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WO2019013573A9 - Procédé de préparation de 2-hydroxy-gamma-butyrolactone ou de 2,4-dihydroxy-butyrate - Google Patents

Procédé de préparation de 2-hydroxy-gamma-butyrolactone ou de 2,4-dihydroxy-butyrate

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Publication number
WO2019013573A9
WO2019013573A9 PCT/KR2018/007923 KR2018007923W WO2019013573A9 WO 2019013573 A9 WO2019013573 A9 WO 2019013573A9 KR 2018007923 W KR2018007923 W KR 2018007923W WO 2019013573 A9 WO2019013573 A9 WO 2019013573A9
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WIPO (PCT)
Prior art keywords
enzyme
seq
amino acid
acid sequence
hydroxy
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Korean (ko)
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WO2019013573A2 (fr
WO2019013573A3 (fr
Inventor
박성훈
김용환
이성국
심증엽
유태현
수만라마
응웬하이남
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UNIST Academy Industry Research Corp
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UNIST Academy Industry Research Corp
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Publication of WO2019013573A9 publication Critical patent/WO2019013573A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01046—Homoserine O-succinyltransferase (2.3.1.46)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01039—Homoserine kinase (2.7.1.39)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02004—Aspartate kinase (2.7.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/08—Phosphoric triester hydrolases (3.1.8)
    • C12Y301/08001—Aryldialkylphosphatase (3.1.8.1), i.e. paraoxonase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/0102—Diaminopimelate decarboxylase (4.1.1.20)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01031—Phosphoenolpyruvate carboxylase (4.1.1.31)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01001—Acetate-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 by the method of efficiently producing homoserine from sugar is provided.
  • the strain may be a homoserine and a producing strain and a homoserine producing strain.
  • microorganism for producing the mutant strain at least one microorganism selected from the group consisting of E. coli, yeast, Corynebacterium, etc. may be used.
  • 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 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)
  • DLB 2,4-dihydroxy-butyrate
  • the primer sequences usable at this time are shown in Table 18. < tb >< TABLE >
  • the 4 step may be to remove all of the five genes kgtP, dsdx, and aci.
  • 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 microbial mutants 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 can be selected depending on the intended use of the compound.
  • D-hydroxy-2-oxo-reductase 4-dihydroxy- Hydroxy-2-oxo-reductase
  • 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,
  • an enzyme consisting of the amino acid sequence of SEQ ID NO: 21 preferably an enzyme consisting of the amino acid sequence of SEQ ID NO: 20, and / or an enzyme comprising the amino acid sequence of SEQ ID NO: An enzyme comprising an amino acid sequence.
  • 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-hydroxygalactybutyrolactone or 2,4-dihydroxybutyrate (2-hydroxybutyrolactone), including the above- , 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.
  • trans-amino tyrosinase mutant enzyme consisting of 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 strain which overexpresses 2-hydroxy gamma butyrolactone (HGBL) and its precursor, 2,4-dihydroxybutanoic acid, 2-hydroxy gamma butyrolactone (HGBL) and its precursor 2,4 - dihydroxybutanoic acid.
  • 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.
  • Figure 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 can be catalyzed by homoserine ine deaminase or homoserine transaminase enzyme, and the homoserine diamine catalyzes the reaction.
  • 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
  • ⁇ -KG ( ⁇ -KG / glutamic acid pair is most widely used for this purpose in the transaminase reaction)
  • ⁇ -KG which is an amino acceptor from glutamic acid produced from ⁇ -KG, is regenerated and homoser it is desirable to use aspartate transaminase for the production of aspartic acid, a biosynthetic precursor.
  • 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).
  • 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 approximately 5 minutes and then 10 mM pyruvate was added to initiate the reaction. After 10 minutes of reaction, 12 mM perchloric acid was added to stop the reaction, followed by centrifugation at 10,000 g for 5 minutes.
  • 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. The gene sequences of these mutants were examined and a total of five mutants were obtained and designated as TA4-1 to TA4-5, respectively.
  • 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.
  • 0HB reductase belongs to 2-hydroxy acid dehydrogenase, which includes lactate dehydrogenase (EC1.1.1.27, EC1.1.1.28), malate dehydrogenase (EC1.1.1.37, EC1.1.12.2, EC1.1.299) and branched chain (D), and (L) -2-hydroxyacid dehydrogenase (ECl.ll 272, EC1.1.1.345).
  • L-form isomer In order to produce L-form isomer, it is expected to be highly active against 0HB from Alcaligenes eutrophus H16, Cupriavidus basi lensis, Achromobacter xylosoxidans, Burkholderiaglumae, Escherichia fergusonii, Escherichia coli, Lactobacillus llus ali and Escherichia coli K-12 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 the expression of the enzyme, and E. coli strain DH5a was used as a host for cloning and plasmid preservation.
  • 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. Cultivation was carried out at 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 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 plasmid pG_Tf2 which expresses Chaperon, was further introduced.
  • recombinant BL21 (pET-Xx-LDH) strain was aerobically cultured in LB medium containing 50 Ug / mL kanamycin.
  • the culture temperature is 30 2 C
  • stirring speed was put in a 250 mL medium was 100 rpm in 1 L flask.
  • 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, 0.5 M NaCl, 10 mM imidazole) and soni- Lt; / RTI > The cell lysate was centrifuged at 25,000 g for 30 minutes at 4 ° C for 30 min to remove the non-soluble portion, and the soluble portion was collected for enzyme activity analysis or additional enzyme separation.
  • 25 mM phosphate buffer solution pH 7.0
  • binding buffer (20 mM pH 7.0 phosphate buffer, 0.5 M NaCl, 10 mM imidazole) and soni- Lt; / RTI &gt
  • the cell lysate was centrifuged at 25,000 g for 30 minutes at 4 ° C for 30 min to remove the non-soluble portion, and the soluble portion was collected 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 ⁇ / 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
  • 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.
  • Biological half-life experiments were carried out 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 actonase 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 signal sequence was attached to the N-terminus to be expressed in the periplasmic space.
  • the ponl (G3C9) gene was synthesized and then PCR amplified (FP, agacatat at ggc t aaac t gacagcg, RP, t acat ac t cgagt t acagct cacagt aaagagc 111 g) Jl Cloning into pACYC- Duet plasmid Respectively.
  • the tac promoter was used for the expression of ponl (G3C9) (Fig. 18). This plasmid was named pACYC_Ponl.
  • the obtained Ponl plasmids were introduced into the strains for producing the two strains, L-DHB and D-DHB, respectively. In this case, it is not preferable to increase the extracellular transport rate of DHB. Therefore, EcW16 was used as a host cell (Table 19), 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 remarkably 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.

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Abstract

La présente invention concerne : une voie de biosynthèse nouvellement démontrée utilisant la production d'homosérine à titre d'étape intermédiaire dans la préparation de 2-hydroxy-gamma-butyrol-acétone (HGBL) et d'un précurseur de celle-ci, l'acide 2,4-dihydroxybutanoïque; et une souche recombinée de gène comprenant ladite voie. Une HGBL ayant un groupe hydroxyle substitué en position 2 peut être utilisée comme intermédiaire important, pouvant être employé comme résine pour photoréserve, matière première pour produit médical, et matériau pour revêtir une surface métallique.
PCT/KR2018/007923 2017-07-12 2018-07-12 Procédé de préparation de 2-hydroxy-gamma-butyrolactone ou de 2,4-dihydroxy-butyrate Ceased WO2019013573A2 (fr)

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