WO2013085361A2 - Mutant microorganism having a high 4-hydroxybutyric acid production capacity, and method for preparing 4-hydroxybutyric acid using same - Google Patents

Mutant microorganism having a high 4-hydroxybutyric acid production capacity, and method for preparing 4-hydroxybutyric acid using same Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
gene
hydroxybutyric acid
encoding
gene encoding
involved
Prior art date
Application number
PCT/KR2012/010665
Other languages
French (fr)
Korean (ko)
Other versions
WO2013085361A3 (en
Inventor
이상엽
최솔
이정욱
박시재
Original Assignee
한국과학기술원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한국과학기술원 filed Critical 한국과학기술원
Publication of WO2013085361A2 publication Critical patent/WO2013085361A2/en
Publication of WO2013085361A3 publication Critical patent/WO2013085361A3/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • 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.

Landscapes

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

Abstract

The present invention relates to a mutant microorganism having a high 4-hydroxybutyric acid production capacity and to a method for preparing 4-hydroxybutyric acid using the mutant microorganism. More particularly, the present invention relates to a mutant microorganism having a high 4-hydroxybutyric acid production capacity, in which genes for synthesizing 4-hydroxybutyric acid from succinic acid are amplified, genes in an anaplerotic pathway are amplified, and competitive genes are deleted so as to increase the 4-hydroxybutyric acid production capacity. The present invention also relates to a method for preparing 4-hydroxybutyric acid with a high yield rate by culturing the mutant microorganism under microaerobic conditions. The present invention enables the preparation of a mutant microorganism having a high 4-hydroxybutyric acid production capacity and the use of the mutant microorganism, and therefore is useful in the preparation of 4-hydroxybutyric acid with a high yield rate.

Description

4-하이드록시부티릭산 고생성능을 가지는 변이 미생물 및 이를 이용한 4-하이드록시부티릭산의 제조방법Mutant microorganism having high performance of 4-hydroxybutyric acid and preparation method of 4-hydroxybutyric acid using the same
본 발명은 4-하이드록시부티릭산 고생성능을 가지는 변이 미생물 및 이를 이용한 4-하이드록시부티릭산의 제조방법에 관한 것으로, 더욱 상세하게는 4-하이드록시부티릭산의 생성능을 증가시키기 위해 숙신산으로부터 4-하이드록시부티릭산을 합성하는 유전자가 증폭되어 있고, 보충 회로(anaplerotic pathway) 유전자가 증폭되어 있으며, 경쟁 유전자들이 결실되어 있는 4-하이드록시부티릭산 고생성능을 가지는 변이 미생물 및 이를 미 호기성(micro-aerobic) 조건하에서 배양하여 4-하이드록시부티릭산을 고수율로 제조하는 방법에 관한 것이다.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-하이드록시부티릭산은 생물학적으로 1,4-부탄디올 (1,4-butanediol) 또는 γ-부틸로락톤(gamma-butyrolactone)과 같은 중요한 C4 화합물의 전구체로 쓰일 수 있으며, 또한 다양한 폴리하이드록시알카노에이트(polyhydroxyalkanoate)의 단량체로 이용되고 있다. 또한 Sodium 형태의 4-하이드록시부티릭산은 Xyrem이라는 약으로써, 기면증 환자에 쓰입니다. 현재 1g에 약 18 ＄에 판매되고 있다.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.
현재 미생물을 통한 4-하이드록시부티릭산 생산의 경우 고분자로써만 국한되어 있을 뿐, 4-하이드록시부티릭산을 단독적으로 생산하지는 못하고 있다. 고분자의 형태로 4-하이드록시부티릭산을 생성하게 되면, 상기에서 언급한 1,4-부탄디올 및 γ-부틸로락톤을 생물학적으로 생산할 때, 고분자를 단량체로 끊어야 하기 때문에 효율적이지 못한 문제점이 있다. 따라서, 4-하이드록시부티릭산을 고분자가 아닌 단독적으로 생산하는 균주를 개발하고, 이에 1,4-부탄디올이나 및 γ-부틸로락톤에 필요한 효소를 추가적으로 도입하면, 1,4-부탄디올 및 γ-부틸로락톤 단량체를 쉽게 생산할 수 있을 것으로 보고 이에 대한 연구가 진행중이다.Currently, 4-hydroxybutyric acid production through microorganisms is limited only to polymers and does not produce 4-hydroxybutyric acid alone. When 4-hydroxybutyric acid is produced in the form of a polymer, when the 1,4-butanediol and γ-butyrolactone are biologically produced, there is a problem in that 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 γ-butylolactone, 1,4-butanediol and γ- Butylolactone monomers can be easily produced and research is being conducted.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 예의 노력한 결과, 4-하이드록시부티릭산을 생산하는 미생물에서, 숙신산으로부터 4-하이드록시부티릭산의 합성에 관여하는 유전자를 증폭시키고, 추가적으로 4-하이드록시부티릭산의 전구체인 숙신산 생산에 관여하는 보충 회로(anaplerotic pathway) 유전자를 증폭시키고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들을 약화 또는 결실시키고, 산화적 회로(oxidative pathway)에 관여하는 유전자들을 약화 또는 결실시키거나, 변이시켜 증폭시킬 경우, 4-하이드록시부티릭산 고생성능을 가지는 변이 미생물을 제조할 수 있다는 사실을 확인하고 본 발명을 완성하게 되었다.Accordingly, 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.
발명의 요약Summary of the Invention
본 발명의 목적은 4-하이드록시부티릭산의 고생성능을 가지는 변이 미생물 및 상기 변이 미생물의 제조방법을 제공하는데 있다.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.
본 발명의 다른 목적은 상기 변이 미생물을 미 호기성(micro-aerobic) 조건에서 배양하여, 4-하이드록시부티릭산을 고수율로 제조하는 방법을 제공하는데 있다. 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.
상기 목적을 달성하기 위하여, 본 발명은 4-하이드록시부티릭산의 생합성에 관여하는 코에이 트랜스퍼라아제(CoA transferase)를 코딩하는 유전자(cat1), 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD), 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자(sucD) 및 4-하이드록시부티릭산 디하이드로게나아제(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자(4hbD 또는 yqhD)로 구성된 군에서 선택되는 하나 이상이 증폭되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물 및 상기 변이 미생물의 제조방법을 제공한다.In order to achieve the above object, 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.
본 발명에 있어서, 상기 변이 미생물은 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 하나 이상이 추가로 증폭되어 있는 것을 특징으로 한다. In the present invention, the mutant microorganism is a gene encoding a phosphoenolpyruvate carboxykinase ( pckA ), phosphoenolpyruvate carboxylase (phosphoenolppyruvate carboxylase) involved in the anaplerotic pathway (phosphoenolpyrutave carboxylase) ) Is one or more selected from the group consisting of gene ( ppc ) and gene coding for pyruvate carboxylase ( pyc ) ( pyc ) is further amplified.
본 발명에 있어서, 상기 변이 미생물은 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실되어 있는 것을 특징으로 한다.In the present invention, 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.
본 발명에 있어서, 상기 변이 미생물은 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 또는 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)로 구성된 군에서 선택되는 하나 이상이 추가로 약화 또는 결실되어 있는 것을 특징으로 한다.In the present invention, 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.
본 발명에 있어서, 상기 변이 미생물은 4-하이드록시부티릭산 생합성에 관여하는 효소를 코딩하는 유전자의 발현이 증가되도록 lac 오페론 억제인자를 코딩하는 유전자(lacI)가 추가로 결실되어 있는 것을 특징으로 한다.In the present invention, 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. .
본 발명에 있어서, 상기 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자 (sucD)는 strong promoter를 함유하는 발현벡터 형태로 도입된 것을 특징으로 한다.In the present invention, 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.
본 발명에 있어서, 상기 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)는 strong promoter를 함유하는 발현벡터 형태로 도입된 것을 특징으로 한다.In the present invention, the gene ( pckA ), phosphoenolpyruvic acid carboxylase (phosphoenolpyruvate carboxylase) coding for the phosphoenolpyruvate carboxykinase involved in the anaplerotic pathway (coding) 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.
본 발명에 있어서, 상기 strong promoter는 trc promoter, tac promoter, T7 promoter, lac promoter 및 trp promoter로 구성된 군에서 선택되는 것을 특징으로 한다.In the present invention, the strong promoter is selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter and trp promoter.
본 발명에 있어서, 상기 미생물은 상기 미생물은 바실러스 속(Bacillus sp.), 코리네박테리움 속(Corynebacterium sp.), 에스케리치아 속(Escherichia sp.), 피치아 속(Pichia sp.), 슈도모나스 속(Pseudomonas sp.) 및 사카로마이세스 속(Saccharomyces sp.)으로 구성된 군에서 선택되는 것을 특징으로 한다.In the present invention, 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.
본 발명은 또한, (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)가 증폭되어 있고, (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)가 약화 또는 결실되어 있으며, (C) 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 유전자가 증폭되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물 및 상기 변이 미생물의 제조방법을 제공한다.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) encoding a (phosphoenolpyruvate carboxykinase), phospho yen come pyruvate carboxylic la dehydratase gene encoding the (phosphoenolpyrutave carboxylase) gene (ppc) and f Rubik acid carboxylic la kinase (pyruvate carboxylase) encoding the (pyc The present invention provides a mutant microorganism having a 4-hydroxybutyric acid producing ability and a method for producing the mutant microorganism, characterized in that the gene selected from the group consisting of amplified.
본 발명은 또한, (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)가 증폭되어 있고, (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)가 약화 또는 결실되어 있으며, (C) 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물 및 상기 변이 미생물의 제조방법을 제공한다.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.
본 발명은 또한, (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)가 증폭되어 있고, (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)가 약화 또는 결실되어 있고, (C) 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 유전자가 증폭되어 있으며, (D) 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물 및 상기 변이 미생물의 제조방법을 제공한다.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 carboxykinase ( pckA ), gene encoding phosphoenolpyrutave carboxylase ( ppc ) and gene encoding pyruvate carboxylase ( pyc ) Genes selected from the group consisting of amplified, (D) of the genes involved in the oxidative pathway (i) coding for mutated citrate synthase (mutate) to remove the inhibition by NADH Gene ( gltA ) is amplified; (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.
본 발명은 또한, 상기 변이 미생물을 배양하여 4-하이드록시부티릭산을 생성시킨 다음, 4-하이드록시부티릭산을 회수하는 것을 특징으로 하는 4-하이드록시부티릭산의 제조방법을 제공한다.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.
본 발명의 다른 특징 및 구현예는 다음의 상세한 설명 및 첨부된 특허청구범위로부터 더욱 명백해 질 것이다.Other features and embodiments of the present invention will become more apparent from the following detailed description and the appended claims.
도 1은 글루코즈로부터 4-하이드록시부티릭산의 합성경로를 나타내는 모식도이다.1 is a schematic diagram showing a synthetic route of 4-hydroxybutyric acid from glucose.
발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments
본 발명에서 "약화"란 해당 유전자의 일부 염기를 변이, 치환, 또는 삭제시키거나 일부 염기를 도입시켜 해당유전자에 의해 발현되는 효소의 활성을 감소시키는 것을 포괄하는 개념으로, 해당 유전자의 효소가 관여하는 생합성경로의 일부 또는 상당부분을 차단하는 모든 것을 포함한다. In the present invention, 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.
본 발명에서 '결실'이란 해당 유전자의 일부 또는 전체 염기를 변이, 치환, 또는 삭제시키거나, 일부 염기를 도입시켜 해당유전자가 발현되지 않도록 하거나 발현되더라도 효소활성을 나타내지 못하도록 하는 것을 포괄하는 개념으로, 해당 유전자의 효소가 관여하는 생합성경로를 차단하는 모든 것을 포함한다. In the present invention, 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.
본 발명에서 "증폭"이란 해당 유전자의 일부 염기를 변이, 치환, 또는 삭제시키거나, 일부 염기를 도입시키거나, 또는 동일한 효소를 코딩하는 다른 미생물 유래의 유전자를 도입시켜 대응하는 효소의 활성을 증가시키는 것을 포괄하는 개념이다.In the present invention, "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.
도 1은 글루코즈로부터 4-하이드록시부티릭산의 합성경로를 나타내는 모식도이다. 도 1에 도시된 바와 같이, 4-하이드록시부티릭산의 합성에 관여하는 유전자(cat1, sucCD, sucD, yqhD 또는 4hbD) 유전자를 증폭시키면 4-하이드록시부티릭산의 생성능을 높일 수 있음을 확인하고자 하였다. 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.
본 발명의 일 실시예에서는 먼저 대장균의 경우, 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제 (CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자 (sucD)가 존재하지 않기 때문에 Clostridium kluyveri 유래의 sucD를 도입하였다. 그 결과, 본래 야생형 대장균은 4-하이드록시부티릭산을 생산하지 못하지만 sucD를 도입시킨 변이 미생물은 4-하이드록시부티릭산을 생산하는 것을 확인하였다. In one embodiment of the present invention, in the case of Escherichia coli, 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.
그 다음, 4-하이드록시부티릭산 디하이드로게나아제 (4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자인 E. coli 유래의 yqhD와 C. kluyveri 유래의 4hbD를 각각 sucD와 같이 증폭하였다. 그 결과 sucD만 증폭했을때보다 두 경우 모두 4-하이드록시부티릭산의 생산이 증가하는 것을 확인할 수 있었다. 이 결과를 통해 yqhD 및 4hbD가 4-하이드록시부티릭산에 효과가 있음을 입증하였다.That the following 4-hydroxy-butyric acid dehydrogenase (4-hydroxybutyrate dehydrogenase) gene of E. coli origin of 4hbD of yqhD and C. kluyveri derived encoding was amplified as each sucD. As a result, the production of 4-hydroxybutyric acid was increased in both cases than when amplifying only sucD . These results demonstrated that yqhD and 4hbD are effective for 4-hydroxybutyric acid.
본 발명의 다른 실시예에서는 C. kluyveri 유래의코에이-트랜스퍼라아제를 코딩하는 유전자(cat1)과 E. coli 유래의숙시닐-코에이 합성효소를 코딩하는 유전자 (sucCD)를 각각 sucD 및 4hbD와 같이 증폭하였다. 그 결과 sucD와 4hbD만 증폭했을때보다, 두 경우 모두 4-하이드록시부티릭산의 생산이 증가하는 것을 확인할 수 있었다. 이 결과를 통해 cat1 및 yqhD가 4-하이드록시부티릭산에 효과가 있음을 입증하였다.In another embodiment of the present invention, 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.
따라서, 본 발명은 일 관점에서, 4-하이드록시부티릭산의 생합성에 관여하는 코에이 트랜스퍼라아제(CoA transferase)를 코딩하는 유전자(cat1), 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD), 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자(sucD) 및 4-하이드록시부티릭산 디하이드로게나아제(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자(4hbD 또는 yqhD)로 구성된 군에서 선택되는 하나 이상이 증폭되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물에 관한 것이다.Accordingly, the present invention, in one aspect, 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) It relates to a mutant microorganism having a 4-hydroxybutyric acid producing ability, characterized in that one or more selected from the group consisting of genes ( 4hbD or yqhD ) encoding a) is amplified.
본 발명에서는 또한, 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있는 변이 미생물에 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들을 약화 또는 결실시킬 경우 4-하이드록시부티릭산의 생성능을 증가시킬 수 있을 것으로 예측하였다. In the present invention, 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.
본 발명의 일 실시예에서는 앞서 언급한 4-하이드록시부티릭산의 합성에 관여하는 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자(sucD), 숙시닐-코에이 합성효소 (succinyl-CoA synthetase)를 코딩하는 유전자(sucCD) 및 4-하이드록시부티릭산 디하이드로게나아제 (4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자(yqhD)를 함께 증폭시킨 균주에 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 또는 yneI)를 결실시킨 변이 미생물을 제조하였다. 그 결과 숙신산 세미알데히드 디하이드로게나아제 (succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 또는 yneI)를 결실시키지 않은 균주보다 4-하이드록시부티릭산 생성능이 향상되었음을 확인하였다.In one embodiment of the present invention, the gene encoding the CoA-dependent succinate semialdehyde dehydrogenase ( sucD ) involved in the synthesis of the aforementioned 4-hydroxybutyric acid ( sucD ), succinyl Succinic acid succinic acid in a strain amplified together with a gene encoding sucinyl -CoA synthetase ( sucCD ) and a gene encoding 4-hydroxybutyrate dehydrogenase ( yqhD ) Mutant microorganisms in which genes ( gabD or yneI) encoding aldehyde dehydrogenase were deleted 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 ( gabD or yneI) encoding succinate semialdehyde dehydrogenase.
본 발명의 또 다른 실시예에서는 gabD 및 yneI 유전자가 결실된 균주에 젖산디하이드로게나아제(lactate dehydrogenase)를 코딩하는 유전자(ldhA)를 결실시키고, 4-하이드록시부티릭산의 합성에 관여하는 유전자(sucCD, sucD 또는 yqhD) 유전자를 증폭시킨 변이 미생물을 제조하였다. 그 결과 젖산 디하이드로게나아제(lactate dehydrogenase)를 코딩하는 유전자(ldhA)를 결실 시키지 않은 균주보다 4-하이드록시부티릭산 생성능이 향상되었음을 확인하였다.In another embodiment of the present invention, 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. As a result, it was confirmed that 4-hydroxybutyric acid production ability was improved compared to the strain that did not delete the gene ( ldhA) encoding lactate dehydrogenase.
본 발명의 또 다른 실시예에서는 gabD, yneI 및 ldhA 유전자가 결실된 균주에 알코올디하이드로게나아제 (alcohol dehydrogenase)를 코딩하는 유전자(adhE)를 결실시키고, 4-하이드록시부티릭산의 합성에 관여하는 유전자(sucCD, sucD 또는 yqhD) 유전자를 증폭시킨 변이 미생물을 제조하였다. 그 결과 알코올 디하이드로게나아제 (alcohol dehydrogenase)를 코딩하는 유전자(adhE)를 결실 시키지 않은 균주보다 4-하이드록시부티릭산 생성능이 향상되었음을 확인하였다.In another embodiment of the present invention, 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 ).
본 발명의 또 다른 실시예에서는 gabD, yneI, ldhA 및 adhE가 결실된 균주에 피루빅산-포름산 리아제 (pyruvate formate lyase)를 코딩하는 유전자(pflB)를 결실시키고, 4-하이드록시부티릭산의 합성에 관여하는 유전자(sucCD,sucD또는 yqhD) 유전자를 증폭시킨 변이 미생물을 제조하였다. 그 결과 피루빅산-포름산 리아제 (pyruvate formate lyase)를 코딩하는 유전자(pflB)를 결실 시키지 않은 균주보다 4-하이드록시부티릭산 생성능이 향상되었음을 확인하였다. In another embodiment of the present invention, 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.
본 발명의 또 다른 실시예에서는 gabD, yneI, ldhA, adhE 및 pflB가 결실된 균주에 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)를 결실시키고, 4-하이드록시부티릭산의 합성에 관여하는 유전자(sucCD, sucD 또는 yqhD) 유전자를 증폭시킨 변이 미생물을 제조하였다. PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)를 결실시킨 변이 미생물을 제조하였다. 그 결과 ptsI 또는 ptsG를 결실시키지 않은 균주보다 4-하이드록시부티릭산 생성능이 향상되었음을 확인하였다.In another embodiment of the present invention, a gene encoding a PEP-protein phosphotransferase of PTS system ( ptsI or ptsG ) of a PTS system in a strain deleted gabD, yneI, ldhA , adhE and pflB. ), And a mutant microorganism having amplified a gene ( sucCD , sucD or yqhD ) gene involved in the synthesis of 4-hydroxybutyric acid was prepared. Mutant microorganisms were deleted in which the gene ( ptsI or ptsG ) encoding PEP-protein phosphotransferase of PTS system of the PTS system was deleted. As a result, it was confirmed that 4-hydroxybutyric acid producing ability was improved compared to the strain without deleting ptsI or ptsG .
따라서, 본 발명에서 상기 4-하이드록시부티릭산의 합성에 관여하는 유전자(cat1, sucCD, sucD, yqhD 또는 4hbD)가 증폭되어 있는 변이 미생물 또는 상기 4-하이드록시부티릭산의 합성에 관여하는 유전자(cat1, sucCD, sucD, yqhD 또는 4hbD) 및 보충 회로(anaplerotic pathway)에 관여하는 유전자(pckA, ppc 또는 pyc)가 증폭되어 있는 변이 미생물은 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 또는 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)로 구성된 군에서 선택되는 하나 이상이 추가로 약화 또는 결실되어 있는 것을 특징으로 한다.Therefore, in the present invention, the gene involved in the synthesis of the 4-hydroxybutyric acid ( cat1 , sucCD , sucD , yqhD or 4hbD ) 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 consisting of genes ( ptsI or ptsG ) encoding a protein phosphotransferase of PTS system is further weakened or deleted.
본 발명에서는 또한, 4-하이드록시부티릭산의 생산을 강화하기 위해, 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있는 변이 미생물에 보충 회로(anaplerotic pathway)에 관여하는 유전자(pckA, ppc 또는 pyc)를 추가로 증폭시킬 경우, 4-하이드록시부티릭산의 생성능을 증가시킬 수 있을 것으로 예측하였다. In the present invention, in order to enhance the production of 4-hydroxybutyric acid, 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.
본 발명의 일 실시예에서는 앞서 언급한 4-하이드록시부티릭산의 합성에 관여하는 숙시닐-코에이 합성효소 (succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)를 증폭시키고, 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD, yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)로 구성된 군에서 선택되는 하나 이상을 결실시킨 균주에 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)를 각각 증폭시킨 변이 미생물을 제조하였다. 그 결과 pckA, ppc 또는 pyc 유전자를증폭시키지않은균주보다 4-하이드록시부티릭산 생성능이 향상되었음을 확인하였다.In one embodiment of 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 or more selected from the group consisting of (pckA), Sports yen come pyruvate kinase carboxylic La (phosphoenolpyrutave carboxylase) to the mutant microorganism in which each amplify the gene (ppc) and f Rubik carboxylic acid la dehydratase gene (pyc) encoding the (pyruvate carboxylase) was prepared encoding. As a result, it was confirmed that 4-hydroxybutyric acid production ability was improved compared to the strain without amplifying the pckA, ppc or pyc gene.
따라서, 본 발명에서 상기 4-하이드록시부티릭산의 합성에 관여하는 유전자(cat1, sucCD, sucD, yqhD 또는 4hbD)가 증폭되어 있는 변이 미생물은 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 하나 이상이 추가로 증폭되어 있는 것을 특징으로 한다. Therefore, in the present invention, 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 encoding the acid phosphoenolpyruvate carboxykinase ( pckA ), the gene encoding the phosphoenolpyrutave carboxylase ( ppc ) and the pyruvate carboxylase Characterized in that one or more selected from the group consisting of gene ( pyc ) is further amplified.
본 발명에 있어서, 상기 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자 (sucD)는 strong promoter를 함유하는 발현벡터 형태로 도입되고, 상기 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)는 strong promoter를 함유하는 발현벡터 형태로 도입된 것을 특징으로 한다. In the present invention, 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.
상기 strong promoter는 trc promoter, tac promoter, T7 promoter, lac promoter, trp promoter 등을 예시할 수 있다. The strong promoter may exemplify a trc promoter, a tac promoter, a T7 promoter, a lac promoter, a trp promoter, and the like.
본 발명에서는 또한, 4-하이드록시부티릭산의 생산을 강화하기 위해, 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있는 변이 미생물 또는 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있고, 보충 회로(anaplerotic pathway)에 관여하는 유전자(pckA, ppc 또는 pyc)가 증폭되어 있는 변이 미생물에 산화적 회로(oxidative pathway)에 관여하는 유전자 중 NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)를 추가로 증폭하거나 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)를 추가로 약화 또는 결실시킬 경우, 4-하이드록시부티릭산의 생성능을 증가시킬 수 있을 것으로 예측하였다. In the present invention, in order to enhance the production of 4-hydroxybutyric acid, 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.
본 발명의 일 실시예에서는 앞서 설명한 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있는 변이 미생물 또는 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있고, 보충 회로(anaplerotic pathway)에 관여하는 유전자(pckA, ppc 또는 pyc)가 증폭되어 있는 변이 미생물에 산화적 회로(oxidative pathway)에 관여하는 유전자 중 NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)를 추가로 증폭시켜 CS16/p99SC4CD/p15PpcGltAR163L를 제조하였고, 여기에 추가로 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자(arcA)가 결실된 CS28/p99SC4CD/p15PpcGltAR163L를 제조하고, 이들의 4-하이드록시부티릭산의 생성능을 증가되었음을 확인하였다.In one embodiment of the present invention, a mutant microorganism or 4 in which a gene involved in biosynthesis of 4-hydroxybutyric acid described above has been amplified and the genes involved in the competition pathway of 4-hydroxybutyric acid production are weakened or deleted. -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, In addition, a gene ( gltA ) encoding a mutated citrate synthase to remove the NADH inhibition among genes involved in the oxidative pathway in a mutant microorganism with amplification of ppc or pyc ). 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.
따라서, 본 발명의 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있는 변이 미생물 또는 4-하이드록시부티릭산의 생합성에 관여하는 유전자가 증폭되어 있고, 4-하이드록시부티릭산 생산의 경쟁 경로에 관여하는 유전자들이 약화 또는 결실되어 있고, 보충 회로(anaplerotic pathway)에 관여하는 유전자(pckA,ppc또는 pyc)가 증폭되어 있는 변이 미생물은 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실되어 있는 것을 특징으로 한다. Therefore, 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. ; (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.
본 발명에 있어서, 상기 돌연변이된 시트레이트 합성효소(citrate synthase)는 4-하이드록시부티릭산의 생성능을 향상시키기 위한 것으로서, NADH에 의한 저해가 제거된 것이라면 돌연변이의 위치나 종류는 특별히 제한되지 않는다.In the present invention, 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.
본 발명에 있어서, 상기 미생물은 글루코즈로부터 4-하이드록시부티릭산을 생성하는 미생물이면 제한없이 사용할 수 있으며, 바실러스 속(Bacillus sp.), 코리네박테리움 속(Corynebacterium sp.), 에스케리치아 속(Escherichia sp.), 피치아 속(Pichia sp.), 슈도모나스 속(Pseudomonas sp.), 사카로마이세스 속(Saccharomyces sp.)등을 예시할 수 있다.In the present invention, 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.
본 발명은 또 다른 관점에 있어서, 상기 변이 미생물을 배양하여 4-하이드록시부티릭산을 생성시킨 다음, 배양액으로부터 4-하이드록시부티릭산을 회수하는 것을 특징으로 하는 4-하이드록시부티릭산의 제조방법에 관한 것이다.In still another aspect, 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.
본 발명에서, 변이 미생물의 배양 및 4-하이드록시부티릭산의 수득 과정은 종래 발효공정에서 통상적으로 알려진 배양방법(회분식 배양, 유가식 배양) 및 4-하이드록시부티릭산의 분리 및 정제방법을 사용하여 수행할 수 있다.In the present invention, 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.
본 발명에 있어서, 4-하이드록시부티릭산의 생물공학적 생산은 세포 내 혹은 세포 외(in vivo or in vitro)에서 수행될 수 있다. In the present invention, the biotechnological production of 4-hydroxybutyric acid can be carried out intracellularly or extracellularly ( in vivo or in vitro ).
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
특히, 하기 실시예에서는 본 발명에 따른 유전자를 제거하기 위하여 특정 벡터와 숙주세포로 4-하이드록시부티릭산 생성 미생물인 에스케리치아 속(Escherichia sp.) 미생물만을 예시하였으나, 다른 종류의 벡터와 4-하이드록시부티릭산 생성 미생물들을 사용하는 것 역시 당업자에게 자명한 사항이라 할 것이다.In particular, the following examples illustrate only Escherichia sp. Microorganisms, 4-hydroxybutyric acid producing microorganisms, as specific vectors and host cells in order to remove the gene according to the present invention. The use of hydroxybutyric acid producing microorganisms will also be apparent to those skilled in the art.
실시예 1: 4-하이드록시부티릭산 생산에 관여하는 유전자들의 증폭과 증폭된 변이 미생물의 제조 Example 1 Amplification of Genes Involved in 4-Hydroxybutyric Acid Production and Preparation of Amplified Mutant Microorganisms
1-1: 플라스미드 p99SucD의 제작1-1: Construction of the plasmid p99SucD
항시적, 생합성적 Clostridium kluyver i(DSM 555)의CoA-dependent succinate semialdehyde dehydrogenase를 코딩하는 유전자 sucD를 trc promoter의 강한 유전자 발현을 진행하는 pTrc99a (Pharmacia Biotech, Uppsala, Sweden) 발현벡터에 클로닝하였다. C.kluyveri의 게놈 DNA를 주형으로 하고, 합성된 서열번호 1과 2의 프라이머를 이용하여, PCR을 수행함으로써, sucD 절편을 제조하였다. 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). By the genomic DNA of C.kluyveri as a template, using primers of the synthesized SEQ ID NO: 1 and 2, perform the PCR, to prepare a fragment sucD.
이때, PCR 조건은 다음과 같다. step 1 : 95℃, 2 min; step 2 : 95℃, 20 sec; step 3 : 55℃, 30 sec; step 4 : 72℃, 1 min; step 2 ~ step 4 28회 반복; step 5 : 72℃, 7 min; step 6 : 4℃ 유지.At this time, 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.
[서열번호 1]: 5'-GCGATAGAATTCATGAGTAATGAAGTATCTATAAAAGAATT AATTG-3'[SEQ ID NO 1]: 5'-GCGATAGAATTCATGAGTAATGAAGTATCTATAAAAGAATT AATTG-3 '
[서열번호 2]: 5'-GCATATGAGCTCTTATCCCCATATTTCCTCATAGCTA-3'[SEQ ID NO 2]: 5'-GCATATGAGCTCTTATCCCCATATTTCCTCATAGCTA-3 '
다음으로 제조된 sucD 절편및 pTrc99a 플라스미드에 제한효소(EcoRI와 SacI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 sucD 절편 및 pTrc99a 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p99SucD를 제작하였다.Next, the prepared 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. Was produced.
1-2: 플라스미드 p99SYn의 제작1-2: Construction of the plasmid p99SYn
항시적, 생합성적 E. coli W3110(ATCC 39936) (derived from E. coli K-12, λ-, F-, prototrophic)의 NAD(P) dependent alcohol dehydrogenase를 코딩하는 유전자 yqhD를 상기 1-1에서 제조된 p99SucD에 클로닝 하였다. 발현은 yqhD 유전자 앞의 RBS(ribosome binding site)에 의해 sucD 유전자 앞에 있는 trc promoter의 영향을 받는다. E.coli W3110(ATCC39936)(derivedfromE.coliK-12, λ-, F-,prototrophic)의 게놈 DNA를 주형으로 하고, 합성된 서열번호 3과 4의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, yqhD 절편을 제조하였다. The gene yqhD encoding NAD (P) dependent alcohol dehydrogenase of constitutive and biosynthetic E. coli W3110 (ATCC 39936) (derived from E. coli K-12, λ − , 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, λ − , 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.
[서열번호 3]: 5'-ATTGATGAGCTCGGAGCAAGTAATGAACAACTTTAAT-3'[SEQ ID NO 3]: 5'-ATTGATGAGCTCGGAGCAAGTAATGAACAACTTTAAT-3 '
[서열번호 4]: 5'-CTTGTCTCTAGATTAGCGGGCGGCTTCGTATA-3'[SEQ ID NO 4]: 5'-CTTGTCTCTAGATTAGCGGGCGGCTTCGTATA-3 '
다음으로 제조된 yqhD 절편 및 p99SucD 플라스미드에 제한효소(SacI과 XbaI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 yqhD 절편 및 p99SucD 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p99SYn을 제작하였다.Next, 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.
1-3: 플라스미드 p99SC4의 제작1-3: Construction of plasmid p99SC4
항시적, 생합성적 Clostridium kluyveri(DSM 555)의 4-hydroxybutyrate dehydrogenase를 코딩하는 유전자 4hbD를 상기 1-1에서 제작된 p99SucD에 클로닝 하였다. 발현은 4hbD 유전자 앞의 RBS(ribosome binding site)에 의해 sucD 유전자 앞에 있는 trc promoter의 영향을 받는다. C.kluyveri의 게놈 DNA를 주형으로 하고, 합성된 서열번호 5과 6의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 4hbD 절편을 제조하였다.The gene 4hbD encoding 4-hydroxybutyrate dehydrogenase of the constitutive and biosynthetic Clostridium kluyveri (DSM 555) 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. To the genomic DNA of C.kluyveri as a template and using the primers of the synthesized SEQ ID NO: 5 and 6, by carrying out PCR in the same conditions as 1-1, to prepare a fragment 4hbD.
[서열번호 5]: 5'-GATATAGAGCTCACAGGAAACAATGAAGTTATTAAAATTGG CACCTG-3'[SEQ ID NO 5]: 5'-GATATAGAGCTCACAGGAAACAATGAAGTTATTAAAATTGG CACCTG-3 '
[서열번호 6]: 5'-GCCAGCTCTAGATTAATATAACTTTTTATATGTGTTTACTA TGTCTTC-3'[SEQ ID NO: 6]: 5'-GCCAGCTCTAGATTAATATAACTTTTTATATGTGTTTACTA TGTCTTC-3 '
다음으로 제조된 4hbD 절편 및 p99SucD 플라스미드에 제한효소(SacI 과 XbaI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 4hbD 절편 및 p99SucD 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p99SC4를 제작하였다. Next, 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. Was produced.
1-4: 플라스미드 p99SC4C1의 제작1-4: Construction of the plasmid p99SC4C1
항시적, 생합성적 Clostridium kluyveri(DSM 555)의CoA transferase 를 코딩하는 유전자 cat1을 상기 1-3에서 제작된 p99SC4에 클로닝 하였다. 발현은 cat1 유전자앞의 RBS(ribosome binding site)에 의해 SucD 유전자 앞에 있는 trc promoter의 영향을 받는다. C.kluyveri의 게놈 DNA를 주형으로 하고, 합성된 서열번호 7과 8의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, cat1 절편을 제조하였다. The gene cat1 encoding CoA transferase of constitutive and biosynthetic Clostridium kluyveri (DSM 555) 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. To the genomic DNA of C.kluyveri as a template and using the primers of the synthesized SEQ ID NO: 7 and 8, by carrying out PCR in the same conditions as 1-1, to prepare a fragment cat1.
[서열번호 7]: 5'-GTTATATCTAGAACAGGAAACAATGAGTAAAGGGATAAAGA ATTCACAA-3'[SEQ ID NO: 7]: 5'-GTTATATCTAGAACAGGAAACAATGAGTAAAGGGATAAAGA ATTCACAA-3 '
[서열번호 8]: 5'-ATAATCTCTAGAACAGGAAACAATGAACTTACATGAATATCA GGCA-3'[SEQ ID NO 8]: 5'-ATAATCTCTAGAACAGGAAACAATGAACTTACATGAATATCA GGCA-3 '
다음으로 제조된 cat1 절편 및 p99SC4 플라스미드에 제한효소(XbaI과 SbfI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 cat1 절편 및 p99SC4 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p99SC4C1을 제작하였다.Next, 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. Was produced.
1-5: 플라스미드 p99SC4CD의 제작 1-5: Construction of plasmid p99SC4CD
항시적, 생합성적 E. coli W3110(ATCC 39936) (derived from E.coliK-12, λ-, F-,prototrophic)의 succinyl-CoA synthase beta subunit를 코딩하는 유전자(sucC)와 succinyl-CoA synthase alpha subunit를 코딩하는 유전자(sucD)를 상기 1-3에서 제작된 p99SC4에 클로닝 하였다. sucC와 sucD의 경우 오페론으로써 게놈상에서 한 promoter에 의해 조절되며 같이 붙어있다. 또한 두 유전자는 각각 succinyl-CoA synthase의 한 부분을 코딩하고 발현된 단백질이 합쳐져 succinyl-CoA synthase의 기능을 한다. 이하 두 유전자를 합쳐서 sucCD라 하고 succinyl-CoA synthase beta subunit과 succinyl-CoA synthase alpha subunit를 합쳐서 succinyl-CoA synthase라 한다. 발현은 sucCD 유전자 앞의 RBS(ribosome binding site)에 의해 sucD 유전자 앞에 있는 trc promoter의 영향을 받는다. E.coli W3110(ATCC39936)(derivedfromE.coliK-12, λ-, F-,prototrophic)의 게놈 DNA를 주형으로 하고, 합성된 서열번호 9과 10의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, sucCD 절편을 제조하였다. Constitutive, biosynthetic ever E. coli W3110 (ATCC 39936) ( derived from E.coli K-12, λ -, 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. In addition, the two genes each encode a portion of the succinyl-CoA synthase and the expressed proteins combine to function as succinyl-CoA synthase. Hereinafter, the two genes are called sucCD , and the 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. The genomic DNA of E. coli W3110 (ATCC39936) (derived from E. coli K-12, λ − , 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.
[서열번호 9]: 5'-ATAATCTCTAGAACAGGAAACAATGAACTTACATGAATATCA GGCA-3'[SEQ ID NO 9:]: 5'-ATAATCTCTAGAACAGGAAACAATGAACTTACATGAATATCA GGCA-3 '
[서열번호 10]: 5'-ATACTTCCTGCAGGTTATTTCAGAACAGTTTTCAGTGCT-3'[SEQ ID NO 10]: 5'-ATACTTCCTGCAGGTTATTTCAGAACAGTTTTCAGTGCT-3 '
다음으로 제조된 sucCD 절편 및 p99SC4 플라스미드에 제한효소(XbaI과 SbfI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 sucCD 절편 및 p99SC4 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p99SC4CD을 제작하였다.Next, the 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. Was produced.
1-6: 플라스미드 p99SYnCD의 제작1-6: Construction of the plasmid p99SYnCD
항시적, 생합성적 E. coli W3110(ATCC 39936) (derived from E.coliK-12, λ-, F-,prototrophic)의 succinyl-CoA synthase를 코딩하는 유전자(sucCD)를 상기 1-2에서 제작된 p99SYn에 클로닝 하였다. 발현은 sucCD 유전자 앞의 RBS(ribosome binding site)에 의해 sucD 유전자 앞에 있는 trc promoter의 영향을 받는다. E.coli W3110(ATCC39936)(derived from E.coliK-12, λ-, F-,prototrophic)의 게놈 DNA를 주형으로 하고, 상기 1-5에서 제조한 합성된 서열번호 9과 10의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, sucCD 절편을 제조하였다. A succinyl-CoA synthase coding gene ( sucCD ) of constitutive and biosynthetic E. coli W3110 (ATCC 39936) (derived from E. coli K-12, λ − , 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, λ − , 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.
다음으로 제조된 sucCD 절편 및 p99SYn 플라스미드에 제한효소(XbaI과 SbfI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 sucCD 절편 및 p99SYn 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p99SYnCD를 제작하였다Next, the 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. Produced
1-7: WL3110/p99SucD 균주의 제조1-7: Preparation of WL3110 / p99SucD Strain
본 발명자가 한국공개특허 제2009-0018781호에서 개시한 방법으로 제조한 WL3110 균주(W3110 ΔlacI)에 상기 1-1에서 제조된 p99SucD 벡터를 도입시켜 WL3110/p99SucD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar) 고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.The present inventors introduced the p99SucD vector prepared in 1-1 to the WL3110 strain (W3110 Δ 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).
참고로, WL3110 균주(W3110 ΔlacI)는 다음의 방법으로 제조할 수 있다.For reference, the WL3110 strain (W3110 ΔlacI ) can be prepared by the following method.
서열번호 11과 12의 프라이머, 주형으로써 플라스미드인 pECmulox를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, lacI유전자가 결실된 PCR 산물을 제조하였다. Using SEQ ID NO: 11 and 12 primers, 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.
다음으로, 제조된 PCR 산물을 정제한 후, λ recombinase를 함유하는 전기충격용 대장균(W3110)에 electroporation하여 WL3110(W3110 ΔlacI)를 제조하였다.Next, the purified PCR product was purified, and then electroporated to E. coli (W3110) containing λ recombinase to prepare WL3110 (W3110 Δ lacI ).
[서열번호 11] : 5'-GTGAAACCAGTAACGTTATACGATRTCGCAGAGTATGCCG GTGTCTCTTAGATTGGCAGCATTACACGTCTTG-3'[SEQ ID NO 11]: 5'-GTGAAACCAGTAACGTTATACGATRTCGCAGAGTATGCCG GTGTCTCTTAGATTGGCAGCATTACACGTCTTG-3 '
[서열번호 12] : 5'-TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC TGCATTAATGCACTTAACGGCTGACATGGG-3'[SEQ ID NO 12]: 5'-TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC TGCATTAATGCACTTAACGGCTGACATGGG-3 '
1-8: WL3110/p99SYn 균주의 제조1-8: Preparation of WL3110 / p99SYn Strain
WL3110 균주(W3110 ΔlacI)에 상기 1-2에서 제조된 p99SYn 벡터를 도입시켜 WL3110/p99SucD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.The WL3110 / p99SucD strain was prepared by introducing the p99SYn vector prepared in 1-2 into the WL3110 strain (W3110 Δ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
1-9: WL3110/p99SC4 균주의 제조1-9: Preparation of WL3110 / p99SC4 Strain
WL3110 균주(W3110 ΔlacI)에 상기 1-3에서 제조된 p99SC4 벡터를 도입시켜 WL3110/p99SC4 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.The WL3110 / p99SC4 strain was prepared by introducing the p99SC4 vector prepared in 1-3 into the WL3110 strain (W3110 Δ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
1-10: WL3110/p99SC4C1 균주의 제조1-10: Preparation of WL3110 / p99SC4C1 Strain
WL3110 균주(W3110 ΔlacI)에 상기 1-4에서 제조된 p99SC4C1 벡터를 도입시켜 WL3110/p99SC4C1 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.The WL3110 / p99SC4C1 strain was prepared by introducing the p99SC4C1 vector prepared in 1-4 into the WL3110 strain (W3110 Δ lacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
1-11: WL3110/p99SC4CD 균주의 제조1-11: Preparation of WL3110 / p99SC4CD Strain
WL3110 균주(W3110 ΔlacI)에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 WL3110/p99SC4CD균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.The WL3110 / p99SC4CD strain was prepared by introducing the p99SC4CD vector prepared in 1-5 into the WL3110 strain (W3110 ΔlacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
1-12: WL3110/p99SYnCD 균주의 제조1-12: Preparation of WL3110 / p99SYnCD Strain
WL3110 균주(W3110 ΔlacI)에 상기 1-6에서 제조된 p99SYnCD 벡터를 도입시켜 WL3110/p99SYnCD균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.The WL3110 / p99SYnCD strain was prepared by introducing the p99SYnCD vector prepared in 1-6 into the WL3110 strain (W3110 ΔlacI ). Next, the recombinant strains were selected while culturing the prepared strains in agar (agar) solid medium containing ampicillin (ampicillin).
실시예 2: 4-하이드록시부티릭산 생산 유전자들이 증폭된 변이 미생물을 이용한 4-하이드록시부티릭산의 제조 Example 2: Preparation of 4-hydroxybutyric acid using mutant microorganisms amplified with 4-hydroxybutyric acid producing genes
실시예 1에서 제조된 하기 표 1의 변이 미생물들을 4g/L (NH4)2HPO4, 6.67g/LKH2PO4, 0.8g/Lcitricacid, 0.8g/LMgSO4·7H2O, 0.5%(v/v)tracemetalsolution으로 구성되는 최소 MR배지(Jung, Y.K., Kim, T.Y., Park, S.J., and Lee, S.Y., Biotechnol. Bioeng., 105(1):161-171,2010)에서 플라스크 컬쳐하였다. Trace metal solution은 1리터당 5M HCl: 10g FeSO4·7H2O, 2.25gZnSO4·7H2O, 1gCuSO4·5H2O, 0.5gMnSO4·5H2O, 0.23gNa2B4O7·10H2O, 2gCaCl2·2H2O 및 0.1g (NH4)6Mo7O24을 포함한다. 포도당(100g/l) 저장 용액 및 NaHCO3(60g/l)는 별도로 멸균시켰고, 각각 최종 농도 10g/l 및 6g/l까지 멸균된 배지에 추가 하였다. 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. 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.
4-하이드록시부티릭산 생산을 위해 먼저 엠피실린(ampicillin)(50㎍/㎖)이 첨가된 LB 평판배지에서 선별하였다. 형질전환 균주를 10㎖ LB 배지에 접종하여 37℃에서 12시간동안 전배양을 수행하였다. 배양액중 3mL을 동일한 배지 100mL을 포함하는 anaerobic flask에 첨가한 후, 5시간 동안 세포의 농도를 높이기 위해 37℃, 220 rpm으로 배양하였다. 다음으로 micro-aerobic 조건으로 전환하기 위해 질소가스를 10분정도 charging 하였으며, 동시에 클로닝된 유전자들의 발현 유도를 위해 IPTG(Isopropyl-1-thio-β-D-galactopyranoside) 1M용액 100㎕와 포도당 용액 (500g/L) 1mL를 추가 하였다. 그 후 microaerobic 조건을 유지시켜 주기 위해 flask 가지에 주사바늘을 꽂아 공기가 들어갈 수 있도록 하였다. micro-aerobic 조건으로 전환한 후 24시간 동안 37℃, 220 rpm으로 배양하였다. 그 후 배양액을 원심분리를 이용하여 세포를 분리한 후에, 해리된 상등액을 4-하이드록시부티릭산 분석을 위해 packed column(Supelco Carbopack™BAW/6.6% PEG20M,2m× 2mm ID, Bellefonte, PA, USA)이 장착된 gas chromatography(Agillent 6890N GC System, Agilent Technologies Inc., CA, USA)로 측정하고, 그 결과를 표 1에 나타내었다.For 4-hydroxybutyric acid production was first screened in LB plate medium with ampicillin (50 μg / ml) added. The transformed strains were inoculated in 10 ml LB medium and precultured at 37 ° C. for 12 hours. 3mL of the culture solution was added to an anaerobic flask containing 100mL of the same medium, and then cultured at 37 ° C. and 220 rpm to increase the concentration of cells for 5 hours. Next, the nitrogen gas was charged for 10 minutes to convert to micro-aerobic conditions. At the same time, 100 μl of ITG (Isopropyl-1-thio-β-D-galactopyranoside) 1M solution and glucose solution ( 1 g of 500 g / L) was added. Then, to maintain microaerobic conditions, the needle was inserted into the flask branch to allow air to enter. After switching to the micro-aerobic condition was incubated at 37 ℃, 220 rpm for 24 hours. The cells were then separated by centrifugation of the culture medium, and the dissociated supernatants were packed in a packed column (Supelco Carbopack ™ 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.
표 1
Figure PCTKR2012010665-appb-T000001
 Table 1
Figure PCTKR2012010665-appb-T000001
표 1로부터, sucD, yqhD, 4hbD, cat1 및 sucCD 유전자들이 증폭된 변이 미생물들 모두 글루코즈를 기질로 한4-하이드록시부티릭산 생성능이 향상되었음을 알 수 있었다.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.
실시예 3: 4-하이드록시부티릭산 생산의 증가를 위해 경쟁 회로들의 유전자가 결실된 변이 미생물의 제조Example 3: Preparation of Mutant Microorganisms Deleting Genes of Competition Circuits for Increased 4-hydroxybutyric Acid Production
염색체 상의 경쟁회로 유전자들의 결실은 이중교차상동 재조합(double-crossover homologous recombination)에 의해서 수행되었다(Datsenko, K. A., ＆ Wanner, B. L. Proc. Natl. Acad. Sci., 97:6640-6645, 2000). 결실을 위한 절편은 (1) 결실을 원하는 유전자의 상동서열, (2) lox71-chloramphenicol marker (CmR)-lox66 및 (3) 결실을 원하는 유전자의 상동서열을 포함한다.Deletion of competing genes on the chromosome was performed by double-crossover homologous recombination (Datsenko, KA, & Wanner, BL Proc. Natl. Acad. Sci., 97: 6640-6645, 2000). 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) ~ (3)은 5‘ → 3’ 방향이며, (1)과 (3)은 다른 서열이어야 되고, (1)의 서열이 (3)의 서열보다 왼쪽 즉 5‘ 방향이다. (2)는 lox71-CmR-lox66 카세트를 포함하는 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278:78-85, 2008)를 PCR을 수행하여 제조할 수 있다. 즉, pACYC184 (NEW England Biolabs)를 주형으로 하여, 서열번호 13 및 14의 프라이머로 PCR을 수행하여 얻은 PCR 산물을 제한 효소 HindIII와 SmaI으로 자르고 pUG6(Guldener U, Heck S, Fielder T, Beinhauer J ＆ Hegemann JH, Nucleic Acids Res, 24:2519-2524,1996; NCBI GenBank: AF298793.1)을 제한 효소 HindIII와 EcoRV로 자른 DNA 산물과 Ligation함으로써, pECmulox를 제조할 수 있다.(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. . In other words, using pACYC184 (NEW England Biolabs) as a template, 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.
[서열번호 13] : 5'-ATATAAGCTTTACCGTTCGTATAGCATACATTATAC GAAGTTATTGCCCTGAACCGACGACCG-3'[SEQ ID NO 13]: 5'-ATATAAGCTTTACCGTTCGTATAGCATACATTATAC GAAGTTATTGCCCTGAACCGACGACCG-3 '
[서열번호 14] : 5'-AATTCCCGGGTACCGTTCGTATAATGTATGCTATACG AAGTTATCATCACCCGACGCACTTTGC-3'[SEQ ID NO 14]: 5'-AATTCCCGGGTACCGTTCGTATAATGTATGCTATACG AAGTTATCATCACCCGACGCACTTTGC-3 '
상기 서열번호 13 및 14의 프라이머에 각각 (1)과 (3)의 서열을 포함시켜 PCR을 수행하면 결실을 위한 절편을 완성할 수 있다. 또한 유전자와의 이중교차상동 재조합을 강화하기 위해, 즉 결실하고 싶은 유전자의 상동서열 개수를 늘리기 위해 한번 완성된 결실을 위한 절편을 주형으로 하여 프라이머에 추가적으로 상동서열을 더 붙여 PCR을 통해 상동서열이 늘어난 PCR 절편을 얻을 수 있다. By performing PCR by including the sequences of (1) and (3) in the primers of SEQ ID NOs: 13 and 14, respectively, 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.
또 다른 방법으로는 (1), (2), (3)을 각각 별도의 프라미어를 이용해서 PCR을 수행한 후 (1)의 Sense 프라이머와 (3)의 Antisense 프라이머를 이용하여 Overlapping PCR을 통해 PCR 산물을 얻을 수 있다. 이 방법의 장점은 (1)과 (3)을 별도로 PCR을 수행하기 때문에 유전자의 상동서열 개수를 원하는 만큼 늘릴 수 있다.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.
상기 PCR 산물들은 λ recombinase를 포함하는 전기충격용 대장균 세포(electrocompetent cells)에 트랜스포메이션시켰다. 콜로니들은 클로로암페니콜(Cm; 34μg/ml)을 포함하는 Luria-Bertani (LB) agar (Sambrook, J., Fritsch E. F., ＆ Maniatis, T., Molecular cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2000) 플레이트에서 선택되었다. CmR의 성공적인 유전자 대체는 direct colony PCR에 의해서 확인되었다. 상기 항생제 마커는 차후에 temperature-sensitive replication origin과 IPTG-inducible cre recombinase를 포함하는 helper plasmid pJW168 (Lucigen, USA)에 의해 제거되었다. The PCR products were transformed into electrocompetent cells containing λ 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 μg / 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.
3-1: CS01 균주의 제조3-1: Preparation of CS01 Strain
서열번호 15 및 16의 프라이머로, 주형으로써 플라스미드 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278:78-85, 2008)를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, NAD(P)-dependent succinate semialdehyde dehydrogenase를 코딩하는 gabD가 결실된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 1-7에서 제조한 WL3110 균주(W3110 ΔlacI)에 electroporation하여 CS01 균주(W3110 ΔlacI ΔgabD )를 제조하였다.With primers of SEQ ID NOs: 15 and 16, the same conditions as in 1-1 above using 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 that was gabD -deleted encoding NAD (P) -dependent succinate semialdehyde dehydrogenase. Next, the purified PCR product was purified, and then electroporated to the WL3110 strain (W3110 ΔlacI ) prepared in 1-7 to prepare a CS01 strain (W3110 Δ lacI gabD ).
[서열번호 15]:5'-CCATTCTGCGCAACTGGTTCAATTTGATGATGGAGCATCAG GACGATTT AGACACTATAGAACGCGGCCG-3'[SEQ ID NO: 15]: 5'-CCATTCTGCGCAACTGGTTCAATTTGATGATGGAGCATCAG GACGATTT AGACACTATAGAACGCGGCCG-3 '
[서열번호 16]:5'-GGTCATACACGCCGTCCTGCACATACAGGCGGTTGGCGCAG ACGCAGGTTCCGCATAGGCCACTAGTGGA-3’[SEQ ID NO 16]: 5'-GGTCATACACGCCGTCCTGCACATACAGGCGGTTGGCGCAG ACGCAGGTTCCGCATAGGCCACTAGTGGA-3 ’
3-2: CS03균주의 제조3-2: Production of CS03 Strain
서열번호 17 및 18의 프라이머로, 주형으로써 플라스미드 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278:78-85,2008)를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, aldehyde dehydrogenase(특히 succinate semialdehyde dehydrogenase 라고 알려짐)를 코딩하는 yneI 유전자가결실된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-1에서 제조된 CS01 균주에 electroporation하여 CS03 균주(W3110 ΔlacI ΔgabD ΔyneI)를 제조하였다.With primers of SEQ ID NOs: 17 and 18, the same conditions as in 1-1 above using 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 yneI gene encoding aldehyde dehydrogenase (particularly known as succinate semialdehyde dehydrogenase) was deleted. Next, the purified PCR product was purified and then electroporated to the CS01 strain prepared in the above 3-1 to prepare a CS03 strain (W3110 Δ lacI Δ gabD Δ yne I ).
[서열번호 17]: 5'-GTCCGGCAATGCTGAAGGCGGAACCTACGCTGGTGGAAAA TCAGCAGGCGGACACTATAGAACGCGGCCG-3'[SEQ ID NO: 17]: 5'-GTCCGGCAATGCTGAAGGCGGAACCTACGCTGGTGGAAAA TCAGCAGGCGGACACTATAGAACGCGGCCG-3 '
[서열번호 18]: 5'-AGGGTTTTCTCCACCTGATGATGCAGCTCATCACGTAAATC AAAACGAGCCCGCATAGGCCACTAGTGGA-3’[SEQ ID NO 18]: 5'-AGGGTTTTCTCCACCTGATGATGCAGCTCATCACGTAAATC AAAACGAGCCCGCATAGGCCACTAGTGGA-3 ’
3-3: CS05 균주의 제조3-3: Preparation of CS05 Strain
서열번호 19 및 20의 프라이머, 주형으로써 플라스미드 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278:78-85, 2008)를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA)유전자가 결실된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-2에서 제조된 CS03 균주에 electroporation하여 CS05 균주(W3110 ΔlacI ΔgabD ΔyneI ΔldhA)를 제조하였다.Using primers of SEQ ID NOs: 19 and 20, 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 By performing the PCR, a PCR product was prepared in which the gene ( ldhA ) gene encoding lactate dehydrogenase was deleted. Next, the purified PCR product was purified, and then electroporated to the CS03 strain prepared in 3-2 to prepare a CS05 strain (W3110 Δ lacI Δ gabD Δ yne I Δ ldhA ).
[서열번호 19]: 5'-CACAAAACAGTACGACAAGAAGTACCTGCAACAGGTGAAC GAGTCCTTTGGACACTATAGAACGCGGCCG-3'[SEQ ID NO 19]: 5'-CACAAAACAGTACGACAAGAAGTACCTGCAACAGGTGAAC GAGTCCTTTGGACACTATAGAACGCGGCCG-3 '
[서열번호 20]: 5'-AGGTTTCGCCTTTTTCCAGATTGCTTAAGTTTTGCAGCGT AGTCTGAGAACCGCATAGGCCACTAGTGGA-3'[SEQ ID NO 20]: 5'-AGGTTTCGCCTTTTTCCAGATTGCTTAAGTTTTGCAGCGT AGTCTGAGAACCGCATAGGCCACTAGTGGA-3 '
3-4: CS06 균주의 제조3-4: Preparation of CS06 Strain
서열번호 21 및 22의 프라이머, 주형으로써 플라스미드 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278:78-85,2008)를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE)가 결실된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-3에서 제조된 CS05 균주에 electroporation하여 CS06 균주(W3110 ΔlacI ΔgabD ΔyneI ΔldhA ΔadhE)를 제조하였다.Under the same conditions as 1-1 using the primers of SEQ ID NOs: 21 and 22, 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. Next, the purified PCR product was purified and then electroporated to the CS05 strain prepared in 3-3 to prepare a CS06 strain (W3110 Δ lacI Δ gabD Δ yne I Δ ldhA Δ adhE ).
[서열번호 21]: 5'-TGAACTTAACGCACTCGTAGAGCGTGTAAAAAAAGCCCAG CGTGAATATGGACACTATAGAACGCGGCCG-3'[SEQ ID NO: 21]: 5'-TGAACTTAACGCACTCGTAGAGCGTGTAAAAAAAGCCCAG CGTGAATATGGACACTATAGAACGCGGCCG-3 '
[서열번호 22]: 5'-GCTTTTTTCTCAGCTTTAGCCGGAGCAGCTTCTTTCTTCG CTGCAGTTTCCCGCATAGGCCACTAGTGGA-3'[SEQ ID NO 22]: 5'-GCTTTTTTCTCAGCTTTAGCCGGAGCAGCTTCTTTCTTCG CTGCAGTTTCCCGCATAGGCCACTAGTGGA-3 '
3-5: CS07 균주의 제조3-5: Preparation of the CS07 Strain
서열번호 23 및 24의 프라이머, 주형으로써 플라스미드 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278:78-85,2008)를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB)가 결실된 PCR 산물을 제조하였다. 그 후, 이중교차상동 재조합을 강화하기 위해 상기 PCR 산물을 주형으로 하여, 서열번호 25 및 26의 프라이머를 이용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, pflB 유전자와 상동서열 개수가 증가된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-4에서 제조된 CS06 균주에 electroporation하여 CS07 균주(W3110 ΔlacI ΔgabD ΔyneI ΔldhA ΔadhE ΔpflB)를 제조하였다.Under the same conditions as 1-1 using the primers SEQ ID NOs: 23 and 24, plasmid pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85,2008) as a template By performing PCR, a 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. Next, the purified PCR product was purified and then electroporated to the CS06 strain prepared in 3-4 to prepare a CS07 strain (W3110 Δ lacI Δ gabD Δ yne I Δ ldhA Δ adhE Δ pflB ).
[서열번호 23]: 5'-TCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTT TTACCAAAGGGACACTATAGAACGCGGCCG-3'[SEQ ID NO 23]: 5'-TCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTT TTACCAAAGGGACACTATAGAACGCGGCCG-3 '
[서열번호 24]: 5'-CGATACCACACGCCATGGTGCGGATAACGTCACGGTCGTG CAGCGCCATCCCGCATAGGCCACTAGTGGA-3'[SEQ ID NO: 24]: 5'-CGATACCACACGCCATGGTGCGGATAACGTCACGGTCGTG CAGCGCCATCCCGCATAGGCCACTAGTGGA-3 '
[서열번호 25]: 5'-TGTCGAAGTACGCAGTAAATAAAAAATCCACTTA[SEQ ID NO 25]: 5'-TGTCGAAGTACGCAGTAAATAAAAAATCCACTTA
AGAAGG TAGGTGTTACATGTCCGAGCTTAATGAAAAGTT-3'AGAAGG TAGGTGTTACATGTCCGAGCTTAATGAAAAGTT-3 '
[서열번호 26]: 5'-CACGGTCGTGCAGCGCCATCAGAGAGGCTTCGTAGCTGTAC TTGTCGTGCATGTAGTGGATGATGTTCAGTGC-3’[SEQ ID NO 26]: 5'-CACGGTCGTGCAGCGCCATCAGAGAGGCTTCGTAGCTGTAC TTGTCGTGCATGTAGTGGATGATGTTCAGTGC-3 '
3-6: CS10 균주의 제조3-6: Preparation of CS10 Strain
서열번호 27과 28, 서열번호 29과 30, 서열번호 31과 32의 프라이머를 이용하고, 주형으로써 각각 E. coli W3110(ATCC 39936) (derived from E. coli K-12, λ-, F-, prototrophic)의 게놈 DNA, 플라스미드인 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278: 78-85, 2008), E. coli W3110(ATCC 39936) (derived from E. coli K-12, λ-, F-, prototrophic)의 게놈 DNA를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 각각 PCR 절편을 얻고, 세 개의 PCR 절편을 주형으로 하고 서열번호 27 및 32의 프라이머를 이용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, PEP-protein phosphotransferase of PTS system을 코딩하는 유전자 (ptsI) 가 결실된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-5에서 제조된 CS07 균주에 electroporation하여 CS10 균주(W3110 ΔlacI ΔgabD ΔyneI ΔldhA ΔadhE ΔpflB ΔptsI )를 제조하였다.SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, using the primers of SEQ ID NOS: 31 and 32, each as a template E. coli W3110 (ATCC 39936) ( derived from E. coli K-12, λ -, 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, λ − , 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. By performing PCR under the same conditions as in 1-1 using the primers of 32, a PCR product in which the gene ( ptsI ) encoding the PEP-protein phosphotransferase of PTS system was deleted was prepared. Next, the purified PCR product was purified and then electroporated to the CS07 strain prepared in 3-5 to prepare a CS10 strain (W3110 Δ lacI Δ gabD Δ yne I Δ ldhA Δ adhE Δ pflB Δ ptsI ).
[서열번호 27] : 5'- CGCAACAATTGCACGTCATTT-3'[SEQ ID NO 27]: 5'- CGCAACAATTGCACGTCATTT-3 '
[서열번호 28] : 5'-CTATAGTGTCACCtaCAGCAACAGAAGTGTAGCACG-3'[SEQ ID NO 28]: 5'-CTATAGTGTCACCtaCAGCAACAGAAGTGTAGCACG-3 '
[서열번호 29] : 5'-ACACTTCTGTTGCTGTAGGTGACACTATAGAACGCGG-3'[SEQ ID NO 29]: 5'-ACACTTCTGTTGCTGTAGGTGACACTATAGAACGCGG-3 '
[서열번호 30] : 5'-GCATCTCGTGGATTACCGCATAGGCCACTAGTGGA-3'[SEQ ID NO 30]: 5'-GCATCTCGTGGATTACCGCATAGGCCACTAGTGGA-3 '
[서열번호 31] : 5'-TGGCCTATGCGGTAATCCACGAGATGCGGCCC-3'[SEQ ID NO 31]: 5'-TGGCCTATGCGGTAATCCACGAGATGCGGCCC-3 '
[서열번호 32] : 5'-AGATTTTACCAATGGTGCCGTC-3'[SEQ ID NO 32]: 5'-AGATTTTACCAATGGTGCCGTC-3 '
3-7: CS16 균주의 제조3-7: Preparation of CS16 Strain
서열번호 33 및 34의 프라이머, 주형으로써 플라스미드 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278: 78-85, 2008)를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, PEP-protein phosphotransferase of PTS system을 코딩하는 유전자 (ptsG) 가 결실된 PCR 산물을 제조하였다. 그 후, 이중교차상동 재조합을 강화하기 위해 상기 PCR 산물을 주형으로 하여, 서열번호 35 및 36의 프라이머를 이용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, ptsG 유전자와 상동서열 개수가 증가된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-5에서 제조된 CS07 균주에 electroporation하여 CS16 균주(W3110 ΔlacI ΔgabD ΔyneI ΔldhA ΔadhE ΔpflB ΔptsG)를 제조하였다.Using the primers of SEQ ID NOS: 33 and 34, template plasmid pECmulox (Kim, JM, Lee, KH & Lee, SY, FEMS Microbiol. Lett ., 278: 78-85, 2008) under the same conditions as in 1-1 above By performing PCR, a PCR product in which the gene ( ptsG ) encoding the PEP-protein phosphotransferase of PTS system was deleted was prepared. Thereafter, using the PCR product as a template to enhance double-cross homologous recombination, PCR was performed under the same conditions as in 1-1 using primers SEQ ID NOs: 35 and 36, thereby increasing the number of ptsG genes and homologous sequences. 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 Δ lacI Δ gabD Δ yne I Δ ldhA Δ adhE Δ pflB Δ ptsG ).
[서열번호 33]: 5'-CCTGTACACGGCGAGGCTCTCCCCCCTTGCCACGCGTGAGAACGTAAAAAGACACTATAGAACGCGGCCG-3'[SEQ ID NO: 33]: 5'-CCTGTACACGGCGAGGCTCTCCCCCCTTGCCACGCGTGAGAACGTAAAAAGACACTATAGAACGCGGCCG-3 '
[서열번호 34]: 5'-GAGAGAAGGTCTGGATTGCAGAACCAATCGGCGGCCAAATGAAGGACAGCCCGCATAGGCCACTAGTGGA-3'[SEQ ID NO 34]: 5'-GAGAGAAGGTCTGGATTGCAGAACCAATCGGCGGCCAAATGAAGGACAGCCCGCATAGGCCACTAGTGGA-3 '
[서열번호 35]: 5'-TGGCACTGAATTATTTTACTCTGTGTAATAAATAAAGGGCGCTTAGATGCCCTGTACACGGCGAGGCTCT-3'[SEQ ID NO 35]: 5'-TGGCACTGAATTATTTTACTCTGTGTAATAAATAAAGGGCGCTTAGATGCCCTGTACACGGCGAGGCTCT-3 '
[서열번호 36]: 5'-GAAACCGTAAATGCCAAACGCAACTACCGGGTTCTGGTAAGCAGCCCACTGAGAGAAGGTCTGGATTGCAGA-3'[SEQ ID NO: 36]: 5'-GAAACCGTAAATGCCAAACGCAACTACCGGGTTCTGGTAAGCAGCCCACTGAGAGAAGGTCTGGATTGCAGA-3 '
3-8: CS03/p99SC4CD 균주의 제조3-8: Preparation of CS03 / p99SC4CD Strain
실시예 3-2에서 제조된 CS03 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS03/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
3-9: CS05/p99SC4CD 균주의 제조3-9: Preparation of CS05 / p99SC4CD Strain
실시예 3-3에서 제조된 CS05 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS05/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
3-10: CS06/p99SC4CD 균주의 제조3-10: Preparation of CS06 / p99SC4CD Strain
실시예 3-4에서 제조된 CS06 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS06/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
3-11: CS07/p99SC4CD 균주의 제조3-11: Preparation of CS07 / p99SC4CD Strain
실시예 3-5에서 제조된 CS07 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS07/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
3-12: CS10/p99SC4CD 균주의 제조3-12: Preparation of CS10 / p99SC4CD Strain
실시예 3-6에서 제조된 CS10 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS10/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
3-13: CS16/p99SC4CD 균주의 제조3-13: Preparation of CS16 / p99SC4CD Strain
실시예 3-6에서 제조된 CS16 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS16/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
실시예 4: 4-하이드록시부티릭산 생산의 증가를 위해 경쟁 회로들의 유전자가 결실된 변이 미생물을 이용한 4-하이드록시부티릭산의 제조Example 4 Preparation of 4-hydroxybutyric Acid Using Mutant Microorganisms Deleting Genes of Competition Circuits for Increased 4-hydroxybutyric Acid Production
실시예 3에서 제조된 표 2의 변이 균주들을 실시예 2와 동일한 방법으로 배양하였다. 그 후 배양액을 실시예 2와 동일한 방법으로 처리하고, GC 분석을 통해 4-하이드록시부티릭산의 농도를 측정하고, 표 2에 나타내었다.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.
표 2
Figure PCTKR2012010665-appb-T000002
 TABLE 2
Figure PCTKR2012010665-appb-T000002
표 2로부터, gabD, yneI, ldhA, adhE, pflB, ptsI 및 ptsG 유전자로 구성된 군에서 선택되는 하나 이상의 유전자가 추가로 결실된 변이 미생물들 모두 4-하이드록시부티릭산 생성능이 향상되었음을 알 수 있었다.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.
실시예 5: 보충회로(anaplerotic pathway) 유전자가 증폭된 변이 미생물의 제조Example 5 Preparation of Mutant Microorganisms Amplified with an Anaplerotic Pathway Gene
5-1: 플라스미드 p15PckA의 제작5-1: Construction of the plasmid p15PckA
항시적, 생합성적 Mannheimia succiniciproducens(KCTC 0769BP)의 phosphoenolpyruvate carboxykinase를 코딩하는 유전자 pckA를 tac promoter로 강한 유전자 발현을 진행하는 pTac15K(p15A origin, low copies, KmR; KAISTMBELstock) 발현벡터에 클로닝하였다. Always, biosyntheticMannheimia succiniciproducens(KCTC 0769BP) gene coding for phosphoenolpyruvate carboxykinasepckAPTac15K (p15A origin, low copies, Km)R; KAISTMBELstock) cloned into the expression vector.
참고로, pTac15k의 제조방법은 다음과 같다. pHCE IIB(NcoI) ((주)바이오리더스)를 AatII와 NheI으로 자르고 pACYC177(New England Biolabs)도 AaII와 NheI으로 자른 후, pHCE IIB(NcoI)의 pHCD promoter를 포함하는 절편이랑 pACYC177의 p15A origin과 암피실린 항생제를 포함하는 절편과 중합시켜, pHNC15라는 벡터를 만들었다. 암피실린을 카나마이신으로 만들기 위해 pHNC15를 FspI으로 자르고 pUC4K (GE Healthcare Life Sciences)를 pstI으로 자른 후, Fill-in 하고, 중합시켜 pHNC15에서 암피실린 대신 카나마이신이 들어간 pHNC15K 벡터를 완성하였다. 다음으로 pHNC15K에서 promoter를 pHCE 대신 pTac으로 바꾸기 위해 pHNC15K를 NheI으로 자른 후 filling in 한 후, EcoRI으로 잘랐다, 이 때 생긴 절편은 pHNC15K에서 pHCE부분이 제거된 절편이다. tac promoter를 얻기 위해 pKK223-3 (Pharmacia Biotech)를 sphI으로 자르고 filling in 한 후 EcoRI으로 잘랐다. 이 때 생긴 절편 중 tac promoter를 포함한 절편과 위에서 언급한 절편을 중합하여 pTac15K을 완성하였다.For reference, the manufacturing method of pTac15k is as follows. 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. To make ampicillin kanamycin, 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. Next, in order to change the promoter into pTac instead of pHCE in pHNC15K, the 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. To obtain a tac promoter, 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.
M. succiniciproducens(KCTC 0769BP)의 게놈 DNA를 주형으로 하고, 합성된 서열번호 37과 38의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, pckA 절편을 제조하였다. 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.
[서열번호 37]: 5'-ATATTAGAATTCATGACAGATCTTAATCAATT-3'[SEQ ID NO: 37]: 5'-ATATTAGAATTCATGACAGATCTTAATCAATT-3 '
[서열번호 38]: 5'-TGCTACGAGCTCTTATGCTTTAGGACCGGCAGC-3'[SEQ ID NO: 38]: 5'-TGCTACGAGCTCTTATGCTTTAGGACCGGCAGC-3 '
다음으로 제조된 pckA 절편 및 pTac15k 플라스미드에 제한효소(EcoRI와 SacI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 pckA 절편 및 pTac15k 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p15PckA를 제작하였다.Next, 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.
5-2: 플라스미드 p15Pyc의 제작5-2: Construction of the plasmid p15Pyc
항시적, 생합성적 Corynebacterium glutamicum(ATCC 13032)의 phosphoenolpyruvate carboxykinase를 코딩하는 유전자 pyc를 tac promoter로 강한 유전자 발현을 진행하는 pTac15K(p15A origin, low copies, KmR; KAISTMBELstock) 발현벡터에 클로닝하였다. C. glutamicum(ATCC 13032)의 게놈 DNA를 주형으로 하고, 합성된 서열번호 39와 40의 프라이머를 이용하여, PCR을 수행함으로써, pyc 절편을 제조하였다. PCR 조건은 step 4의 시간이 1min 45 sec인 점을 제외하고는 실시예 1-1과 동일하다.Always, biosyntheticCorynebacterium glutamicum(ATCC 13032) gene coding for phosphoenolpyruvate carboxykinasepycPTac15K (p15A origin, low copies, Km)R; KAISTMBELstock) cloned into the expression vector.C. glutamicumBy using genomic DNA of (ATCC 13032) as a template and PCR using the primers of SEQ ID NOs: 39 and 40 synthesized,pycSections were prepared. PCR conditions were the same as in Example 1-1 except that the time of step 4 was 1min 45 sec.
[서열번호 39]: 5'-ATCGGAGCTCGTGTCGACTCACACATCTTC-3'[SEQ ID NO 39]: 5'-ATCGGAGCTCGTGTCGACTCACACATCTTC-3 '
[서열번호 40]: 5'-GCTGTCTAGATTAGGAAACGACGACGATCA-3'[SEQ ID NO 40]: 5'-GCTGTCTAGATTAGGAAACGACGACGATCA-3 '
다음으로 제조된 pyc 절편 및 pTac15k 플라스미드에 제한효소(SacI와 XbaI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 pckA 절편 및 pTac15k 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p15Pyc를 제작하였다.Next, 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. Was produced.
5-3: 플라스미드 p15CY의 제작5-3: Construction of plasmid p15CY
항시적, 생합성적 Corynebacterium glutamicum(ATCC 13032)의 phosphoenolpyruvate carboxykinase를 코딩하는 유전자 pyc를 상기 5-1에서 제작된 p15PckA에 클로닝 하였다. 발현은 pyc 유전자 앞의 RBS(ribosome binding site)에 의해 pckA 유전자 앞에 있는 tac promoter의 영향을 받는다. C. glutamicum의 게놈 DNA를 주형으로 하고, 합성된 서열번호 41과 40의 프라이머를 이용하여, 상기 5-2와 동일한 조건으로 PCR을 수행함으로써, pyc 절편을 제조하였다. Always, biosyntheticCorynebacterium glutamicum(ATCC 13032) gene coding for phosphoenolpyruvate carboxykinasepycWas cloned into p15PckA prepared in 5-1. Expression ispycBy RBS (ribosome binding site) in front of genepckAAffected by the tac promoter in front of the gene.C. glutamicumBy 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,pycSections were prepared.
[서열번호 41]: 5'-ATTAGAGAGCTCACAGGAAACAGTGTCGACTCACACATCTT CAAC-3'[SEQ ID NO 41]: 5'-ATTAGAGAGCTCACAGGAAACAGTGTCGACTCACACATCTT CAAC-3 '
다음으로 제조된 pyc 절편 및 실시예 5-1에서 제작된 p15PckA 플라스미드에 제한효소(SacI 와 XbaI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 pyc 절편 및 p15PckA 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p15CY를 제작하였다.Next, 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. By polymerization, the recombinant pplasmid vector p15CY was produced.
5-4: 플라스미드 p15Ppc의 제작5-4: Construction of plasmid p15Ppc
항시적, 생합성적 E. coli(W3110)의 phosphoenolpyruvate carboxylase를 코딩하는 유전자 ppc를 tac promoter로 강한 유전자 발현을 진행하는 pTac15K(p15A origin, low copies, KmR; KAISTMBELstock) 발현벡터에 클로닝하였다. E. coli(W3110)의 게놈 DNA를 주형으로 하고, 합성된 서열번호 42와 43의 프라이머를 이용하여, PCR을 수행함으로써, ppc 절편을 제조하였다. PCR 조건은 step 4의 시간이 1min 45 sec인 점을 제외하고는 실시예 1-1과 동일하다.Always, biosyntheticE. coli(W3110) gene coding for phosphoenolpyruvate carboxylaseppcPTac15K (p15A origin, low copies, Km)R; KAISTMBELstock) cloned into the expression vector.E. coliBy using the genomic DNA of (W3110) as a template and PCR using the primers of SEQ ID NOs: 42 and 43 synthesized,ppcSections were prepared. PCR conditions were the same as in Example 1-1 except that the time of step 4 was 1min 45 sec.
[서열번호 42]: 5'-ATTGTAGAATTCATGAACGAACAATATTCCGCATT-3'[SEQ ID NO: 42]: 5'-ATTGTAGAATTCATGAACGAACAATATTCCGCATT-3 '
[서열번호 43]: 5'-ATAGTGGAGCTCTTAGCCGGTATTACGCATACCT-3'[SEQ ID NO: 43]: 5'-ATAGTGGAGCTCTTAGCCGGTATTACGCATACCT-3 '
다음으로 제조된 ppc 절편 및 pTac15k 플라스미드에 제한효소(EcoRI와 SacI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 ppc 절편 및 pTac15k 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p15Ppc를 제작하였다.Next, 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. Was produced.
5-5: CS10/p99SC4CD/p15PckA 균주의 제조5-5: Preparation of CS10 / p99SC4CD / p15PckA Strains
실시예 3-12에서 제조된 CS10/p99SC4CD 균주에 상기 5-1에서 제작된 p15PckA 벡터를 도입시켜 CS10/p99SC4CD/p15PckA 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-6: CS10/p99SC4CD/p15Pyc 균주의 제조5-6: Preparation of CS10 / p99SC4CD / p15Pyc Strains
실시예 3-12에서 제조된 CS10/p99SC4CD 균주에 상기 5-2에서 제작된 p15Pyc 벡터를 도입시켜 CS10/p99SC4CD/p15Pyc 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-7: CS10/p99SC4CD/p15CY 균주의 제조5-7: Preparation of CS10 / p99SC4CD / p15CY Strains
실시예 3-12에서 제조된 CS10/p99SC4CD 균주에 상기 5-3에서 제작된 p15CY 벡터를 도입시켜 CS10/p99SC4CD/p15CY 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-8: CS10/p99SC4CD/pTac15K 균주의 제조5-8: Preparation of CS10 / p99SC4CD / pTac15K Strains
실시예 3-12에서 제조된 CS10/p99SC4CD 균주에 pTac15K 벡터를 도입시켜 CS10/p99SC4CD/pTac15K 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-9: CS16/p99SC4CD/p15PckA 균주의 제조5-9: Preparation of CS16 / p99SC4CD / p15PckA Strains
실시예 3-13에서 제조된 CS16/p99SC4CD 균주에 상기 5-1에서 제작된 p15PckA 벡터를 도입시켜 CS16/p99SC4CD/p15PckA 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-10: CS16/p99SC4CD/p15Ppc 균주의 제조5-10: Preparation of CS16 / p99SC4CD / p15Ppc Strains
실시예 3-13에서 제조된 CS16/p99SC4CD 균주에 상기 5-4에서 제작된 p15Ppc 벡터를 도입시켜 CS16/p99SC4CD/p15Ppc 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-11: CS16/p99SC4CD/p15Pyc 균주의 제조5-11: Preparation of CS16 / p99SC4CD / p15Pyc Strain
실시예 3-13에서 제조된 CS16/p99SC4CD 균주에 상기 5-2에서 제작된 p15Pyc 벡터를 도입시켜 CS16/p99SC4CD/p15Pyc 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
5-12: CS16/p99SC4CD/pTac15K 균주의 제조5-12: Preparation of CS16 / p99SC4CD / pTac15K Strains
실시예 3-13에서 제조된 CS16/p99SC4CD 균주에 pTac15K 벡터를 도입시켜 CS16/p99SC4CD/pTac15K 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar)고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
실시예 6: 보충회로(anaplerotic pathway) 유전자가 증폭된 변이 미생물을 이용한 4-하이드록시부티릭산의 제조 Example 6 Preparation of 4-Hydroxybutyric Acid Using Mutant Microorganisms Amplified with an Anaplerotic Pathway Gene
실시예 5에서 제조된 표 3의 변이 균주들을 실시예 2와 동일한 방법으로 배양하였다. 다른 점은 생산균주에 따라 엠피실린(ampicilin)만 들어간 LB 평판배지나 엠피실린(ampicilin)과 카나마이신(kanamycin)이 같이 들어간 LB 평판배지에서 선별하였다. 그 후 배양액을 실시예 2와 마찬가지 방법으로 처리하고 GC 분석을 통해 4-하이드록시부티릭산의 농도를 측정하고, 표 3에 기재하였다.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.
표 3
Figure PCTKR2012010665-appb-T000003
 TABLE 3
Figure PCTKR2012010665-appb-T000003
표 3으로부터, 단순히 strong promoter를 이용한 미생물보다는 보충회로 유전자인 pckA, pyc 및 ppc 유전자들이 증폭된 변이 미생물들 모두 4-하이드록시부티릭산 생성능이 향상되었음을 알 수 있었다.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.
실시예 7: 산화적 회로(oxidative pathway) 유전자가 증폭되거나 강화된 변이 미생물의 제조Example 7 Preparation of Mutant Microorganisms Amplified or Enhanced Oxidative Pathway Genes
7-1: 플라스미드 p15PpcGltAR163L의 제작7-1: Construction of the plasmid p15PpcGltAR163L
항시적, 생합성적 E. coli(W3110)의 citrate synthase를 코딩하는 유전자 gltA를 tac promoter로 강한 유전자 발현을 진행하는 pTac15K(p15A origin, low copies, KmR; KAISTMBELstock) 발현벡터에 클로닝하였다. 여기서 gltA는 NADH에 의해 저해를 받는 기작을 제거하기 위해 돌연변이가 일어난 것을 사용하였다 (Pereira, D.S., Donald, I.J., Hosfield, D.J. ＆ Duckworth, H.W. J. Biol. Chem. 269: 412-417, 1994).Always, biosyntheticE. coli(W3110) gene coding for citrate synthasegltAPTac15K (p15A origin, low copies, Km)R; KAISTMBELstock) cloned into the expression vector. heregltAMutations were used to eliminate mechanisms that are inhibited by NADH (Pereira, D.S., Donald, I.J., Hosfield, D.J. & Duckworth, H.W.J. Biol. Chem.269: 412-417, 1994).
즉, 163번째 아미노산인 arginine(R)이 leucine(L)으로 치환된 돌연변이를 만들기 위해 E. coli(W3110)의 게놈 DNA를 주형으로 하고, 합성된 서열번호 44과 45 및 46와 47의 프라이머를 이용하여, 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 각각의 대해서 PCR 절편을 제조하였다. In other words, genomic DNA of E. coli (W3110) 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. Using the PCR under the same conditions as in 1-1, PCR fragments were prepared for each.
[서열번호 44]: 5'-GCTGTCGGTACCAGGAGACCTTAAATGGCTGATACA-3'[SEQ ID NO: 44]: 5'-GCTGTCGGTACCAGGAGACCTTAAATGGCTGATACA-3 '
[서열번호 45]: 5'-GGAACGCGGCAATTTCACGGTGACGAGGATTGTTAACATCCA-3'[SEQ ID NO 45]: 5'-GGAACGCGGCAATTTCACGGTGACGAGGATTGTTAACATCCA-3 '
[서열번호 46]: 5'-AAATTGCCGCGTTCCTCCTGCTGTCGAAAAT-3'[SEQ ID NO 46]: 5'-AAATTGCCGCGTTCCTCCTGCTGTCGAAAAT-3 '
[서열번호 47]: 5'-TGCTACGAGCTCTTATGCTTTAGGACCGGCAGC-3'[SEQ ID NO 47]: 5'-TGCTACGAGCTCTTATGCTTTAGGACCGGCAGC-3 '
다음으로 제조된 각각의 PCR 절편들을 주형으로 하여 서열번호 44와 47의 프라이머를 이용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 돌연변이가 일어난 gltA 절편을 제조할 수 있었다. 다음으로 제조된 gltA 절편 및 p15Ppc 플라스미드에 제한효소(KpnI와 XbaI)를 처리한 후, T4 DNA ligase를 처리하여, 제한효소로 절단된 돌열변이 gltA 절편 및 p15Ppc 플라스미드를 중합시킴으로써, 재조합 플라스미드인 벡터 p15PpcGltAR163L를 제작하였다.Next, 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. After then process the restriction enzyme (Kpn I and Xba I) to gltA fragments and p15Ppc plasmid prepared by, 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.
7-2: CS28 균주의 제조7-2: Preparation of CS28 Strain
서열번호 48과 49, 서열번호 50과 51, 서열번호 52와 53의 프라이머를 이용하고, 주형으로써 각각 E. coli W3110(ATCC 39936) (derived from E. coli K-12, λ-, F-, prototrophic)의 게놈 DNA, 플라스미드인 pECmulox(Kim, J.M., Lee, K.H. ＆ Lee, S.Y., FEMS Microbiol. Lett., 278: 78-85, 2008), E. coli W3110(ATCC 39936) (derived from E. coli K-12, λ-, F-, prototrophic)의 게놈 DNA를 사용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, 각각 PCR 절편을 얻고, 세 개의 PCR 절편을 주형으로 하고 서열번호 48 및 53의 프라이머를 이용하여 상기 1-1과 동일한 조건으로 PCR을 수행함으로써, aerobic respiration control protein ArcA를 코딩하는 유전자 (arcA) 가 결실된 PCR 산물을 제조하였다. 다음으로, 제조된 PCR 산물을 정제한 후, 상기 3-7에서 제조된 CS16 균주에 electroporation하여 CS28 균주(W3110 ΔlacI ΔgabD ΔyneI ΔldhA ΔadhE ΔpflB ΔptsGΔarcA)를 제조하였다.SEQ ID NO: 48 and 49, SEQ ID NO: 50 and 51, SEQ ID NO: 52 and 53, and using the primers of each mold as E. coli W3110 (ATCC 39936) ( derived from E. coli K-12, λ -, 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, λ − , 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. Next, the purified PCR product was purified, and then electroporated to the CS16 strain prepared in 3-7 to prepare a CS28 strain (W3110 Δ lacI Δ gabD Δ yne I Δ ldhA Δ adhE Δ pflB Δ ptsG Δ arcA ).
[서열번호 48] : 5'- TTTTGACACTGTCGGGTCCT-3'[SEQ ID NO 48]: 5'- TTTTGACACTGTCGGGTCCT-3 '
[서열번호 49] : 5'-GCGTTCTATAGTGTCTTTCAACGTGTTGCGTGTTA-3'[SEQ ID NO 49]: 5'-GCGTTCTATAGTGTCTTTCAACGTGTTGCGTGTTA-3 '
[서열번호 50] : 5'-CGCAACACGTTGAAAGACACTATAGAACGCGGCCG-3'[SEQ ID NO 50]: 5'-CGCAACACGTTGAAAGACACTATAGAACGCGGCCG-3 '
[서열번호 51] : 5'-GATTCGAAATGTTTACCGCATAGGCCACTAGTGGA-3'[SEQ ID NO 51]: 5'-GATTCGAAATGTTTACCGCATAGGCCACTAGTGGA-3 '
[서열번호 52] : 5'-TAGTGGCCTATGCGGCATTTCGAATCTACGCCGGA-3'[SEQ ID NO 52]: 5'-TAGTGGCCTATGCGGCATTTCGAATCTACGCCGGA-3 '
[서열번호 53] : 5'-CGGCATGATGTTTGTGACCC-3'[SEQ ID NO 53]: 5'-CGGCATGATGTTTGTGACCC-3 '
7-3: CS16/p99SC4CD/p15PpcGltAR163L 균주의 제조7-3: Preparation of CS16 / p99SC4CD / p15PpcGltAR163L Strains
실시예 3-13에서 제조된 CS16/p99SC4CD 균주에 상기 7-1에서 제작된 p15PpcGltAR163L 벡터를 도입시켜 CS16/p99SC4CD/p15PpcGltAR163L 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar) 고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
7-4: CS28/p99SC4CD 균주의 제조7-4: Preparation of CS28 / p99SC4CD Strain
실시예 7-2에서 제조된 CS28 균주에 상기 1-5에서 제조된 p99SC4CD 벡터를 도입시켜 CS28/p99SC4CD 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin)이 포함된 아가(agar) 고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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).
7-5: CS28/p99SC4CD/p15PpcGltAR163L 균주의 제조 7-5: Preparation of CS28 / p99SC4CD / p15PpcGltAR163L Strains
실시예 7-4에서 제조된 CS28/p99SC4CD 균주에 상기 7-1에서 제작된 p15PpcGltAR163L 벡터를 도입시켜 CS28/p99SC4CD/p15PpcGltAR163L 균주를 제조하였다. 다음으로 제조된 균주를 암피실린(ampicillin) 및 카나마이신(kanamycin)을 포함된 아가(agar) 고체 배지에서 배양하면서 재조합된 변이 미생물들을 선별하였다.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.
실시예 8: 산화적 회로(oxidative pathway) 유전자가 증폭되거나 강화된 변이 미생물을 이용한 4-하이드록시부티릭산의 제조 Example 8: Preparation of 4-hydroxybutyric acid using mutant microorganisms amplified or enhanced oxidative pathway genes
실시예 7 에서 제조된 표 4의 변이 균주들을 실시예 2와 동일한 방법으로 배양하였다. 다른 점은 생산균주에 따라 엠피실린(ampicilin)만 들어간 LB 평판배지나 엠피실린(ampicilin)과 카나마이신(kanamycin)이 같이 들어간 LB 평판배지에서 선별하였다. 그 후 배양액을 실시예 2와 동일한 방법으로 처리하고 GC 분석을 통해 4-하이드록시부티릭산의 농도를 측정하고, 표 4에 기재하였다.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.
표 4
Figure PCTKR2012010665-appb-T000004
 Table 4
Figure PCTKR2012010665-appb-T000004
표 4로부터, 산화적 회로 유전자인 gltA가 증폭되거나 및 산화적회로 유전자들을 저해하는 조절 단백질을 코딩하는 arcA 유전자가 제거된 변이 미생물들 모두 4-하이드록시부티릭산 생성능이 향상되었음을 알 수 있었다.From Table 4, it was found that all of the mutant microorganisms in which the oxidative circuit gene gltA was amplified and in which the arcA gene encoding the regulatory protein that inhibited the oxidative circuit genes were removed were improved in 4-hydroxybutyric acid production ability .
이상 설명한 바와 같이, 본 발명에 따른 4-하이드록시부티릭산 생성능을 가지는 변이 미생물은 4-하이드록시부티릭산을 고수율로 제조하는데 유용하다.As described above, 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.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.

Claims (25)

  1. 4-하이드록시부티릭산의 생합성에 관여하는 코에이 트랜스퍼라아제(CoA transferase)를 코딩하는 유전자(cat1), 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD), 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자(sucD) 및 4-하이드록시부티릭산 디하이드로게나아제(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자(4hbD 또는 yqhD)로 구성된 군에서 선택되는 하나 이상이 증폭되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.Gene encoding CoA transferase ( Ca1 ) involved in biosynthesis of 4-hydroxybutyric acid ( cat1 ), gene encoding succinyl-CoA synthetase ( sucCD ), nose Genes encoding co-dependent succinate semialdehyde dehydrogenase ( sucD ) and genes encoding 4-hydroxybutyrate dehydrogenase ( 4hbD or yqhD A mutant microorganism having 4-hydroxybutyric acid producing ability, wherein at least one selected from the group consisting of a) is amplified.
  2. 제1항에 있어서, 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 또는 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)로 구성된 군에서 선택되는 하나 이상이 추가로 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.The method of claim 1, wherein the gene encoding succinate semialdehyde dehydrogenase ( gabD or yneI), lactate dehydrogenase involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein of PTS system 4-hydroxybutyric acid producing ability, characterized in that at least one selected from the group consisting of genes ( ptsI or ptsG ) encoding phosphotransferase of PTS system is further weakened or deleted. Mutant microorganisms.
  3. 제2항에 있어서, 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 하나 이상이 추가로 증폭되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.The method of claim 2, wherein the gene encoding phosphoenolpyruvate carboxykinase ( pckA ), phosphoenolppyruvate carboxylase (phosphoenolpyrutave carboxylase) is involved in the anaplerotic pathway (coding) At least one selected from the group consisting of a gene ( ppc ) and a gene encoding a pyruvate carboxylase ( pyc ), wherein the ability to generate 4-hydroxybutyric acid is further amplified. Eggplant is a mutant microorganism.
  4. 제2항 또는 제3항에 있어서, 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.The gene ( gltA ) according to claim 2 or 3, wherein (i) of the genes involved in the oxidative pathway (i) encodes a mutated citrate synthase such that inhibition by NADH is eliminated. Further amplification; (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 a mutant microorganism having a 4-hydroxybutyric acid producing ability, characterized in that further weakened or deleted.
  5. 제1항에 있어서, 상기 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자 (sucD)는 strong promoter를 함유하는 발현벡터 형태로 도입된 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.According to claim 1, wherein the gene encoding the CoA-dependent succinate semialdehyde dehydrogenase ( sucD ) is characterized in that introduced in the form of an expression vector containing a strong promoter -Mutant microorganisms having hydroxybutyric acid producing ability.
  6. 제3항에 있어서, 상기 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)는 strong promoter를 함유하는 발현벡터 형태로 도입된 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.The method of claim 3, wherein the gene encoding a phosphoenolpyruvate carboxykinase ( pckA ), phosphoenolpyruvate carboxylase involved in the anaplerotic pathway (phosphoenolpyrutave carboxylase) Gene encoding ( ppc ) and gene encoding pyruvate carboxylase (pyruvate carboxylase) ( pyc ) has a 4-hydroxybutyric acid generating ability, characterized in that introduced in the form of an expression vector containing a strong promoter Mutant microorganisms.
  7. 제5항 또는 제6항에 있어서, 상기 strong promoter는 trc promoter, tac promoter, T7 promoter, lac promoter 및 trp promoter로 구성된 군에서 선택되는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.The mutant microorganism having the 4-hydroxybutyric acid producing ability according to claim 5 or 6, wherein the strong promoter is selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter, and a trp promoter.
  8. 제1항에 있어서, 상기 미생물은 바실러스 속(Bacillus sp.), 코리네박테리움 속(Corynebacterium sp.), 에스케리치아 속(Escherichia sp.), 피치아 속(Pichia sp.), 슈도모나스 속(Pseudomonas sp.) 및 사카로마이세스 속(Saccharomyces sp.)으로 구성된 군에서 선택되는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.The method of claim 1, wherein the microorganisms are of the genus Bacillus sp., Corynebacterium sp., Escherichia sp., Pichia sp., Pseudomonas genus. Pseudomonas sp.) And Saccharomyces sp.) A mutant microorganism having a 4-hydroxybutyric acid producing ability, characterized in that selected from the group consisting of.
  9. (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)가 증폭되어 있고, (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)가 약화 또는 결실되어 있으며,(A) A gene encoding a succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid ( sucCD ) is amplified, and (B) Production of 4-hydroxybutyric acid Genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), genes encoding lactate dehydrogenase ( ldhA ), pyruvic acid-formic acid lyase (pyruvate formate lyase) ( pflB ), a gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransferase of PTS system (PEP-protein phosphotransferase of PTS system) The gene encoding ptsI or ptsG is weakened or deleted,
    (C) 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc)및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 유전자가 증폭되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.(C) genes encoding phosphoenolpyruvate carboxykinase ( pckA ) involved in the anaplerotic pathway ( pckA ), genes encoding phosphoenolpyruvate carboxylase (phosphoenolpyrutave carboxylase) ppc ) and a mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that a gene selected from the group consisting of a gene encoding pyruvate carboxylase ( pyc ) is amplified.
  10. (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)가 증폭되어 있고,(A) a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid is amplified,
    (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)가 약화 또는 결실되어 있으며,(B) genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), lactate dehydrogenase (lactate dehydrogenase), which are involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransfer of the PTS system The gene encoding PPE -protein phosphotransferase of PTS system ( ptsI or ptsG ) is weakened or deleted,
    (C) 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.(C) amplification of genes involved in the oxidative pathway (i) a gene ( gltA ) encoding a mutated citrate synthase such that inhibition by NADH is eliminated; (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 ) A mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that it is weakened or deleted.
  11. (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)가 증폭되어 있고,(A) a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid is amplified,
    (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)가 약화 또는 결실되어 있고,(B) genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), lactate dehydrogenase (lactate dehydrogenase), which are involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransfer of the PTS system The gene encoding PPE -protein phosphotransferase of PTS system ( ptsI or ptsG ) is weakened or deleted,
    (C) 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc)및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 유전자가 증폭되어 있으며,(C) genes encoding phosphoenolpyruvate carboxykinase ( pckA ) involved in the anaplerotic pathway ( pckA ), genes encoding phosphoenolpyruvate carboxylase (phosphoenolpyrutave carboxylase) ppc ) and a gene selected from the group consisting of the gene ( pyc ) encoding pyruvate carboxylase (amplified), amplified
    (D) 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물.(D) of genes involved in the oxidative pathway (i) amplify the gene ( gltA ) encoding a mutated citrate synthase such that inhibition by NADH is eliminated; (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 ) A mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that it is weakened or deleted.
  12. 4-하이드록시부티릭산의 생합성에 관여하는 코에이 트랜스퍼라아제(CoA transferase)를 코딩하는 유전자(cat1), 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD), 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자(sucD) 및 4-하이드록시부티릭산 디하이드로게나아제(4-hydroxybutyrate dehydrogenase)를 코딩하는 유전자(4hbD 또는 yqhD)로 구성된 군에서 선택되는 하나 이상을 증폭시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.Gene encoding CoA transferase ( Ca1 ) involved in biosynthesis of 4-hydroxybutyric acid ( cat1 ), gene encoding succinyl-CoA synthetase ( sucCD ), nose Genes encoding co-dependent succinate semialdehyde dehydrogenase ( sucD ) and genes encoding 4-hydroxybutyrate dehydrogenase ( 4hbD or yqhD Method for producing a mutant microorganism having a 4-hydroxybutyric acid producing ability, characterized in that for amplifying at least one selected from the group consisting of.
  13. 제12항에 있어서, 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 또는 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)로 구성된 군에서 선택되는 하나 이상이 추가로 약화 또는 결실되어 있는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.The method of claim 12, wherein the gene encoding succinate semialdehyde dehydrogenase ( gabD or yneI ), lactate dehydrogenase involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein of PTS system 4-hydroxybutyric acid producing ability, characterized in that at least one selected from the group consisting of genes ( ptsI or ptsG ) encoding phosphotransferase of PTS system is further weakened or deleted. Method for producing a mutant microorganism having a.
  14. 제12항 또는 제13항에 있어서, 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 하나 이상을 추가로 증폭시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.The gene ( pckA ) encoding phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase according to claim 12 or 13, which is involved in the anaplerotic pathway. 4-hydroxybuty characterized by further amplifying at least one selected from the group consisting of a gene encoding carboxylase ( ppc ) and a gene encoding pyruvate carboxylase ( pyc ). Method for producing a mutant microorganism having lyric acid producing ability.
  15. 제13항에 있어서, 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 추가로 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 추가로 약화 또는 결실시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.14. The method of claim 13, wherein (i) of the genes involved in the oxidative pathway (i) a gene encoding a mutated citrate synthase ( gltA ) to remove the inhibition by NADH is further amplified; (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 a method for producing a mutant microorganism having a 4-hydroxybutyric acid producing ability , characterized in that further weakened or deleted.
  16. 제12항에 있어서, 상기 코에이-의존적 숙신산 세미알데히드 디하이드로게나아제(CoA-dependent succinate semialdehyde dehydrogenase)를 코딩하는 유전자 (sucD)는 strong promoter를 함유하는 발현벡터 형태로 도입시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.The method of claim 12, wherein the gene encoding the coA-dependent succinate semialdehyde dehydrogenase ( sucD ) is characterized in that the introduction of a strong promoter in the form of an expression vector containing 4 -Method for producing mutant microorganisms having hydroxybutyric acid producing ability.
  17. 제14항에 있어서, 상기 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)는 strong promoter를 함유하는 발현벡터 형태로 도입시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.15. The method of claim 14, wherein the gene coding for phosphoenolpyruvate carboxykinase ( pckA ), phosphoenolpyruvate carboxylase involved in the anaplerotic pathway (phosphoenolpyrutave carboxylase) Gene encoding ( ppc ) and gene encoding pyruvate carboxylase (pyruvate carboxylase) ( pyc ) has a 4-hydroxybutyric acid generating ability, characterized in that the introduction into the expression vector containing a strong promoter Method for producing mutant microorganisms.
  18. 제16항 또는 제17항에 있어서, 상기 strong promoter는 trc promoter, tac promoter, T7 promoter, lac promoter 및 trp promoter로 구성된 군에서 선택되는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.18. The method of claim 16 or 17, wherein the strong promoter is a mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter and trp promoter Manufacturing method.
  19. 제12항에 있어서, 상기 미생물은 바실러스 속(Bacillus sp.), 코리네박테리움 속(Corynebacterium sp.), 에스케리치아 속(Escherichia sp.), 피치아 속(Pichia sp.), 슈도모나스 속(Pseudomonas sp.) 및 사카로마이세스 속(Saccharomyces sp.)으로 구성된 군에서 선택되는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.The method of claim 12, wherein the microorganisms are genus Bacillus sp., Corynebacterium sp., Escherichia sp., Pichia sp., Pseudomonas genus. Pseudomonas sp.) And Saccharomyces sp.) A method for producing a mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that it is selected from the group consisting of.
  20. (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)를 증폭시키고, (A) amplifying a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid,
    (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)를 약화 또는 결실시키고,(B) genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), lactate dehydrogenase (lactate dehydrogenase), which are involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransfer of the PTS system Attenuates or deletes the gene ( ptsI or ptsG ) that encodes the enzyme (PEP-protein phosphotransferase of PTS system),
    (C) 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc)및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 유전자를 증폭시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.(C) genes encoding phosphoenolpyruvate carboxykinase ( pckA ) involved in the anaplerotic pathway ( pckA ), genes encoding phosphoenolpyruvate carboxylase (phosphoenolpyrutave carboxylase) ppc ) and a method for producing a mutant microorganism having 4-hydroxybutyric acid producing ability, characterized by amplifying a gene selected from the group consisting of a gene encoding pyruvate carboxylase ( pyc ).
  21. (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)를 증폭시키고, (A) amplifying a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid,
    (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)를 약화 또는 결실시키고,(B) genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), lactate dehydrogenase (lactate dehydrogenase), which are involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransfer of the PTS system Attenuates or deletes the gene ( ptsI or ptsG ) that encodes the enzyme (PEP-protein phosphotransferase of PTS system),
    (C) 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.(C) amplification of genes involved in the oxidative pathway (i) a gene ( gltA ) encoding a mutated citrate synthase such that inhibition by NADH is eliminated; (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 ) A method for producing a mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that it is weakened or deleted.
  22. (A) 4-하이드록시부티릭산의 생합성에 관여하는 숙시닐-코에이 합성효소(succinyl-CoA synthetase)를 코딩하는 유전자(sucCD)를 증폭시키고, (A) amplifying a gene ( sucCD ) encoding succinyl-CoA synthetase involved in the biosynthesis of 4-hydroxybutyric acid,
    (B) 4-하이드록시부티릭산의 생산 경쟁 경로에 관여하는 숙신산 세미알데히드 디하이드로게나아제(succinate semialdehyde dehydrogenase)를 코딩하는 유전자(gabD 및 yneI), 젖산 디하이드로게나아제 (lactate dehydrogenase)를 코딩하는 유전자(ldhA), 피루빅산-포름산 리아제(pyruvate formate lyase)를 코딩하는 유전자(pflB), 알코올 디하이드로게나아제(alcohol dehydrogenase)를 코딩하는 유전자(adhE) 및 PTS 시스템의 PEP-단백질 포스포트랜스퍼라아제(PEP-protein phosphotransferase of PTS system)를 코딩하는 유전자(ptsI 또는 ptsG)를 약화 또는 결실시키고,(B) genes encoding succinate semialdehyde dehydrogenase ( gabD and yneI ), lactate dehydrogenase (lactate dehydrogenase), which are involved in the production competition pathway of 4-hydroxybutyric acid. Gene ( ldhA ), gene encoding pyruvate formate lyase ( pflB ), gene encoding alcohol dehydrogenase ( adhE ) and PEP-protein phosphotransfer of the PTS system Attenuates or deletes the gene ( ptsI or ptsG ) that encodes the enzyme (PEP-protein phosphotransferase of PTS system),
    (C) 보충 회로(anaplerotic pathway)에 관여하는 포스포엔올피루빅산 카르복시키나아제(phosphoenolpyruvate carboxykinase)를 코딩하는 유전자(pckA), 포스포엔올피루브산 카르복실라아제(phosphoenolpyrutave carboxylase)를 코딩하는 유전자(ppc) 및 피루빅산 카르복실라아제(pyruvate carboxylase)를 코딩하는 유전자(pyc)로 구성된 군에서 선택되는 유전자를 증폭시키고,(C) genes encoding phosphoenolpyruvate carboxykinase ( pckA ) involved in the anaplerotic pathway ( pckA ), genes encoding phosphoenolpyruvate carboxylase (phosphoenolpyrutave carboxylase) amplify a gene selected from the group consisting of ppc ) and a gene encoding pyruvate carboxylase ( pyc ),
    (D) 산화적 회로(oxidative pathway)에 관여하는 유전자 중 (i) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭; (ii) 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실; 또는 (iii) NADH에 의한 저해가 제거되도록 돌연변이된 시트레이트 합성효소(citrate synthase)를 코딩하는 유전자(gltA)가 증폭되고, 동시에 유기호흡 조절 단백질(aerobic respiration control protein)을 코딩하는 유전자 (arcA)가 약화 또는 결실시키는 것을 특징으로 하는 4-하이드록시부티릭산 생성능을 가지는 변이 미생물의 제조방법.(D) of genes involved in the oxidative pathway (i) amplify the gene ( gltA ) encoding a mutated citrate synthase such that inhibition by NADH is eliminated; (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 ) A method for producing a mutant microorganism having 4-hydroxybutyric acid producing ability, characterized in that it is weakened or deleted.
  23. 제1항 내지 제3항, 제5항, 제6항 및 제8항 내지 제11항 중 어느 한 항의 변이 미생물을 배양하여 4-하이드록시부티릭산을 생성시킨 다음, 4-하이드록시부티릭산을 회수하는 것을 특징으로 하는 4-하이드록시부티릭산의 제조방법.The mutant microorganism of any one of claims 1 to 3, 5, 6 and 8 to 11 is cultured to produce 4-hydroxybutyric acid, and then 4-hydroxybutyric acid Method for producing 4-hydroxybutyric acid, characterized in that the recovery.
  24. 제4항의 변이 미생물을 배양하여 4-하이드록시부티릭산을 생성시킨 다음, 4-하이드록시부티릭산을 회수하는 것을 특징으로 하는 4-하이드록시부티릭산의 제조방법.The method for producing 4-hydroxybutyric acid according to claim 4, wherein the mutant microorganism is cultured to produce 4-hydroxybutyric acid, and then 4-hydroxybutyric acid is recovered.
  25. 제7항의 변이 미생물을 배양하여 4-하이드록시부티릭산을 생성시킨 다음, 4-하이드록시부티릭산을 회수하는 것을 특징으로 하는 4-하이드록시부티릭산의 제조방법.The method of producing 4-hydroxybutyric acid, wherein the mutant microorganism of claim 7 is produced to produce 4-hydroxybutyric acid, and then 4-hydroxybutyric acid is recovered.
PCT/KR2012/010665 2011-12-07 2012-12-07 Mutant microorganism having a high 4-hydroxybutyric acid production capacity, and method for preparing 4-hydroxybutyric acid using same WO2013085361A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20110130183 2011-12-07
KR10-2011-0130183 2011-12-07

Publications (2)

Publication Number Publication Date
WO2013085361A2 true WO2013085361A2 (en) 2013-06-13
WO2013085361A3 WO2013085361A3 (en) 2013-10-17

Family

ID=48575027

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2012/010665 WO2013085361A2 (en) 2011-12-07 2012-12-07 Mutant microorganism having a high 4-hydroxybutyric acid production capacity, and method for preparing 4-hydroxybutyric acid using same

Country Status (2)

Country Link
KR (1) KR101587618B1 (en)
WO (1) WO2013085361A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9084467B2 (en) 2010-02-11 2015-07-21 Metabolix, Inc. Process for gamma-butyrolactone production
CN108588105A (en) * 2018-05-03 2018-09-28 成都理工大学 Coli expression carrier, construction method and its application

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102097065B1 (en) * 2013-08-23 2020-04-03 삼성전자주식회사 A microorganism producing 4-hydroxybutyrate and a method for producing 4-hydroxybutyrate in anaerobic condition using the same
US10240140B2 (en) 2014-10-30 2019-03-26 Samyang Corporation Expression system for psicose epimerase and production for psicose using the same
KR102120995B1 (en) * 2018-10-12 2020-06-18 경희대학교 산학협력단 Transformed methanotrophs for producing 4-Hydroxybutyric acid and uses thereof
KR102105689B1 (en) * 2018-12-17 2020-04-28 울산과학기술원 Lactate dehydrogenase mutants and preparing method of 2-hydroxy-carboxylic acid
KR102339122B1 (en) * 2020-03-11 2021-12-14 경희대학교 산학협력단 Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from methane by metabolic engineered methanotrophs

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010006076A2 (en) * 2008-07-08 2010-01-14 Opx Biotechnologies Inc. Methods, compositions and systems for biosynthetic bio production of 1,4-butanediol
KR100957772B1 (en) * 2006-03-23 2010-05-12 주식회사 엘지화학 Mutants Having a Producing Ability of 4?hydroxybutyrate and Method for Preparing 4HB Using the Same
WO2010141920A2 (en) * 2009-06-04 2010-12-09 Genomatica, Inc. Microorganisms for the production of 1,4-butanediol and related methods
KR101042242B1 (en) * 2007-09-07 2011-06-17 한국과학기술원 Mutants having a producing ability of 1,4-butanediol and method for preparing 1,4-butanediol using the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100957772B1 (en) * 2006-03-23 2010-05-12 주식회사 엘지화학 Mutants Having a Producing Ability of 4?hydroxybutyrate and Method for Preparing 4HB Using the Same
KR101042242B1 (en) * 2007-09-07 2011-06-17 한국과학기술원 Mutants having a producing ability of 1,4-butanediol and method for preparing 1,4-butanediol using the same
WO2010006076A2 (en) * 2008-07-08 2010-01-14 Opx Biotechnologies Inc. Methods, compositions and systems for biosynthetic bio production of 1,4-butanediol
WO2010141920A2 (en) * 2009-06-04 2010-12-09 Genomatica, Inc. Microorganisms for the production of 1,4-butanediol and related methods

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9084467B2 (en) 2010-02-11 2015-07-21 Metabolix, Inc. Process for gamma-butyrolactone production
US10786064B2 (en) 2010-02-11 2020-09-29 Cj Cheiljedang Corporation Process for producing a monomer component from a genetically modified polyhydroxyalkanoate biomass
CN108588105A (en) * 2018-05-03 2018-09-28 成都理工大学 Coli expression carrier, construction method and its application

Also Published As

Publication number Publication date
KR101587618B1 (en) 2016-01-22
KR20130064037A (en) 2013-06-17
WO2013085361A3 (en) 2013-10-17

Similar Documents

Publication Publication Date Title
WO2013085361A2 (en) Mutant microorganism having a high 4-hydroxybutyric acid production capacity, and method for preparing 4-hydroxybutyric acid using same
WO2009125924A2 (en) Mutant microorganism with high ability of producing putrescine and preparation of putrescine using same
WO2019164348A1 (en) Novel l-tryptophan export protein and method for producing l-tryptophan using same
WO2020204427A1 (en) Novel l-tryptophan export protein variant and method for producing l-tryptophan using same
WO2012087039A2 (en) Variant polypeptide having homoserine acetyltransferase activity and microorganism expressing same
WO2012053794A2 (en) Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same
WO2017069578A1 (en) Corynebacterium sp. microorganism having l-isoleucine producing ability and method for producing l-isoleucine by using same
WO2021125896A1 (en) Mutant of inner membrane protein, and method for producing target product by using same
WO2017014532A1 (en) Microorganism for producing putrescine or ornithine and method for producing putrescine or ornithine by using same
WO2021150029A1 (en) Method for producing l-amino acid by using microorganism containing nadp dependent glyceraldehyde-3-phosphate dehydrogenase
WO2022163951A1 (en) Novel protein variant and method for producing l-lysine using same
WO2021167414A1 (en) Purine nucleotide-producing microorganism and purine nucleotide production method using same
WO2021049866A1 (en) L-threonine export protein variant and method for production of l-threonine using same
WO2018131898A2 (en) Novel use of methylomonas sp. dh-1 strain
WO2020226341A1 (en) Microorganism producing l-amino acid and method for producing l-amino acid by using same
WO2021085999A1 (en) L-methionine producing microorganism to which protein encoded by foreign metz gene is introduced and method for producing l-methionine using same
WO2011037414A2 (en) Recombinant mutant microorganism with increased alcohol production ability, and preparation method of alcohol using same
WO2018182361A1 (en) Method for preparing corynebacterium mutant strain, using crispr/cas system, recombinase, and single-stranded oligodeoxyribonucleic acid
WO2021060696A1 (en) Modified polypeptide of dihydrodipicolinate reductase, and method for producing l-threonine by using same
WO2019031804A9 (en) E. coli and corynebacterium glutamicum shuttle vector for regulating expression of target gene
WO2022164118A1 (en) Prephenate dehydratase variant and method for producing branched-chain amino acid using same
WO2019017706A2 (en) Putrescine-producing microorganism and putrescine-producing method using same
WO2016122058A1 (en) Method for analyzing activity of human phenylalanine hydroxylase using cellular slime molds
WO2022225075A1 (en) Novel variant, and method for producing xmp or gmp using same
WO2015060649A1 (en) Microorganism producing o-succinyl homoserine and method for producing o-succinyl homoserine by using same

Legal Events

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

Ref document number: 12854628

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 12854628

Country of ref document: EP

Kind code of ref document: A2