WO2013085361A2 - Microorganisme mutant présentant une grande capacité de production d'acide 4-hydroxybutyrique et procédé de préparation d'acide 4-hydroxybutyrique l'utilisant - Google Patents

Microorganisme mutant présentant une grande capacité de production d'acide 4-hydroxybutyrique et procédé de préparation d'acide 4-hydroxybutyrique l'utilisant Download PDF

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WO2013085361A2
WO2013085361A2 PCT/KR2012/010665 KR2012010665W WO2013085361A2 WO 2013085361 A2 WO2013085361 A2 WO 2013085361A2 KR 2012010665 W KR2012010665 W KR 2012010665W WO 2013085361 A2 WO2013085361 A2 WO 2013085361A2
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gene
hydroxybutyric acid
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이상엽
최솔
이정욱
박시재
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한국과학기술원
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids

Definitions

  • the present invention relates to a mutant microorganism having a high performance of 4-hydroxybutyric acid and a method for producing 4-hydroxybutyric acid using the same, and more particularly, to increase the production capacity of 4-hydroxybutyric acid from 4 succinic acid.
  • -A mutant microorganism having a high performance of 4-hydroxybutyric acid in which a gene for synthesizing hydroxybutyric acid is amplified, an amplification of a supplemental pathway gene, and competition genes are deleted, and a microaerobic -aerobic) to the 4-hydroxybutyric acid is produced in high yield by culturing under conditions.
  • 4-Hydroxybutyric acid can be used as a precursor of important C4 compounds such as 1,4-butanediol or gamma-butyrolactone biologically and also various polyhydroxyal It is used as a monomer of the polyhydroxyalkanoate.
  • Sodium 4-hydroxybutyric acid is a drug called Xyrem that is used in patients with narcolepsy. It is currently sold for about 18 kilograms per 1g.
  • 4-hydroxybutyric acid production through microorganisms is limited only to polymers and does not produce 4-hydroxybutyric acid alone.
  • 4-hydroxybutyric acid is produced in the form of a polymer
  • 1,4-butanediol and ⁇ -butyrolactone are biologically produced
  • the polymer has to be broken down into monomers. Therefore, by developing a strain that produces 4-hydroxybutyric acid alone rather than a polymer, and additionally introducing enzymes necessary for 1,4-butanediol and ⁇ -butylolactone, 1,4-butanediol and ⁇ - Butylolactone monomers can be easily produced and research is being conducted.
  • the present inventors have made efforts to solve the above problems, and as a result, in the microorganism producing 4-hydroxybutyric acid, amplification of the gene involved in the synthesis of 4-hydroxybutyric acid from succinic acid, and further 4-hydroxy Amplifys the complementary pathway genes involved in the production of succinic acid, the precursor of butyric acid, attenuates or deletes genes involved in the competitive pathway of 4-hydroxybutyric acid production, and participates in the oxidative pathway When the attenuated genes are attenuated or deleted, or mutated and amplified, it was confirmed that the mutant microorganisms having 4-hydroxybutyric acid high performance could be prepared and completed the present invention.
  • An object of the present invention is to provide a mutant microorganism having high performance of 4-hydroxybutyric acid and a method for producing the mutant microorganism.
  • Another object of the present invention is to provide a method for producing 4-hydroxybutyric acid in high yield by culturing the mutant microorganisms in micro-aerobic conditions.
  • the present invention is a gene encoding the CoA transferase (CoA transferase) involved in the biosynthesis of 4-hydroxybutyric acid ( cat1 ), succinyl-CoA synthetase Gene encoding su ) ( sucCD ), gene encoding CoA-dependent succinate semialdehyde dehydrogenase ( sucD ), and 4-hydroxybutyric acid dehydrogenase (4-hydroxybutyrate) It provides a mutant microorganism having 4-hydroxybutyric acid generating ability and a method for producing the mutant microorganism, characterized in that at least one selected from the group consisting of genes ( 4hbD or yqhD ) encoding dehydrogenase is amplified.
  • the mutant microorganism is a gene encoding a phosphoenolpyruvate carboxykinase ( pckA ), phosphoenolpyruvate carboxylase (phosphoenolppyruvate carboxylase) involved in the anaplerotic pathway (phosphoenolpyrutave carboxylase) )
  • pckA phosphoenolpyruvate carboxykinase
  • phosphoenolpyruvate carboxylase phosphoenolppyruvate carboxylase
  • pyc phosphoenolppyruvate carboxylase
  • the mutant microorganism is a gene ( gltA ) encoding a citrate synthase mutated to remove (i) NADH inhibition of genes involved in the oxidative pathway (addition) Amplified by; (ii) the gene encoding the aerobic respiration control protein ( arcA ) is further attenuated or deleted; Or (iii) a gene that encodes a mutated citrate synthase ( gltA ) is further amplified so that inhibition by NADH is eliminated and at the same time a gene encoding an aerobic respiration control protein ( arcA ) is further weakened or deleted.
  • the mutant microorganism is a gene encoding a succinate semialdehyde dehydrogenase ( gabD or yneI ) involved in the production competition pathway of 4-hydroxybutyric acid ( gabD or yneI ), lactic acid dehydrogenase ( the gene encoding lactate dehydrogenase ( ldhA ), the gene encoding pyruvate formate lyase ( pflB ), the gene encoding alcohol dehydrogenase ( adhE ) and the PTS system.
  • At least one selected from the group consisting of genes ( ptsI or ptsG ) encoding PEP-protein phosphotransferase of PTS system is further weakened or deleted.
  • the mutant microorganism is characterized in that the gene encoding the lac operon inhibitor ( lacI ) is further deleted so that the expression of the gene encoding the enzyme involved in 4-hydroxybutyric acid biosynthesis is increased. .
  • the gene encoding the CoA-dependent succinate semialdehyde dehydrogenase ( sucD ) is characterized in that it is introduced in the form of an expression vector containing a strong promoter.
  • the gene ( ppc ) and the gene encoding pyruvate carboxylase (pyruvate carboxylase) ( pyc ) is characterized in that it was introduced in the form of an expression vector containing a strong promoter.
  • the strong promoter is selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter and trp promoter.
  • the microorganism is the genus Bacillus ( Bacillus sp.), Corynebacterium sp. ( Coynebacterium sp.), Escherichia sp. ( Esherichia sp.), Pichia sp., Pseudomonas Genus ( Pseudomonas sp.) And Saccharomyces sp.
  • the present invention also provides (A) a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid, (B) 4-hydroxy Genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), genes encoding lactate dehydrogenase ( ldhA ), blood involved in the production pathway of oxybutyric acid Genes encoding pyruvate formate lyase ( pflB ), genes encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransferase (PEP-protein) of PTS system phosphotransferase system PTS of) a gene and (ptsI or ptsG) has been inactivated or deleted encoding, (C) supplementary circuit (phospho yen acid carboxy key olpi Rubik involved in anaplerotic pathway) better gene (pckA) en
  • the present invention also provides (A) a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid, (B) 4-hydroxy Genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), genes encoding lactate dehydrogenase ( ldhA ), blood involved in the production pathway of oxybutyric acid Genes encoding pyruvate formate lyase ( pflB ), genes encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransferase (PEP-protein) of PTS system
  • the gene encoding the phosphotransferase of PTS system ( ptsI or ptsG ) is weakened or deleted, and (C) NADH inhibition of genes involved in the oxidative pathway is eliminated.
  • Amplifying the gene ( gltA ) encoding a mutated citrate synthase preferably; (ii) the gene encoding the aerobic respiration control protein ( arcA ) is weakened or deleted; Or (iii) the gene encoding the mutated citrate synthase ( gltA ) is amplified so that inhibition by NADH is eliminated and at the same time the gene encoding the aerobic respiration control protein ( arca ) It provides a mutant microorganism having a 4-hydroxybutyric acid producing ability, and a method for producing the mutant microorganism, characterized in that the weakened or deleted.
  • the present invention also provides (A) a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid, (B) 4-hydroxy Genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), genes encoding lactate dehydrogenase ( ldhA ), blood involved in the production pathway of oxybutyric acid Genes encoding pyruvate formate lyase ( pflB ), genes encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransferase (PEP-protein) of PTS system phosphotransferase of PTS system ( ptsI or ptsG ) encoding a weakened or deleted, and (C) phosphoenolpyruvic acid carboxykinase involved in the anaplerotic pathway ( gene encoding phosphoenolpyruvate carboxykin
  • the present invention also provides a method for producing 4-hydroxybutyric acid, wherein the mutant microorganism is cultured to produce 4-hydroxybutyric acid, and then 4-hydroxybutyric acid is recovered.
  • 1 is a schematic diagram showing a synthetic route of 4-hydroxybutyric acid from glucose.
  • the term "attenuation” refers to a concept encompassing a mutation, substitution or deletion of some bases of a gene or introduction of some bases to reduce the activity of an enzyme expressed by the gene of interest. It includes everything that blocks some or much of the biosynthetic pathway.
  • the term 'deletion' is a concept encompassing a part or whole base of the gene, mutating, substituting or deleting, or introducing some base so that the gene is not expressed or does not exhibit enzymatic activity even when expressed. It includes everything that blocks the biosynthetic pathways involved in the enzymes of the gene.
  • amplification means that some bases of the gene are mutated, substituted or deleted, introduced into some bases, or introduced into a gene from another microorganism encoding the same enzyme to increase the activity of the corresponding enzyme. It is a concept that encompasses letting.
  • FIG. 1 is a schematic diagram showing a synthetic route of 4-hydroxybutyric acid from glucose. As shown in Figure 1, to amplify the gene ( cat1 , sucCD , sucD , yqhD or 4hbD ) gene involved in the synthesis of 4-hydroxybutyric acid to increase the ability to produce 4-hydroxybutyric acid It was.
  • sucD derived from Clostridium kluyveri is introduced because there is no gene (sucD) encoding CoA-dependent succinate semialdehyde dehydrogenase. It was. As a result, it was confirmed that wild-type E. coli does not produce 4-hydroxybutyric acid, but that the mutant microorganism into which sucD was introduced produces 4-hydroxybutyric acid.
  • the gene encoding the coei-transferase from C. kluyveri ( cat1 ) and the gene encoding the succinyl- coei synthase ( sucCD ) from E. coli are sucD and 4hbD , respectively. Amplified as As a result, the production of 4-hydroxybutyric acid was increased in both cases than when amplifying only sucD and 4hbD . These results demonstrate that cat1 and yqhD are effective on 4-hydroxybutyric acid.
  • gene ( cat1 ) succinyl-CoA synthetase coding for CoA transferase (CoA transferase) involved in the biosynthesis of 4-hydroxybutyric acid Gene ( sucCD ), gene encoding CoA-dependent succinate semialdehyde dehydrogenase ( sucD ) and 4-hydroxybutyrate dehydrogenase (4-hydroxybutyrate dehydrogenase)
  • sucCD 4-hydroxybutyric acid Gene
  • sucD CoA-dependent succinate semialdehyde dehydrogenase
  • 4-hydroxybutyrate dehydrogenase 4-hydroxybutyrate dehydrogenase
  • 4-hydroxybutyric acid is attenuated or deleted when the genes involved in the competition pathway of 4-hydroxybutyric acid production in a mutant microorganism in which the gene involved in the biosynthesis of 4-hydroxybutyric acid is amplified. It was predicted that the ability to produce can be increased.
  • a gene ( ldhA) encoding lactate dehydrogenase ( ldhA) is deleted in a strain from which the gabD and yneI genes are deleted, and a gene involved in the synthesis of 4-hydroxybutyric acid ( sucCD , sucD or yqhD ) genes were amplified.
  • sucCD , sucD or yqhD 4-hydroxybutyric acid
  • a gene encoding an alcohol dehydrogenase ( adhE ) is deleted in a strain deleted gabD, yneI and ldhA genes, and is involved in the synthesis of 4-hydroxybutyric acid. Mutant microorganisms amplifying the gene ( sucCD , sucD or yqhD ) gene were prepared. As a result, it was confirmed that 4-hydroxybutyric acid production ability was improved compared to the strain that did not delete the gene encoding acohol dehydrogenase ( adhE ).
  • the gene coding for pyruvate formate lyase ( pflB) is deleted in a strain that is gabD, yneI, ldhA and adhE deleted, and 4-hydroxybutyric acid is synthesized.
  • a mutant microorganism was prepared by amplifying a gene ( sucCD , sucD or yqhD ) gene. As a result, it was confirmed that 4-hydroxybutyric acid production ability was improved compared to the strain that did not delete the gene ( pflB) encoding pyruvate formate lyase.
  • the gene involved in the synthesis of the 4-hydroxybutyric acid is amplified microorganism or gene involved in the synthesis of the 4-hydroxybutyric acid ( mutant microorganisms with amplified genes ( pckA , ppc or pyc ) involved in the cationic pathway ( cat1 , sucCD , sucD , yqhD or 4hbD ) and the complementary pathway (succinic acid semi Coding a gene encoding aldehyde dehydrogenase ( gabD or yneI ), gene encoding lactic acid dehydrogenase ( ldhA ), pyruvate formate lyase Gene ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransferase (PEP) of PTS system at least one selected from the group
  • genes involved in the biosynthesis of 4-hydroxybutyric acid are amplified, and genes involved in the competition pathway of 4-hydroxybutyric acid production It was predicted that additional amplification of genes involved in the anaplerotic pathway ( pckA, ppc or pyc ) in attenuated or deleted mutant microorganisms could increase the ability to produce 4-hydroxybutyric acid.
  • the present invention amplifies the gene ( sucCD) encoding the succinyl-CoA synthetase involved in the synthesis of the aforementioned 4-hydroxybutyric acid (succinyl-CoA synthetase), succinic acid semialdehyde dehydro Gene encoding succinate semialdehyde dehydrogenase ( gabD , yneI ), gene encoding lactate dehydrogenase ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), the gene encoding alcohol dehydrogenase ( adhE ) and the gene encoding PEP-protein phosphotransferase of PTS system ( ptsI or ptsG ) gene encoding a phosphonate yen olpi Rubik acid carboxy-kinase (phosphoenolpyruvate carboxykinase) in strains having deletions of one
  • the mutant microorganism in which the gene ( cat1 , sucCD , sucD , yqhD or 4hbD ) involved in the synthesis of the 4-hydroxybutyric acid is amplified is phosphoenolpyruvic involved in the complementary pathway.
  • the gene coding for the CoA-dependent succinate semialdehyde dehydrogenase ( sucD ) is introduced in the form of an expression vector containing a strong promoter, and the supplementary circuit (anaplerotic) gene coding for phosphoenolpyruvate carboxykinase ( pckA ), gene coding for phosphoenolpyrutave carboxylase ( ppc ) and pyruvic acid carba
  • the gene encoding pyruvate carboxylase ( pyc ) is characterized in that it is introduced in the form of an expression vector containing a strong promoter.
  • the strong promoter may exemplify a trc promoter, a tac promoter, a T7 promoter, a lac promoter, a trp promoter, and the like.
  • genes involved in the biosynthesis of 4-hydroxybutyric acid are amplified, and genes involved in the competition pathway of 4-hydroxybutyric acid production Attenuated or deleted mutant microorganisms or genes involved in the biosynthesis of 4-hydroxybutyric acid are amplified, genes involved in the competition pathway of 4-hydroxybutyric acid production are attenuated or deleted, and an anaplerotic Citrate synthase mutated to remove NADH inhibition among mutant microorganisms in which the gene involved in the pathway ( pckA, ppc or pyc ) is amplified. Further amplifies the gene encoding gltA or encodes an aerobic respiration control protein ( a It was predicted that further weakening or deletion of rcA could increase the ability of 4-hydroxybutyric acid.
  • -Genes involved in the biosynthesis of hydroxybutyric acid have been amplified, genes involved in the competitive pathway of 4-hydroxybutyric acid production are weakened or deleted, and genes involved in the anaplerotic pathway ( pckA,
  • CS16 / p99SC4CD / p15PpcGltAR163L was prepared by amplification, which further involved in the competitive pathway of 4-hydroxybutyric acid production. It was confirmed that the gene (arcA) is prepared the deletion of CS28 / p99SC4CD / p15PpcGltAR163L, increase their 4-hydroxy butyric acid producing ability of the hydroxy.
  • a mutant microorganism or 4-hydroxybuty is a gene in which the gene involved in the biosynthesis of 4-hydroxybutyric acid of the present invention is amplified and the genes involved in the competition pathway of 4-hydroxybutyric acid production is weakened or deleted. Genes involved in the biosynthesis of lactic acid are amplified, genes involved in the competitive pathway of 4-hydroxybutyric acid production are weakened or deleted, and genes involved in the anaplerotic pathway ( pckA, ppc or pyc ).
  • the mutant microorganism, which is amplified is further amplified by (i) a gene encoding a mutated citrate synthase ( gltA ) among the genes involved in the oxidative pathway to remove the inhibition by NADH.
  • the gene encoding the aerobic respiration control protein ( arcA ) is further attenuated or deleted; Or (iii) a gene that encodes a mutated citrate synthase ( gltA ) is further amplified so that inhibition by NADH is eliminated and at the same time a gene encoding an aerobic respiration control protein ( arcA ) is further weakened or deleted.
  • the mutated citrate synthase is for improving the ability to produce 4-hydroxybutyric acid, and the position or type of mutation is not particularly limited as long as the inhibition by NADH is eliminated.
  • the microorganism may be used without limitation as long as it is a microorganism that generates 4-hydroxybutyric acid from glucose, Bacillus sp., Corynebacterium sp., Escherichia sp. ( Escherichia sp.), Pichia sp., Pseudomonas sp., Saccharomyces sp.
  • the present invention provides a method for producing 4-hydroxybutyric acid, wherein the mutant microorganism is cultured to produce 4-hydroxybutyric acid, and then 4-hydroxybutyric acid is recovered from the culture solution. It is about.
  • the culturing of the mutant microorganism and the process of obtaining 4-hydroxybutyric acid use a culture method (batch culture, fed-batch culture) and the separation and purification method of 4-hydroxybutyric acid commonly known in the conventional fermentation process. Can be done.
  • the biotechnological production of 4-hydroxybutyric acid can be carried out intracellularly or extracellularly ( in vivo or in vitro ).
  • the gene sucD which encodes CoA-dependent succinate semialdehyde dehydrogenase of constitutive and biosynthetic Clostridium kluyver i (DSM 555), was cloned into pTrc99a (Pharmacia Biotech, Uppsala, Sweden).
  • pTrc99a Pharmacia Biotech, Uppsala, Sweden.
  • PCR conditions are as follows. step 1: 95 ° C, 2 min; step 2: 95 ° C., 20 sec; step 3: 55 ° C., 30 sec; step 4: 72 ° C., 1 min; step 2 to step 4 repeat 28 times; step 5: 72 ° C., 7 min; step 6: Maintain 4 ° C.
  • sucD fragment and pTrc99a plasmid were treated with restriction enzymes ( Eco RI and Sac I), followed by treatment with T4 DNA ligase to polymerize the sucD fragment and pTrc99a plasmid digested with the restriction enzyme, thereby recombining the vector p99SucD as a recombinant plasmid.
  • restriction enzymes Eco RI and Sac I
  • T4 DNA ligase to polymerize the sucD fragment and pTrc99a plasmid digested with the restriction enzyme, thereby recombining the vector p99SucD as a recombinant plasmid.
  • E. coli W3110 ATCC 39936 (derived from E. coli K-12, ⁇ ⁇ , F ⁇ , prototrophic) is described in 1-1 above. Cloned to the prepared p99SucD. Expression is affected by the trc promoter in front of the sucD gene by the ribosomal binding site (RBS) in front of the yqhD gene.
  • the genomic DNA of E. coli W3110 (ATCC39936) (derived from E. coli K-12, ⁇ ⁇ , F ⁇ , prototrophic) was used as a template, and the primers of SEQ ID NOs: 3 and 4 were used. By performing PCR under the same conditions, yqhD fragments were prepared.
  • the prepared yqhD fragment and p99SucD plasmid were treated with restriction enzymes ( Sac I and Xba I), followed by treatment with T4 DNA ligase to polymerize the restriction enzyme cleaved yqhD fragment and p99SucD plasmid, a vector p99SYn as a recombinant plasmid was produced.
  • the gene 4hbD encoding 4-hydroxybutyrate dehydrogenase of the constitutive and biosynthetic Clostridium kluyveri was cloned into p99SucD prepared in 1-1 above. Expression is affected by the trc promoter in front of the sucD gene by the ribosomal binding site (RBS) in front of the 4hbD gene.
  • RBS ribosomal binding site
  • the prepared 4hbD fragment and the p99SucD plasmid were treated with restriction enzymes ( Sac I and Xba I), followed by treatment with T4 DNA ligase to polymerize the 4hbD fragment and the p99SucD plasmid digested with the restriction enzyme to obtain a recombinant plasmid vector p99SC4.
  • restriction enzymes Sac I and Xba I
  • T4 DNA ligase to polymerize the 4hbD fragment and the p99SucD plasmid digested with the restriction enzyme to obtain a recombinant plasmid vector p99SC4.
  • the gene cat1 encoding CoA transferase of constitutive and biosynthetic Clostridium kluyveri was cloned into p99SC4 prepared in 1-3. Expression is affected by the trc promoter in front of the SucD gene by the ribosomal binding site (RBS) in front of the cat1 gene.
  • RBS ribosomal binding site
  • the cat1 fragment and the p99SC4 plasmid were treated with restriction enzymes ( Xba I and Sbf I), followed by treatment with T4 DNA ligase to polymerize the cat1 fragment and p99SC4 plasmid digested with the restriction enzyme, thereby yielding the vector p99SC4C1 as a recombinant plasmid.
  • restriction enzymes Xba I and Sbf I
  • T4 DNA ligase to polymerize the cat1 fragment and p99SC4 plasmid digested with the restriction enzyme, thereby yielding the vector p99SC4C1 as a recombinant plasmid.
  • E. coli W3110 (ATCC 39936) (derived from E.coli K-12, ⁇ -, F -, prototrophic) of succinyl-CoA synthase beta subunit gene (sucC) and succinyl-CoA synthase encoding the
  • the gene encoding the alpha subunit ( sucD ) was cloned into p99SC4 prepared in 1-3.
  • sucC and sucD are operons that are regulated by a promoter in the genome and attached together.
  • the two genes each encode a portion of the succinyl-CoA synthase and the expressed proteins combine to function as succinyl-CoA synthase.
  • sucCD succinyl-CoA synthase beta subunit and the succinyl-CoA synthase alpha subunit are combined to be called succinyl-CoA synthase.
  • Expression is affected by the trc promoter in front of the sucD gene by the ribosomal binding site (RBS) in front of the sucCD gene.
  • RBS ribosomal binding site
  • the genomic DNA of E. coli W3110 (ATCC39936) (derived from E. coli K-12, ⁇ ⁇ , F ⁇ , prototrophic) was used as a template, and the primers 1-1 and 10 were synthesized using the primers of SEQ ID NOs. By performing PCR under the same conditions, sucCD fragments were prepared.
  • sucCD fragment and p99SC4 plasmid were treated with restriction enzymes ( Xba I and Sbf I) and then treated with T4 DNA ligase to polymerize the sucCD fragment and p99SC4 plasmid digested with the restriction enzyme, thereby recombining the vector p99SC4CD as a recombinant plasmid.
  • restriction enzymes Xba I and Sbf I
  • T4 DNA ligase to polymerize the sucCD fragment and p99SC4 plasmid digested with the restriction enzyme, thereby recombining the vector p99SC4CD as a recombinant plasmid.
  • a succinyl-CoA synthase coding gene ( sucCD ) of constitutive and biosynthetic E. coli W3110 (ATCC 39936) (derived from E. coli K-12, ⁇ ⁇ , F ⁇ , prototrophic ) was prepared in 1-2. Cloned into p99SYn. Expression is affected by the trc promoter in front of the sucD gene by the ribosomal binding site (RBS) in front of the sucCD gene.
  • Genomic DNA of E. coli W3110 (ATCC39936) (derived from E. coli K-12, ⁇ ⁇ , F ⁇ , prototrophic) was used as a template, and the primers of the synthesized SEQ ID NOs. 9 and 10 prepared in 1-5 above were used. By using the PCR under the same conditions as in the above 1-1, sucCD fragments were prepared.
  • sucCD fragment and p99SYn plasmid were treated with restriction enzymes ( Xba I and Sbf I), and then treated with T4 DNA ligase to polymerize the sucCD fragment and p99SYn plasmid digested with the restriction enzyme, thereby recombining the vector p99SYnCD as a recombinant plasmid.
  • the present inventors introduced the p99SucD vector prepared in 1-1 to the WL3110 strain (W3110 ⁇ lacI ) prepared by the method disclosed in Korean Patent Publication No. 2009-0018781 to prepare a WL3110 / p99SucD strain. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the WL3110 strain (W3110 ⁇ lacI ) can be prepared by the following method.
  • plasmid pECmulox as a template by performing the PCR in the same conditions as 1-1, to prepare a PCR product in which the lacI gene was deleted.
  • the purified PCR product was purified, and then electroporated to E. coli (W3110) containing ⁇ recombinase to prepare WL3110 (W3110 ⁇ lacI ).
  • the WL3110 / p99SucD strain was prepared by introducing the p99SYn vector prepared in 1-2 into the WL3110 strain (W3110 ⁇ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the WL3110 / p99SC4 strain was prepared by introducing the p99SC4 vector prepared in 1-3 into the WL3110 strain (W3110 ⁇ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the WL3110 / p99SC4C1 strain was prepared by introducing the p99SC4C1 vector prepared in 1-4 into the WL3110 strain (W3110 ⁇ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the WL3110 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the WL3110 strain (W3110 ⁇ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the WL3110 / p99SYnCD strain was prepared by introducing the p99SYnCD vector prepared in 1-6 into the WL3110 strain (W3110 ⁇ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • Example 2 Preparation of 4-hydroxybutyric acid using mutant microorganisms amplified with 4-hydroxybutyric acid producing genes
  • the mutant microorganisms of Table 1 prepared in Example 1 were prepared using 4 g / L (NH 4 ) 2 HP 4 , 6.67 g / LKH 2 PO 4 , 0.8 g / Lcitricacid, 0.8 g / LMgSO 4 .7H 2 O, 0.5% (v / v) Flask culture in a minimum MR medium (Jung, YK, Kim, TY, Park, SJ, and Lee, SY, Biotechnol. Bioeng . , 105 (1): 161-171,2010) consisting of tracemetal solutions.
  • a minimum MR medium Jung, YK, Kim, TY, Park, SJ, and Lee, SY, Biotechnol. Bioeng . , 105 (1): 161-171,2010
  • Trace metal solution contains 5M HCl per liter: 10g FeSO 4 ⁇ 7H 2 O, 2.25gZnSO 4 ⁇ 7H 2 O, 1gCuSO 4 ⁇ 5H 2 O, 0.5gMnSO 4 ⁇ 5H 2 O, 0.23gNa 2 B 4 O 7 ⁇ 10H 2 O, 2gCaCl 2 .2H 2 O and 0.1g (NH 4 ) 6 Mo 7 O 24 .
  • Glucose (100 g / l) stock solution and NaHCO 3 (60 g / l) were sterilized separately and added to sterile media to final concentrations of 10 g / l and 6 g / l, respectively.
  • the cells were then separated by centrifugation of the culture medium, and the dissociated supernatants were packed in a packed column (Supelco Carbopack TM BAW / 6.6% PEG20M, 2m ⁇ 2mm ID, Bellefonte, PA, USA) for 4-hydroxybutyric acid analysis. ) was measured by gas chromatography (Agillent 6890N GC System, Agilent Technologies Inc., CA, USA), and the results are shown in Table 1.
  • Table 1 shows that all of the mutant microorganisms amplified with the sucD, yqhD, 4hbD, cat1 and sucCD genes improved the ability to produce 4-hydroxybutyric acid based on glucose.
  • Fragments for deletion include (1) homologous sequences of genes for deletion, (2) lox71-chloramphenicol marker (CmR) -lox66, and (3) homologous sequences for genes for deletion.
  • (1) to (3) are in the 5 ' ⁇ 3' direction, and (1) and (3) must be different sequences, and the sequence of (1) is to the left or 5 'direction than the sequence of (3).
  • (2) may be prepared by PCR of pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85, 2008) comprising the lox71-CmR-lox66 cassette. .
  • PCR products obtained by PCR with primers SEQ ID NOs: 13 and 14 were cut with restriction enzymes HindIII and SmaI and pUG6 (Guldener U, Heck S, Fielder T, Beinhauer J & PECmulox can be prepared by Ligation of Hegemann JH, Nucleic Acids Res , 24: 2519-2524,1996; NCBI GenBank: AF298793.1) with DNA products cut with restriction enzymes HindIII and EcoRV.
  • fragments for deletion can be completed. Also, in order to enhance double-cross homologous recombination with genes, that is, to increase the number of homologous sequences of genes to be deleted, a fragment for the deletion is used as a template, and additional homologous sequences are added to the primers. Extended PCR fragments can be obtained.
  • Another method is to perform PCR using (1), (2), and (3) using separate primers, and then perform overlapping PCR using the Sense primer (1) and the Antisense primer (3). PCR products can be obtained.
  • the advantage of this method is that PCR is performed separately for (1) and (3), so that the number of homologous sequences of genes can be increased as desired.
  • the PCR products were transformed into electrocompetent cells containing ⁇ recombinase.
  • Colonies were Luria-Bertani (LB) agar (Sambrook, J., Fritsch EF, & Maniatis, T., Molecular cloning: a laboratory manual, 3rd edition, Cold Spring) containing chloroamphenicol (Cm; 34 ⁇ g / ml) Harbor Laboratory Press, 2000) plate.
  • Successful gene replacement of Cm R was confirmed by direct colony PCR.
  • the antibiotic marker was subsequently removed by helper plasmid pJW168 (Lucigen, USA), including temperature-sensitive replication origin and IPTG-inducible cre recombinase.
  • plasmid pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85, 2008) as a template under the same conditions as in 1-1 above
  • a PCR product was prepared in which the gene ( ldhA ) gene encoding lactate dehydrogenase was deleted.
  • the purified PCR product was purified, and then electroporated to the CS03 strain prepared in 3-2 to prepare a CS05 strain (W3110 ⁇ lacI ⁇ gabD ⁇ yne I ⁇ ldhA ).
  • plasmid pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85,2008) as a template PCR was performed to prepare a PCR product in which the gene encoding alcohol dehydrogenase ( adhE ) was deleted.
  • the purified PCR product was purified and then electroporated to the CS05 strain prepared in 3-3 to prepare a CS06 strain (W3110 ⁇ lacI ⁇ gabD ⁇ yne I ⁇ ldhA ⁇ adhE ).
  • PCR product was prepared in which the gene ( pflB ) encoding pyruvate formate lyase was deleted. Thereafter, the PCR product was used as a template to enhance double-cross homologous recombination, and PCR was performed under the same conditions as in 1-1 using primers of SEQ ID NOs: 25 and 26, thereby increasing the number of homologous sequences with the pflB gene. PCR products were prepared.
  • the purified PCR product was purified and then electroporated to the CS06 strain prepared in 3-4 to prepare a CS07 strain (W3110 ⁇ lacI ⁇ gabD ⁇ yne I ⁇ ldhA ⁇ adhE ⁇ pflB ).
  • E. coli W3110 (ATCC 39936) (derived from E. coli K-12, ⁇ -, F -, genomic DNA of prototrophic, plasmid pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85, 2008), E. coli W3110 (ATCC 39936) (derived from E.
  • PCR was carried out using genomic DNA of coli K-12, ⁇ ⁇ , F ⁇ , prototrophic) under the same conditions as in 1-1 above to obtain PCR fragments, and three PCR fragments were used as the template, respectively.
  • PCR was carried out using genomic DNA of coli K-12, ⁇ ⁇ , F ⁇ , prototrophic
  • genomic DNA of coli K-12, ⁇ ⁇ , F ⁇ , prototrophic
  • the purified PCR product was purified and then electroporated to the CS07 strain prepared in 3-5 to prepare a CS10 strain (W3110 ⁇ lacI ⁇ gabD ⁇ yne I ⁇ ldhA ⁇ adhE ⁇ pflB ⁇ ptsI ).
  • PCR products were prepared. Next, the purified PCR product was purified, and then electroporated to the CS07 strain prepared in 3-5 to prepare a CS16 strain (W3110 ⁇ lacI ⁇ gabD ⁇ yne I ⁇ ldhA ⁇ adhE ⁇ pflB ⁇ ptsG ).
  • the CS03 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS03 strain prepared in Example 3-2. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the CS05 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS05 strain prepared in Example 3-3. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the CS06 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS06 strain prepared in Example 3-4. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the CS07 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS07 strain prepared in Example 3-5. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the CS10 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS10 strain prepared in Example 3-6. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the CS16 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS16 strain prepared in Example 3-6. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • Example 3 The variant strains of Table 2 prepared in Example 3 were cultured in the same manner as in Example 2. Thereafter, the culture solution was treated in the same manner as in Example 2, the concentration of 4-hydroxybutyric acid was measured by GC analysis, and is shown in Table 2.
  • Table 2 shows that all of the mutant microorganisms additionally deleted from one or more genes selected from the group consisting of gabD, yneI, ldhA, adhE, pflB, ptsI and ptsG genes improved 4-hydroxybutyric acid production ability.
  • KCTC 0769BP biosynthetic Mannheimia succiniciproducens gene coding for phosphoenolpyruvate carboxykinase pckA PTac15K (p15A origin, low copies, Km) R ; KAISTMBELstock) cloned into the expression vector.
  • pHCE IIB (NcoI) (BioLeaders) was cut into AatII and NheI, and pACYC177 (New England Biolabs) was also cut into AaII and NheI, followed by fragments containing the pHCD promoter of pHCE IIB (NcoI) Polymerization with sections containing ampicillin antibiotics resulted in a vector called pHNC15.
  • pHNC15 was cut with FspI, pUC4K (GE Healthcare Life Sciences) was cut with pstI, filled-in, and polymerized to complete the pHNC15K vector containing kanamycin instead of ampicillin at pHNC15.
  • pHNC15K was cut into NheI and then filled in, and then cut into EcoRI.
  • the resulting fragment is the one in which pHCE is removed from pHNC15K.
  • pKK223-3 (Pharmacia Biotech) was cut with sphI, filled in and cut with EcoRI.
  • PTac15K was completed by polymerizing the fragments containing the tac promoter and the above-mentioned fragments.
  • a pckA fragment was prepared by using genomic DNA of M. succiniciproducens (KCTC 0769BP) as a template and performing PCR under the same conditions as in 1-1 above using the synthesized primers of SEQ ID NOs: 37 and 38.
  • the prepared pckA fragment and the pTac15k plasmid were treated with restriction enzymes ( EcoRI and SacI ), and then treated with T4 DNA ligase to polymerize the pckA fragment and pTac15k plasmid digested with the restriction enzyme, thereby preparing a vector p15PckA as a recombinant plasmid. It was.
  • biosynthetic Corynebacterium glutamicum (ATCC 13032) gene coding for phosphoenolpyruvate carboxykinase pyc PTac15K (p15A origin, low copies, Km) R ; KAISTMBELstock) cloned into the expression vector.
  • C. glutamicum By using genomic DNA of (ATCC 13032) as a template and PCR using the primers of SEQ ID NOs: 39 and 40 synthesized, pyc Sections were prepared. PCR conditions were the same as in Example 1-1 except that the time of step 4 was 1min 45 sec.
  • the treated pyc fragment and the pTac15k plasmid were treated with restriction enzymes ( Sac I and Xba I), followed by treatment with T4 DNA ligase to polymerize the pckA fragment and pTac15k plasmid digested with the restriction enzyme, thereby recombining the vector p15Pyc as a recombinant plasmid.
  • restriction enzymes Sac I and Xba I
  • biosynthetic Corynebacterium glutamicum (ATCC 13032) gene coding for phosphoenolpyruvate carboxykinase pyc was cloned into p15PckA prepared in 5-1. Expression is pyc By RBS (ribosome binding site) in front of gene pckA Affected by the tac promoter in front of the gene.
  • C. glutamicum By using the genomic DNA of the template as a template, using the primers of SEQ ID NO: 41 and 40 synthesized, PCR is carried out under the same conditions as in the above 5-2, pyc Sections were prepared.
  • the treated pyc fragment and the p15PckA plasmid prepared in Example 5-1 were treated with restriction enzymes ( Sac I and Xba I), and then treated with T4 DNA ligase to digest the pyc fragment and p15PckA plasmid digested with restriction enzymes.
  • restriction enzymes Sac I and Xba I
  • T4 DNA ligase to digest the pyc fragment and p15PckA plasmid digested with restriction enzymes.
  • the recombinant pplasmid vector p15CY was produced.
  • the prepared ppc fragment and pTac15k plasmid were treated with restriction enzymes ( Eco RI and Sac I), and then treated with T4 DNA ligase to polymerize the ppc fragment and pTac15k plasmid digested with the restriction enzyme, thereby recombining the vector p15Ppc as a recombinant plasmid.
  • restriction enzymes Eco RI and Sac I
  • T4 DNA ligase to polymerize the ppc fragment and pTac15k plasmid digested with the restriction enzyme, thereby recombining the vector p15Ppc as a recombinant plasmid.
  • the CS10 / p99SC4CD / p15PckA strain was prepared by introducing the p15PckA vector prepared in 5-1 into the CS10 / p99SC4CD strain prepared in Example 3-12. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS10 / p99SC4CD / p15Pyc strain was prepared by introducing the p15Pyc vector prepared in 5-2 into the CS10 / p99SC4CD strain prepared in Example 3-12. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS10 / p99SC4CD / p15CY strain was prepared by introducing the p15CY vector prepared in 5-3 into the CS10 / p99SC4CD strain prepared in Example 3-12. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS10 / p99SC4CD / pTac15K strain was prepared by introducing a pTac15K vector into the CS10 / p99SC4CD strain prepared in Examples 3-12. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS16 / p99SC4CD / p15PckA strain was prepared by introducing the p15PckA vector prepared in 5-1 into the CS16 / p99SC4CD strain prepared in Example 3-13. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS16 / p99SC4CD / p15Ppc strain was prepared by introducing the p15Ppc vector prepared in 5-4 into the CS16 / p99SC4CD strain prepared in Example 3-13. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS16 / p99SC4CD / p15Pyc strain was prepared by introducing the p15Pyc vector prepared in 5-2 into the CS16 / p99SC4CD strain prepared in Example 3-13. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS16 / p99SC4CD / pTac15K strain was prepared by introducing a pTac15K vector into the CS16 / p99SC4CD strain prepared in Example 3-13. Next, recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the mutant strains of Table 3 prepared in Example 5 were cultured in the same manner as in Example 2. The difference was selected from LB plate medium containing only ampicillin (ampicilin) or LB plate medium containing ampicilin and kanamycin. Thereafter, the culture solution was treated in the same manner as in Example 2, and the concentration of 4-hydroxybutyric acid was measured by GC analysis.
  • Table 3 shows that 4-hydroxybutyric acid production ability was improved in all of the mutant microorganisms amplified by supplementary circuit genes, pckA, pyc and ppc genes , rather than simply using a strong promoter.
  • genomic DNA of E. coli was used as a template to make a mutation in which the 163th amino acid arginine (R) was substituted with leucine (L), and the synthesized primers of SEQ ID NOs 44, 45, 46 and 47 were used.
  • PCR fragments were prepared for each.
  • PCR was performed under the same conditions as in 1-1 using primers of SEQ ID NOs: 44 and 47 using the prepared PCR fragments as templates, thereby producing a mutated gltA fragment.
  • T4 DNA processes the ligase, by the stone Thermal digested with restriction enzyme polymerizing gltA fragments and p15Ppc plasmid, the recombinant plasmid Phosphorus vector p15PpcGltAR163L was constructed.
  • E. coli W3110 ATCC 39936 (derived from E. coli K-12, ⁇ -, F -, genomic DNA of prototrophic, plasmid pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85, 2008), E. coli W3110 (ATCC 39936) (derived from E.
  • PCR was carried out using genomic DNA of coli K-12, ⁇ ⁇ , F ⁇ , prototrophic) under the same conditions as in 1-1 above to obtain PCR fragments, respectively, using three PCR fragments as templates and PCR was carried out using the primers of 53 under the same conditions as in 1-1 to prepare a PCR product in which the gene encoding the aerobic respiration control protein ArcA ( arca ) was deleted.
  • the purified PCR product was purified, and then electroporated to the CS16 strain prepared in 3-7 to prepare a CS28 strain (W3110 ⁇ lacI ⁇ gabD ⁇ yne I ⁇ ldhA ⁇ adhE ⁇ pflB ⁇ ptsG ⁇ arcA ).
  • the CS16 / p99SC4CD / p15PpcGltAR163L strain was prepared by introducing the p15PpcGltAR163L vector prepared in 7-1 into the CS16 / p99SC4CD strain prepared in Example 3-13. Next, the recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • the CS28 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the CS28 strain prepared in Example 7-2. Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
  • the CS28 / p99SC4CD / p15PpcGltAR163L strain was prepared by introducing the p15PpcGltAR163L vector prepared in 7-1 into the CS28 / p99SC4CD strain prepared in Example 7-4. Next, the recombinant strains were selected while culturing the prepared strains in agar solid medium containing ampicillin and kanamycin.
  • Example 8 Preparation of 4-hydroxybutyric acid using mutant microorganisms amplified or enhanced oxidative pathway genes
  • the mutant strains of Table 4 prepared in Example 7 were cultured in the same manner as in Example 2. The difference was selected from LB plate medium containing only ampicillin (ampicilin) or LB plate medium containing ampicilin and kanamycin. Thereafter, the culture solution was treated in the same manner as in Example 2, and the concentration of 4-hydroxybutyric acid was measured by GC analysis.
  • the mutant microorganism having the 4-hydroxybutyric acid producing ability according to the present invention is useful for producing 4-hydroxybutyric acid in high yield.

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Abstract

La présente invention concerne un microorganisme mutant présentant une grande capacité de production d'acide 4-hydroxybutyrique et un procédé de préparation d'acide 4-hydroxybutyrique utilisant ledit microorganisme mutant. La présente invention concerne, plus précisément, un microorganisme mutant présentant une grande capacité de production d'acide 4-hydroxybutyrique, dans lequel les gènes assurant la synthèse de l'acide 4-hydroxybutyrique à partir d'acide succinique sont amplifiés, de même que les gènes d'une voie anaplérotique, tandis que des gènes compétitifs sont délétés afin d'augmenter la capacité de production d'acide 4-hydroxybutyrique. La présente invention concerne également un procédé de préparation d'acide 4-hydroxybutyrique caractérisé par un bon rendement et impliquant la culture de microorganismes mutants dans des conditions microaérobies. La présente invention concerne, donc, la préparation d'un microorganisme mutant présentant une grande capacité de production d'acide 4-hydroxybutyrique et l'utilisation dudit microorganisme mutant et elle peut, par conséquent, être utilisée en vue de la préparation d'acide 4-hydroxybutyrique avec un bon rendement.
PCT/KR2012/010665 2011-12-07 2012-12-07 Microorganisme mutant présentant une grande capacité de production d'acide 4-hydroxybutyrique et procédé de préparation d'acide 4-hydroxybutyrique l'utilisant WO2013085361A2 (fr)

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CN107257856B (zh) 2014-10-30 2021-05-28 株式会社三养社 阿洛酮糖差向异构酶的表达系统和使用其生产阿洛酮糖
KR102120995B1 (ko) * 2018-10-12 2020-06-18 경희대학교 산학협력단 4-하이드록시부티르산 생산용 형질전환 메탄자화균 및 이의 용도
KR102105689B1 (ko) * 2018-12-17 2020-04-28 울산과학기술원 젖산탈수소 효소 돌연변이체 및 이를 이용한 2-하이드록시-카르복실산의 제조방법
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