaaaaaaacgaaat c 223 Psp4- ducA-RP Pr imers sequence
(제거)  (remove)
us- atctaagcggccgcggtatccagcgacgat t t tagcg us- atctaagcggccgcggtatccagcgacgat t t tagcg
224 dsdx-FP  224 dsdx-FP
US— gtacgtctctt tgcgct tacatatctcacctt cccctg  US- gtacgtctctt tgcgct tacatatctcacctt cccctg
225 dsdx-RP  225 dsdx-RP
G-dsdx gcgtgtaaagtcacctaatgc  G-dsdx gcgtgtaaagtcacctaatgc
226 -FP  226 -FP
G-dsdx gggcatcagcagacatatg  G-dsdx gggcatcagcagacatatg
227 -RP  227 -RP
DS- caggggaaggt gagat atgt aagcgcaaagagacgt ac  DS- caggggaaggt gagat atgt aagcgcaaagagacgt ac
228 dsdx-FP  228 dsdx-FP
DS- tagtaatctagacgctcataatgccgattgataac  DS- tagtaatctagacgctcataatgccgattgataac
229 dsdx-RP  229 dsdx-RP
us- atctaagcggccgcggagctgatccgcgagcatac us- atctaagcggccgcggagctgatccgcgagcatac
230 kgtP-FP  230 kgtP-FP
us- acggcaggagacataatggcatgtagtgacgggtcagttgccagac us- acggcaggagacataatggcatgtagtgacgggtcagttgccagac
kgtP 一 231 RP kgtP-231 RP
G- kgtP tggcgtctggatctggaaaacg .  G- kgtP tggcgtctggatctggaaaacg.
232 -FP  232 -FP
G- kgtP tgtgtaagcgcagcgatgc  G- kgtP tgtgtaagcgcagcgatgc
233 -RP  233 -RP
DS— gtctggcaactgacccgtcactacatgccat tatgtctcctgccgt  DS- gtctggcaactgacccgtcactacatgccat tatgtctcctgccgt
kgtP - 234 FPkgtP - 234 FP
DS- ta t agt aatct agagaagc tgt t t t ggcggatga DS-ta agt aatct agagaagc tgt t t t ggcggatga
235 kgtP-RP  235 kgtP-RP
US一 atctaagcggccgcct tatct t tat taaggtaaac  US 1 atctaagcggccgcct tatct t tat taaggtaaac
236 actP-FP us- agtcctgcatgaggtacaagcatcatgtaatctctccccttccccggtcgtctg actP - 237 RP 236 actP-FP us- agtcctgcatgaggtacaagcatcatgtaatctctccccttccccggtcgtctg actP - 237 RP
G- actP ctgtgggat t tcgatagtat  G- actP ctgtgggat t tcgatagtat
238 -FP  238 -FP
G- actP agcaacctgggcgatacctcgac  G- actP agcaacctgggcgatacctcgac
239 -RP  239-RP
DS- c agacgaccggggaaggggagagat t aca t ga t gc 11 gt ac c t c at gcaggac t 240 actP - FP DSC agacgaccggggaaggggagagat t aca t ga t gc 11 gt ac c t c at gcaggac t 240 actP - FP
DS- tagtaatct agaaagc tgct t aagagaagcag 241 actP-RP 최종적으로 transporter와 PLP 생합성 경로가 변화된 DHB 생산 숙주 균주 대장균이 아래 표 19과 같이 얻어졌다. DS-tagtaatct agaaagc tgct t aagagaagcag 241 actP-RP DHB-producing host strain E. coli transformed with transporter and PLP biosynthetic pathway was finally obtained as shown in Table 19 below.
[표 19] . [Table 19].
Figure imgf000067_0001
실시예 6: 광학적으로순수한 S-form 또는 L-form 2, 4-디히드록시 부티르산 (2,4-dihydroxybutyric acid, S-흑은 R-DHB) 생산균주의 제작및 발효 6-1. DHB의 생산을 위한 유전자 재조합 플라스미드 제작
Figure imgf000067_0001
Example 6: Production and fermentation of optically pure S-form or L-form 2, 4-dihydroxybutyric acid (S-black R-DHB) 6-1. 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 PCR증폭한 후 overlap 하고 pBAD plasmid의 Ndel 및 EcoRI site에 ligation 해 주었다. 두 개의 새로운 플라스미드, 즉 L-form DHB를 위한 pBAD_TA4-l_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 0HB 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 Ndel and EcoRI site of pBAD plasmid. Two new plasmids were obtained: pBAD_TA4-l_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 0HB 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, Aeda, Mac I, AthrB, AmetA, MysA, AadhE, ApfJB, MdhA, 과 2개의 프로모터 치환 (APacs: :Pirc), A ppc- -PSP8) , 그리고 metL 유전자를 과발현하기 위한 플라스미드 pUCPK-P rd»e 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. A homozygous homozygous mutant strain, EcW20 (see Table 19), was used to replace the 10 gene deletions pts, Aeda, MacI, AthrB, AmetA, MysA, AadhE, ApfJB, MdhA and two promoter substitutions (APacs: -PSP8), and the plasmid pUCPK-P rd »e for overexpression of 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; MgS047_H20, 2 g; KH2P04, 2 g; (NH4)2S04, 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 사용하였다ᅳ 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; MgS0 4 7_H 2 0, 2 g; KH 2 P0 4, 2 g; (NH 4) 2 S0 4, 10 g; L-methionine, 0.2 g; L L-isoleucine, 0.05 g, trace metal solution, 10 ml) was used. The concentration of glucose was 10 g / L, Kanamycin and ampicillin were 50 mg / L . 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 and glucose consumption decreased and DHB production decreased 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. Ec 20(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 LdM 활성보다 낮기 때문으로 확인했다. (도 22) D-DHB production was investigated in the same way through the EcW20 (pDHB-D) strain in which pDHBD was introduced. As with L-DHB, we have a DHB production plasmid . The non-EcW20 strain produced no DHB at all. In the case of the Ec 20 (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 LdM activity. (Fig. 22)
EcW20(pDHB-L) 및 EcW20(pDHB_D) 균주를 이용하여 생물반응기 실험을 실시하였다 (도 24a) . 반웅기 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 wm의 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. 도 24a 및 24b에서 검은 동그라미 기호의 실선은 세포농도를 의미하며, 흰색 동그라미 기호의 점선은 생성물인 DHB를 의미하고, 역삼각형 기호의 점선은 포도당 농도를 의미한다. Bioreactor experiments were performed using EcW20 (pDHB-L) and EcW20 (pDHB_D) strains (Fig. 24A). The medium 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. In order to maintain aerobic conditions, air was injected at a speed of 1 wm and the agitation speed was appropriately 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. 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, 으 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, there was a difference in cell growth, product production, final concentration and the like for DHB and L-DHB. 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 butyro lactone (HGBL) 생산균주의 제작 및 발효  Example 7: Production and fermentation of 2-hydroxy gamma butyrolactone (HGBL) producing strains
7-1 HGBL의 생산홀 위한 유전자 재조합 플라스미드 및 재조합 균주의 제작  7-1 Production of recombinant plasmids and recombinant strains for production hall of HGBL
DHB를 HGBL로 전환하기 위해서는 l act onase 효소가 필요하다. 락토네이즈 효소로 paraoxonase가 사용되었으며, 이 효소는 칼슘을 필요로 하고 여러 기질에 대하여 활성을 가지고 있다. 이 효소반응은 산성 범위에서 최적이므로 세포막이나 per ipl asmi c space에서 발현하는 것이 필요하다. 또 이 효소가 동물유래이므로 미생물 발현을 위해서는 적절한 변이가 필요하다. 따라서 본 연구에서는 ponl 유전자 변이체 (G3C9)를 사용하고 또한 N—말단에 s ignal sequence를 부착하여 per ip l asmi c space에 발현되도록 하였다. 먼저 ponl(G3C9) 유전자를 합성한 후 PCR 증폭 (FP , tagacacatatggct aaactgacagcg ; RP , t acat act cgagt t acagct cacagt aaagagc 111 g ) s-f Jl 낮은 copy 수를 갖는 pACYC- Duet 플라스미드에 클로닝하였다. ponl(G3C9) 의 발현에는 t ac 프로모터를 사용하였다 (도 18) . 이 플라스미드를 pACYC_Ponl으로 명명하였다.  To convert DHB to HGBL, l act onase enzyme is required. 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 the cell membrane or peripolar space. Since this enzyme is derived from an animal, appropriate mutation is necessary for microbial expression. In this study, the ponl gene mutant (G3C9) was used and the s ignal sequence was attached at the N-terminus to be expressed in the periplank c space. First, the ponl (G3C9) gene was synthesized and then cloned into a pACYC-Duet plasmid having a low copy number of s-f Jl (SEQ ID NO: 16). PCR amplification (FP, tagacacatatggct aaactgacagcg; RP, tacat act cagat t acagct cacagt aaagagc 111 g) A tac promoter was used for the expression of ponl (G3C9) (Fig. 18). This plasmid was named pACYC_Ponl.
얻어진 Ponl pl asmi d를 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), homoser ine에서 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 명명하였다. 7-2 HGBL의 생산을 생물반웅기 운전 The obtained Ponl pl asmi d was 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), and plasmids for producing DHB in homoserine were used for pDHB-L and pDHB- D was used (Fig. 21). These strains were named EcW16 (pHGBL-L) and EcW16 (pHGBL-D), respectively. 7-2 Production of HGBL for Biological Control
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, ponl 발현을 위한 플라스미드 도입과 추가적인 단백질, 특히 ponl 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 transporters, the introduction of plasmids for the expression of ponl, and the production of additional proteins, especially ponl membrane proteins.
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. ponl 발현을 위한 플라스미드 도입과 추가적인 단백질, 특히 ponl 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 ponl expression and the production of additional proteins, particularly ponl membrane proteins, were presumed to be responsible for this. In addition, L-DHB was obtained at a higher concentration than D-DHB because the activity of 2-oxo-reductase was significantly 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 ponl 효소가 L-form 의 DHB에만 활성이 있게 때문으로 추정되었다. The gain D-HGBL was subjected to the same experiments (Fig. 26b) that is conducted after 48 hours incubation the first stage for 24 hours in a two-stage incubation 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 ponl enzyme is active only in DHB of L-form.
화학적 반웅을 통해 2 ,4—di hydroxy butyr ic 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 정치반웅 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 반웅을 이용하는 것이 ponl 효소를 이용하는 것보다 효율적임을 알 수 있었다. Through chemical reaction, we converted 2, 4-dihydroxybutyric acid 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. And 10 hours of political opposition. In this case, L-DHB and D- About 50% of all 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. Therefore, it was found that esterification reaction of DHB was more efficient than using ponl enzyme by using acid catalyst chemical reaction in both D-form and L-form.
¾ :n ¾ : n
원기탁에 의한 수탁증  Deposits by original deposit
:.수. 헤^ :.Number. H ^
50. 휴니스 ¾t 던양 50. Health ¾ t Thunder
주 . 을산, :44919. 대한만국 Λ Note. : 44919. The whole nation Λ
Figure imgf000073_0002
Figure imgf000073_0002
Figure imgf000073_0001
원기탁에 의한 수탁증 박성 Φ
Figure imgf000073_0001
Deposits by original deposits Φ
0 -1 방, 10 빌당,  0 -1 room, 10 billet,
50, 니스 언쨩읍, 50, Nice,
:주군 , 奮산, 민국  : Lieutenant Governor, Excitement, Republic of Korea
I . 미생물≤ 명칭: 기 «'자빼: 의히| 뚜어진 명청: 국제기턱기^이 ^여한 ^탁번호:I. Microorganisms ≤ Name: В «'' Zap: Serve | Tongjin Jungchung: International Tachibanggye ^ ^ Tachan ^ Tak:
EcW20 KCOI12282P EcW20 KCOI12282P
Π:: ,과학적 성 ¾ 분류학샅위치 1 ί애 표서된 :미' 웁애 ^亲 ; 부하였다,. Π ::, Scientificity ¾ Taxonomy Location 1 ί : , Respectively .
f "J 과학적 44 m,수령 및 수탁 f " J Scientific 44 m, receipt and entrustment
본국채기탁기관¾ 상기 1 ¾Λ| _이생뭅 수략받고 20 B년. 6¾ 22 ¾Deposits of Depositary Institutions ¾ above 1 ¾Λ | I have 20 years of life . 6¾ 22 ¾
^령하¾다 ^^
^제기 기관  ^ The filing body
명 짐 : 한극미섕률보 s센터 Jim Jim: Chung Shim Yuryobo Center
주 소 율^ 서대&구 4 4' Address Rate ^ S & D 4 4 '
45 ί-ι^ι^ ' 45 "
2018년 6¾ 22일 1
Figure imgf000074_0001
June 22, 2018 1
Figure imgf000074_0001

Claims

【청구범위】 【청구항 1】 글루코스를 탄소원으로 사용하여 광학적으로 순수한 2 , 4-디히드록시- 부틸레이트 (2 , 4-dihydroxybutyrate) 또는 2-히드록시-감마—부티로락톤 (2- hydroxy ga隱 a butyro l actone)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 : Claims: What is claimed is: 1. A process for preparing optically pure 2, 4-dihydroxybutyrate or 2-hydroxy-gamma-butyrolactone (2-hydroxygal Dihydroxy-butyrate or 2- (4-hydroxybutyrate), comprising the step of performing at least one selected from the following (1) to (4) Method for producing hydroxy-gamma-butyrolactone-producing microorganism variants:
( 1) ptsG, eda, adhE, pflB, lysA, thrBC, met A, Lac I, IdhA, 및 유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 결실, 또는 acs, ppc, 및 metL유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계,  (1) deletion of one or more genes selected from the group consisting of ptsG, eda, adhE, pflB, lysA, thrBC, metA, LacI, IdhA and genes, or one selected from the group consisting of acs, ppc, Overexpressing the above genes, or both,
(2) epd, dxs, 및 pdxj유전자로 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) 및 ducA중 하나 이상의 유전자의 과발현시키는 단계 , 및 (3) and ducA, and
(4) kgtP, dsdx, 및 ac P 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 acP genes.
【청구항 2】  [Claim 2]
제 1항에 있어서, 상기 2, 4-디히드록시 -부틸레이트 또는 2-히드록시- 감마-부티로락톤을 생산하는 미생물은 대장균 0?. coin , 효모 (Yeast ) , 및 코리네박테리움 (Corynebacter ium)으로 이루어진 군에서 선택된 하나 이상인, 방법.  The microorganism producing the 2,4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone according to claim 1, wherein the microorganism produces E. coli 0. coin, yeast, and Corynebacter ium.
【청구항 3】  [Claim 3]
제 1항에 있어서, 상기 미생물을 변이시키는 단계는 ( 1) 단계, 및 (2) 내지 (4) 단계 중 선택된 하나 아상의 단계를 포함하는 것인, 방법.  The method according to claim 1, wherein the step of mutating the microorganism comprises the steps of (1) and (2) to (4).
[청구항 4】  [Claim 4]
거 U항에 있어서, 상기 미생물을 변이시키는 단계는 ( 1) 내지 (4) 단계를 전부 포함하는 것인, 방법.  2. The method according to claim 1, wherein the step of mutating the microorganism includes all steps (1) to (4).
【청구항 5】 제 1항 내지 게 4항 중 어느 한 항에 있어서, 상기 ( 1) 단계는 ptsG, eda, adhE, pflB, lysA, thrB, metA, Lad, ldhA, 및 iclR유전자 전부를 결실시키고, acs, ppc, 및 metL유전자 전부를 과발현시키는 단계를 포함하는 것인, 방법. [Claim 5] The method according to any one of claims 1 to 4, wherein said step (1) deletes all of the ptsG, eda, adhE, pflB, lysA, thrB, metA, Lad, ldhA and iclR genes, And overexpressing all of the metL gene.
[청구항 6】  [Claim 6]
제 1항 내지 게 4항 중 어느 한 항에 있어서, 상기 (2) 단계는 epd, dxs, 및 pdx유전자 전부를 과발현시키는 단계를 포함하는 것인, 방법 .  The method of any one of claims 1 to 4, wherein said step (2) comprises overexpressing all of the epd, dxs, and pdx genes.
【청구항 7】 7.
제 1항 내지 제 4항 중 어느 한 항에 있어서, 상기 (3) 단계는 lpd, 및 ducA유전자 전부를과발현시키는 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.
【청구항 8】  8.
제 1항내지 제 4항 중 어느 한 항에 있어서, 상기 (4) 단계는 kgtP, dsdx, 및 ac 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 ac genes.
【청구항 9】 [Claim 9]
제 1항에 있어서, 상기 미생물 변이체 제조방법은  2. The method according to claim 1,
호모세린으로부터 4-히드록시 -2-옥소 -부틸레이트 (4-hydroxy-2-oxo- butyrate)의 전환을 촉진하는 단계를 추가로 포함하는 것인, 방법.  Further comprising the step of promoting the conversion of 4-hydroxy-2-oxo-butyrate from homoserine.
【청구항 10】  Claim 10
저 19항에 있어서, 상기 호모세린으로부터 4-히드록시 -2-옥소- 부틸레이트의 전환을 촉진하는 단계는 트랜스아미네이즈 (transaminase)를 처리하여 호모세린 (homoser ine)의 알파 위치 아민기를 제거하는 단계를 포함하는 것인, 방법.  In step 19, the step of promoting the conversion of 4-hydroxy-2-oxo-butyrate from the homoserine is a step of treating the transaminase to remove the alpha-position amine group of homoserine The method comprising the steps of:
' '
【청구항 11】 Claim 11
제 10항에 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.
【청구항 12】  Claim 12
제 11항에 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 &gt; and / or &lt; / RTI &gt; How, one.
[청구항 13】  [Claim 13]
겨】 9항에 있어서, 상기 미생물 변이체 제조방법은  9. The method for producing a microorganism variant according to claim 9,
4-히드록시 -2-옥소-부틸레이트를 2, 4-디히드록시-부틸레이트로 전환을 촉진하는 단계를 추가로 포함하는 것인, 방법.  Further comprising the step of promoting conversion of 4-hydroxy-2-oxo-butylate to 2,4-dihydroxy-butyrate.
【청구항 14】  14.
제 13항에 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) Selected one or more.
【청구항 15】  15.
제 13항에 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 단계를포함하는 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.
【청구항 16】  Claim 16
제 15항에 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 &gt; enzymes. &Lt; / RTI &gt;
【청구항 17】  17.
제 15항에 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.
【청구항 18】  Claim 18
제 13항에 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. .
【청구항 19】  Claim 19
제 18항에 있어서, 상기 락톤화의 촉진은 서열번호 22의 아미노산 서열로 이루어진 락토네이즈 ( l actonase)의 발현을 촉진하여 수행되는 것인, -방법 . 19. The method of claim 18, wherein the facilitation of the lactonization comprises contacting the amino acid sequence of SEQ ID NO: Lt; RTI ID = 0.0 &gt; lactonase, &lt; / RTI &gt;
【청구항 20]  [20]
제 18항에 있어서, 상기 2-히드록시-감마-부티로락톤은 (2S)-2- 히드록시 감마 부티로락톤 및 (2R)-2-히드록시 감마 부티로락톤으로  19. The composition of claim 18, wherein the 2-hydroxy-gamma-butyrolactone is (2S) -2-hydroxy gamma butyrolactone and (2R) -2- hydroxy gamma butyrolactone
이루어진 군에서 선택된 하나 이상인, 방법. Wherein said at least one selected from the group consisting of.
【청구항 21]  21,
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:  2, 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone is introduced into the genome of a microorganism producing the mutation of any one of (1) to (4) 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone-producing microorganism variant:
( 1) ptsG, eda, adhE, pflB, lysA, thrBC, metA, Lad, ldhA, 및 /c/ ?유전자로 이루어진 군에서 선택된 하나 이상의 결실, 또는 acs, ppc, 및 metL유전자로 이루어진 군에서 선택된 하나 이상의 유전자를 과발현, 또는 이들 모두를 수행하는 단계;  (1) one or more deletions selected from the group consisting of ptsG, eda, adhE, pflB, lysA, thrBC, metA, Lad, ldhA, and / c / Overexpressing the above genes, or both;
(2) epd, dxs, 및 pdxj유전자로 이루어진 군에서 선택된 하나 ' 이상의 유전자의 과발현, (2) epd, dxs, and over-expression of one 'or more genes selected from the group consisting of pdxj gene,
(3) J d 및 ducA중 하나 이상의 유전자의 과발현,  (3) overexpression of one or more genes of J d and ducA,
(4) kgtP, dsdx, 및 aci 3유전자로 이루어진 군에서 선택돤 하나 이상의 유전자를 결실. (4) deletes one or more genes selected from the group consisting of kgtP, dsdx, and aci 3 genes.
【청구항 22]  [22]
2, 4-디히드록시 -부틸레이트 또는 2-히드록시-감마-부티로락톤을 생산하는 미생물의 게놈 ( genome )에서,  In the genome of microorganisms that produce 2, 4-dihydroxy-butyrate or 2-hydroxy-gamma-butyrolactone,
ptsG, eda, adhE, pflB, lysA, thrBC, metA, Lac I, ldhA, iclR, kgtP, dsd, 및 ac P유전자가 결실되고,  ptsG, eda, adhE, pflB, lysA, thrBC, metA, Lac I, ldhA, iclR, kgtP, dsd,
acs, ppc, metL, epd, dxs, pdxj, 네 c/ucA유전자가 과발현된, 미생물 변이체.  acs, ppc, metL, epd, dxs, pdxj, microbial mutants overexpressing the c / ucA gene.
【청구항 23]  23. The method of claim 22,
제 21항에 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.
【청구항 24】 24.
제 21항에 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.
【청구항 25]  [25]
제 21 내지 제 24항 중 어느 한 항에 있어서, 상기 미생물 변이체는 하기 (1) 내지 (4) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 백터를 추가로 포함하는 것인, 미생물 변이체:  25. The microorganism variant according to any one of claims 21 to 24, wherein the microbial mutant further comprises at least one gene selected from the following (1) to (4) or a recombinant vector comprising the same:
(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,  (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) 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 효소를 암호화하는 유전자.  (4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.
【청구항 26】  26. The method of claim 26,
제 21항에 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.
【청구항 27】  [27]
제 21항 내지 제 24항 및 제 26항 중 어느 한 항의 미생물 변이체를 배양하는 단계를 포함하는, 2-히드록시 감마 부티로락톤 (2-hydroxy ga匪 a butyro lactone) 또는 2,4-디히드록시 부틸레이트 (2 ,4-dihydroxy butanoic acid)의 생산방법. (2-hydroxygalactopyranoside) or 2-hydroxygalactopyranoside, which comprises culturing the microorganism variant according to any one of claims 21 to 24 and 26, 2, 4-dihydroxy butanoic acid acid.
【청구항 28】  28. The method of claim 28,
제 27항에 있어서, 상기 미생물 변이체는 하기 ( 1) 내지 (3) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 백터를 추가로 포함하는 것인, 생산 방법:  28. The production method according to claim 27, wherein the microorganism variant further comprises at least one gene selected from the following (1) to (3) 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 효소로 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 an enzyme consisting of the amino acid sequence of SEQ ID NO: Natri mutational enzyme coding 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,
(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 효소를 암호화하는 유전자.  (4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.
【청구항 29】  Claim 29
제 27항에 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.
【청구항 30]  [30]
제 27항에 있어서, 상기 배양하는 단계 이후에, 당과 메티오닌 (methionine) , 라이신 ( lys ine) , 트레오닌 (threonine), 및 이소류신 ( i soleucine)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 a sugar and methionine, lysine, threonine, and isoleucine is added, &Lt; / RTI &gt;
[청구항 31】  [Claim 31]
제 30항에 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. 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, (2S) -2-hydroxy-gamma-butyrolactone or (2R) -2-hydroxy-gamma-butyrolactone by chemically lowering the pH to 1.0 to 3.0. , Production method.
[청구항 32】  [Claim 32]
제 21항 내지 제 24 및게 26 중 어느 한 항의 미생물 변이체 또는 그 배양물을 포함하는, 2-히드록시 감마 부티로락톤 (2-hydroxy gamma butyrol actone) 또는 2,4-디히드록시 부틸레이트 (2 , 4-dihydroxy butanoic acid)의 생산용 조성물.  (2-hydroxy gamma butyrol actone) or 2,4-dihydroxybutyrate (2-hydroxybutyrate), which comprises a microorganism variant or a culture thereof according to any one of claims 21 to 24 and 26, , 4-dihydroxy butanoic acid).
[청구항 33】  [Claim 33]
제 32항에 있어서, 상기 미생물 변이체는 하기 ( 1) 내지 (3) 중에서 선택된 하나 이상의 유전자 또는 이를 포함하는 재조합 백터를 추가로 포함하는 것인, 생산용 조성물:  The production composition according to claim 32, wherein said microbial mutant further comprises at least one gene selected from the following (1) to (3) or a recombinant vector comprising said gene:
( 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,  (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) 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,
(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 효소를 암호화하는 유전자.  (4) a gene encoding an enzyme consisting of the amino acid sequence of SEQ ID NO: 22.
【청구항 34】  34. The method of claim 34,
서열번호 13의 아미노산 서열로 이루어진, 트랜스아미네이즈  A transaminase comprising the amino acid sequence of SEQ ID NO:
변이효소. Mutation enzyme.
【청구항 35] [35]
서열번호 14의 아미노산 서열로 이루어진, 트랜스아미네이즈 변이효소.  A transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 14.
【청구항 36】  36. The method of claim 36,
서열번호 15의 아미노산 서열로 이루어진, 트랜스아미네이즈 변이효소.  A transaminase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 15.
【청구항 37]  [37]
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:
【청구항 38】  38.
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.
【청구항 39】  39. The method of claim 30,
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.
【청구항 40]  [40]
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.
【청구항 41]  41. The method of claim 41,
서열번호 22의 아미노산 서열로 이루어진, 락토네이즈 변이효소.  A lactonase mutant enzyme comprising the amino acid sequence of SEQ ID NO: 22.
PCT/KR2018/007923 2017-07-12 2018-07-12 Method for preparing 2-hydroxy-gamma-butyrolactone or 2,4-dihydroxy-butyrate WO2019013573A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20170088465 2017-07-12
KR10-2017-0088465 2017-07-12

Publications (3)

Publication Number Publication Date
WO2019013573A2 true WO2019013573A2 (en) 2019-01-17
WO2019013573A3 WO2019013573A3 (en) 2019-03-28
WO2019013573A9 WO2019013573A9 (en) 2019-05-02

Family

ID=65002668

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/007923 WO2019013573A2 (en) 2017-07-12 2018-07-12 Method for preparing 2-hydroxy-gamma-butyrolactone or 2,4-dihydroxy-butyrate

Country Status (2)

Country Link
KR (1) KR102149044B1 (en)
WO (1) WO2019013573A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102021101004B3 (en) 2021-01-19 2022-03-10 Technische Universität Dresden, Körperschaft des öffentlichen Rechts Process for the production of 2,4-dihydroxybutyrate or L-threonine using a microbial pathway
CN115948482A (en) * 2023-02-07 2023-04-11 中国科学院天津工业生物技术研究所 Construction method and application of2, 4-dihydroxybutyric acid biosynthesis pathway

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102547254B1 (en) * 2020-11-06 2023-06-30 주식회사 씨원바이오 Method for Preparing 2-hydroxy-r-butyrolactone Using Enzyme

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112012020299A8 (en) * 2010-02-11 2017-10-03 Metabolix Inc GAMMA-BUTYROLACTONE PRODUCTION PROCESS
ES2800341T3 (en) * 2012-07-11 2020-12-29 Adisseo France Sas Method for the preparation of 2,4-dihydroxybutyrate
US9834491B2 (en) * 2013-03-20 2017-12-05 Cj Cheiljedang Corporation Method for producing bio-based homoserine lactone and bio-based organic acid from O-acyl homoserine produced by microorganisms
CN107771214B (en) * 2015-04-07 2022-01-18 代谢探索者公司 Modified microorganisms for optimized 2,4-dihydroxybutyric acid production with increased 2,4-dihydroxybutyric acid excrements

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102021101004B3 (en) 2021-01-19 2022-03-10 Technische Universität Dresden, Körperschaft des öffentlichen Rechts Process for the production of 2,4-dihydroxybutyrate or L-threonine using a microbial pathway
CN115948482A (en) * 2023-02-07 2023-04-11 中国科学院天津工业生物技术研究所 Construction method and application of2, 4-dihydroxybutyric acid biosynthesis pathway
CN115948482B (en) * 2023-02-07 2024-02-09 中国科学院天津工业生物技术研究所 Construction method and application of 2, 4-dihydroxybutyric acid biosynthesis pathway

Also Published As

Publication number Publication date
WO2019013573A9 (en) 2019-05-02
KR20190007403A (en) 2019-01-22
KR102149044B1 (en) 2020-08-28
WO2019013573A3 (en) 2019-03-28

Similar Documents

Publication Publication Date Title
Riedel et al. Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production
US9121041B2 (en) Method for the preparation of diols
US8945888B2 (en) Method for producing high amount of glycolic acid by fermentation
JP2002511250A (en) Overexpression of pyruvate carboxylase for enhanced production of oxaloacetate-derived biochemicals in microorganisms
US10385322B2 (en) Mutant glutamate dehydrogenase for the conversion of homoserine into 4-hydroxy-2-ketobutyrate
Siedler et al. Reductive whole-cell biotransformation with Corynebacterium glutamicum: improvement of NADPH generation from glucose by a cyclized pentose phosphate pathway using pfkA and gapA deletion mutants
Vo et al. Metabolic engineering of Escherichia coli W3110 for efficient production of homoserine from glucose
JP2024026211A (en) Degradation pathway for pentose and hexose sugars
CN112481288B (en) Method for promoting corynebacterium glutamicum fermentation to produce target product
KR102149044B1 (en) Method of producing 2-hydroxy gamma butyrolactone or 2,4-dihydroxybutanoic acid
CN113710807A (en) Microorganisms and methods for producing oxygenates from hexoses
US20130210097A1 (en) Glycolic acid fermentative production with a modified microorganism
JP6177995B2 (en) Microorganism having L-tryptophan production ability and method for producing L-tryptophan using the same
Zhu et al. Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex
JP2022046736A (en) Methods and microorganisms for producing glycolic acid and/or glyoxylic acid
US20140370557A1 (en) Genetically engineered microbes and methods for producing 4-hydroxycoumarin
Wang et al. Deletion of cg1360 affects ATP synthase function and enhances production of L-valine in Corynebacterium glutamicum
KR20190097250A (en) Conversion of methylglyoxal to hydroxyacetone using a novel enzyme and its application
US11162082B2 (en) Mutant phosphoserine aminotransferase for the conversion of homoserine into 4-hydroxy-2-ketobutyrate
KR20100130564A (en) Recombinant microorganism producing taurine and method for preparing taurine using the same
KR20200026297A (en) Methionine-Producing Yeast
US10947523B2 (en) Biotechnological production of L-tryptophan
US9562224B2 (en) Reduced activity of ubiCA in E. coli
WO2024013212A1 (en) Microbial cell factories producing thiamine
JP2003284579A (en) Method of producing 2-ketobutyric acid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18832195

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18832195

Country of ref document: EP

Kind code of ref document: A2