WO2010064744A1 - Mutant bloqué dans la voie d'oxydation du glycérol pour produire le 1,3-propanediol - Google Patents

Mutant bloqué dans la voie d'oxydation du glycérol pour produire le 1,3-propanediol Download PDF

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WO2010064744A1
WO2010064744A1 PCT/KR2008/007145 KR2008007145W WO2010064744A1 WO 2010064744 A1 WO2010064744 A1 WO 2010064744A1 KR 2008007145 W KR2008007145 W KR 2008007145W WO 2010064744 A1 WO2010064744 A1 WO 2010064744A1
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gene
propanediol
glycerol
mutant
strain
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PCT/KR2008/007145
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English (en)
Inventor
Chul Ho Kim
Jeong-Woo Seo
Sun-Yeon Heo
Mi Young Seo
Baek Rock Oh
Jin-Oh Baek
Pil-Soo Seo
Min Ho Choi
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Korea Research Institute Of Bioscience And Biotechnology
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Priority to PCT/KR2008/007145 priority Critical patent/WO2010064744A1/fr
Priority to CN200880132762.XA priority patent/CN102395671B/zh
Publication of WO2010064744A1 publication Critical patent/WO2010064744A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • 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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01029—Glycerone kinase (2.7.1.29), i.e. dihydroxyacetone kinase

Definitions

  • the present invention relates to a microbial mutant in which a gene encoding a transcription activator or a gene encoding dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3-propanediol using glycerol as a carbon source and to a method for producing 1,3-propanediol using the mutant.
  • 1,3-propanediol can be used as a raw material for synthesizing polyester, poly ether, polyurethanes or the like and is used in various applications, including fibers such as highly functional clothing, carpets, or automobile fabrics, and plastic films.
  • polytrimethylene terephtalate (PTT) which is produced by polymerization of 1,3-propanediol with terephtalic acid has excellent physical properties and a melting point of 228 ° C which is lower than that of polyethylene terephthalate (PET).
  • PET polytrimethylene terephtalate
  • PET polytrimethylene terephtalate
  • polytrimethylene terephtalate (PTT) has a higher utility and is receiving attention as a next-generation fiber material which can substitute for PET.
  • plastics and polymers produced from 1,3-propanediol as a monomer show excellent optical stability compared to that of products produced from butanediol or ethylene glycol.
  • 1,3-propanediol can be used as a polyglycol-type lubricant and a solvent, and thus its commercial value is evaluated to be higher than that of glycerol.
  • 1,3-propanediol can be produced by chemical synthesis or microbial fermentation.
  • Chemical processes for producing 1,3-propanediol include a process of converting i ethylene oxide to 1 ,3 -propanediol by hydroformylation (US Patent No. 3,687,981) and a process of converting acrolein to 1,3 -propanediol by hydration (US Patent No. 5,015,789).
  • such chemical processes have problems in that they require a high-temperature or high-pressure process during the production of 1,3- propanediol, leading to high production costs, and generate waste oil containing environmental pollutants.
  • Biological processes include a process of producing 1,3 -propanediol from glycerol using microorganisms such as Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, Pelobacter or the like, which are facultative anaerobic strains (US Patent No. 5,254,467).
  • microorganisms such as Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, Pelobacter or the like, which are facultative anaerobic strains (US Patent No. 5,254,467).
  • genetic engineering techniques using such microbial strains include a case in which a mutant obtained by amplifying or knocking out the glycerol dehydrogenase gene from Lactobacillus is used to produce 1,3 -propanediol with increased productivity (US 2007/0148749).
  • the present inventors have made many efforts to develop a microorganism producing only 1,3-propanediol in glycerol metabolism by a metabolic engineering method without producing oxidative metabolic byproducts including 2,3-butanediol.
  • the present inventors have constructed a mutant having only a reductive metabolic pathway producing 1,3-propanediol by using a genetic recombination technique to block an oxidative metabolic pathway producing byproducts in the glycerol metabolic pathway and have found that, when the mutant is used to produce 1,3-propanediol, oxidative metabolic byproducts including 2,3-butanediol are not produced, thereby completing the present invention.
  • an object of the present invention to provide a mutant in which the oxidative pathway of glycerol is blocked in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source in order to produce 1,3- propanediol from glycerol without producing byproducts.
  • Another object of the present invention is to provide a method for producing 1,3- propanediol, which comprises culturing the mutant in a medium containing glycerol.
  • the present invention provides a microbial mutant in which a gene encoding a transcription activator or a gene encoding dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source.
  • the present invention also provides a method for preparing 1,3 -propanediol, which comprises producing 1,3 -propanediol by culturing said microbial mutant in a glycerol-containing medium; and recovering said 1,3 -propanediol produced.
  • the present invention also provides a mutant having the ability to produce 1,3- propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3 -propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3- propanediol oxidoreductase is inserted into the chromosome of the mutant (the AK strain).
  • DhaD glycerol dehydrogenase gene
  • DhaR transcription activator gene
  • DhaT 1,3 -propanediol oxidoreductase gene
  • the present invention also provides a mutant having the ability to produce 1,3- propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3- propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3 -propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AK strain).
  • DhaD glyce
  • the present invention also provides a mutant having the ability to produce 1,3- propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3 -propanediol oxidoreductase is inserted into the chromosome of the mutant (the AR strain).
  • DhaR transcription activator gene
  • DhaT 1,3-propanediol oxidoreductase gene
  • DhaBA2 glycerol dehydratase reactivation factor II gene
  • the present invention also provides a mutant having the ability to produce 1 ,3- propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AR strain).
  • DhaR transcription activator gene
  • DhaT 1,3-propanediol oxidore
  • the present invention also provides a method for preparing 1,3 -propanediol, which comprises producing 1,3 -propanediol by culturing said mutant in a glycerol- containing medium; and recovering said 1,3 -propanediol produced.
  • FIG. 1 is a schematic diagram showing a reductive pathway producing 1,3- propanediol and an oxidative pathway producing byproducts in a glycerol metabolic process.
  • FIG. 2 shows a method for preparing a mutant according to the present invention, shown using the structure of the dha regulon.
  • FIG. 3 shows methods for plasmid DNAs for preparing mutants according to the present invention.
  • FIG. 3A shows a method for constructing a plasmid DNA, which is used to prepare an AK strain and comprises a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaK gene amino terminus (dhaK 1 )
  • FIG. 3 B shows a method for constructing a plasmid DNA, which is used to prepare an AR strain and comprises a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaR gene amino terminus (dhaR 1 ).
  • FIG. 4 is a graphic diagram showing analysis results for glycerol metabolites of Klebsiella pneumoniae AK and AR strains.
  • FIG. 5 shows a method for constructing a plasmid DNA containing the DhaT gene and DhaB reactivation enzyme gene of Klebsiella pneumoniae downstream of the lacZ promoter or for constructing a plasmid DNA containing only the DhaT gene downstream of the lacZ promoter.
  • FIG. 6 is a schematic diagram explaining a process for constructing a plasmid DNA containing the 1,3 -propanediol oxidoreductase activity YqhD(E) gene (derived from E. col ⁇ ) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(E) gene downstream of the lacZ promoter.
  • FIG. 7 shows a method for constructing the 1,3 -propanediol oxidoreductase activity YqhD(K) gene (derived from K. pneumoniae) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(K) gene downstream of the lacZ promoter.
  • FIG. 8 is a graphic diagram showing analysis results for glycerol metabolites of recombinant strains derived from Klebsiella pneumoniae Cu, AK and AR according to the present invention.
  • the present invention relates to a microbial mutant in which a gene encoding a transcription activator or a gene encoding dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3- propanediol using glycerol as a carbon source.
  • the gene encoding glycerol dehydrogenase is additionally deleted or inactivated.
  • the glycerol metabolic pathway consists of two metabolic pathways, that is, oxidative and reductive metabolic pathways (FIG. 1).
  • glycerol is oxidized to dehydroxyacetone (DHA) by NAD+ dependent glycerol dehydrogenase while producing NADH, which is then converted to dehydroxyacetone phosphate (DHAP) by DHA kinase.
  • DHA dehydroxyacetone
  • DHAP dehydroxyacetone phosphate
  • DHAP dehydroxyacetone phosphate
  • byproducts including 2,3-butanediol, acetic acid, ethanol, lactic acid and succinic acid are produced.
  • glycerol is converted to 3- hydroxypropionaldehyde by the action of dehyratase, and then reduced to 1,3- propanediol by the action of NADH dependent oxidoreductase while forming NAD+.
  • glycerol dehyratase glycerol dehyratase
  • dhaT 1,3-propanediol reducatse
  • dhaD glycerol dehydrogenase
  • dhaK dehydroxyacetone kinase
  • the mutant of the present invention is a microbial strain in which genes encoding proteins involved in the oxidative pathway of the glycerol metabolic process are deleted or inactivated and which produces only 1,3-propanediol through the reductive pathway without producing byproducts including 2,3-butanediol, ethanol, lactic acid and succinic acid.
  • the proteins involved in the glycerol oxidative pathway of the mutant are preferably glycerol dehydrogenase, a transcription activator and dihydroxyacetone kinase.
  • the microorganism is preferably selected from the group consisting of Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus and Pelobacter.
  • the microorganism is preferably Klebsiella pneumoniae
  • the genes encoding enzymes involved in the glycerol oxidative pathway are a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR) and dihydroxyacetone kinase genes (DhaK, DhaL, DhaM and DhaK 1 ).
  • a Klebsiella pneumoniae mutant (an AK strain) was constructed by deleting the glycerol dehydrogenase gene (DhaD), the transcription activator gene (DhaR), the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2) from the chromosome of the Klebsiella pneumoniae strain.
  • DhaD glycerol dehydrogenase gene
  • DhaR transcription activator gene
  • DhaT 1,3 -propanediol oxidoreductase gene
  • DhaBA2 glycerol dehydratase reactivation factor II gene
  • the mutant was transformed with a recombinant vector comprising the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2), which are genes involved in the reductive pathway of glycerol, thus restoring the reductive pathway.
  • DhaT 1,3 -propanediol oxidoreductase gene
  • DhaBA2 glycerol dehydratase reactivation factor II gene
  • the mutant of the present invention is characterized in that the glycerol dehydrogenase gene (DhaD) and the transcription activator gene (DhaR) are deleted or inactivated.
  • DhaD glycerol dehydrogenase gene
  • DhaR transcription activator gene
  • the lacZ promoter (PlacZ) was inserted upstream of the genes involved in the reductive pathway, such that the genes were no longer regulated by the DhaR regulator, whereby the expression of the genes could be artificially controlled using an inducer.
  • a Klebsiella pneumoniae mutant (an AR strain) was constructed by deleting the transcription activator gene (DhaR), the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2) from the chromosome of the Klebsiella pneumoniae strain.
  • the mutant was transformed with a recombinant vector comprising the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2), which are genes involved in the reductive pathway of glycerol, thus restoring the reductive pathway.
  • DhaT 1,3 -propanediol oxidoreductase gene
  • DhaBA2 glycerol dehydratase reactivation factor II gene
  • the mutant of the present invention is characterized in that the transcription activator gene (DhaR) is deleted or inactivated.
  • DhaR transcription activator gene
  • the present invention relates to a method for producing 1,3- propanediol, which comprises culturing in a glycerol-containing medium a microbial mutant in which a gene encoding transcription activator or a gene encoding dihydroxyacetone kinase in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source is deleted or inactivated.
  • the Klebsiella pneumoniae strain with a deletion of the glycerol dehydrogenase gene (DhaD) and the transcription activator gene (DhaR) and the Klebsiella pneumoniae strain with a deletion of the transcription activator gene (DhaR) produced 1,3 -propanediol in a medium containing glycerol without producing byproducts of the oxidative pathway other than a small amount of acetic acid.
  • the present invention relates to a mutant having the ability to produce 1,3 -propanediol, in which a vector containing a gene encoding 1,3- propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase is inserted into the chromosome of the mutant (the AK strain).
  • DhaD glycerol dehydrogenase gene
  • DhaR transcription activator gene
  • DhaT 1,3-propanediol oxidoreductase gene
  • the DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene of the dha regulon were substituted with the apramycin-resistant gene by a homologous recombination method using a plasmid DNA-cured Klebsiella pneumoniae MGH78578 strain (hereinafter referred to as "Cu") as a parent strain, thus preparing a recombinant strain with a deletion of both oxidative and reductive pathways (hereinafter referred to as an "AK" strain).
  • Cu plasmid DNA-cured Klebsiella pneumoniae MGH78578 strain
  • the DhaB enzyme reactivation factor, DhaT gene and DhaR regulator of the dha regulon were substituted with the apramycin-resistant gene by a homologous recombination method using a plasmid DNA-cured Klebsiella pneumoniae MGH78578 strain (hereinafter referred to as "Cu") as a parent strain, thus preparing a recombinant strain with a deletion of both oxidative and reductive pathways (hereinafter referred to as an "AR" strain).
  • Cu plasmid DNA-cured Klebsiella pneumoniae MGH78578 strain
  • AK and AR strains the proliferation and glycerol metabolic characteristics of the Klebsiella pneumoniae recombinant strains (AK and AR strains) with a deletion of anaerobic metabolic pathways of glycerol were analyzed.
  • the AK and AR strains were cultured in a medium supplemented with glycerol, and the degree of proliferation of the microbial cells was examined.
  • the amount of glycerol present in the culture broth and the production of metabolites including
  • 1,3-propanediol were analyzed by chromatography. As a result, it was shown that oxidation metabolites including 2,3-butanediol other than acetic acid were not produced, and this result was consistent with the genetic background in which the anaerobic metabolic pathways of glycerol in the AK and AR strains were blocked.
  • plasmid DNA was prepared by amplifying a DhaB reactivation enzyme gene ⁇ prfW)-orpC DNA fragment, the dhaT, yqhD (derived from E. colt) or yqhD homologous gene
  • each of the recombinant strains was cultured in a medium supplemented with glycerol, tetracycline and IPTG, while metabolites in the culture broth were analyzed.
  • the recombinant strains derived from Klebsiella pneumoniae Cu completely consumed the added glycerol after a given time of culture, whereas the recombinant strains derived from AK or AR showed glycerol consumption rates slightly slower than those of the Cu-derived recombinant strains, but their ability to produce 1,3- propanediol was found to be restored (FIG. 8).
  • the two yqhD genes derived from E. coli or Klebsiella pneumoniae were shown to restore the reductive pathway of glycerol in the same manner as the dhaT gene. Meanwhile, it was found that the AK- or AR-derived recombinant strains in which the oxidative pathway of glycerol has been blocked still did not produce metabolites of the oxidative pathway other than a small amount of acetic acid (FIG. 8).
  • the present invention relates to a mutant having the ability to produce 1,3 -propanediol, in which a vector containing a gene encoding 1,3- propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3 -propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3- propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AK strain).
  • DhaD gly
  • the present invention relates to a mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase is introduced in a, Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3- propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase is inserted into the chromosome of the mutant (the AR strain).
  • DhaR transcription activator gene
  • DhaT 1,3- propanediol oxidoreductase gene
  • DhaBA2 glycerol dehydratase reactivation factor II gene
  • the present invention relates to a mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AR strain).
  • DhaR transcription activator gene
  • DhaT 1,3-propanediol oxidor
  • the term “deletion” of genes refers to the state in which the genes were deleted from a chromosome or a plasmid, such that proteins encoded by the genes could not be produced.
  • the term “inactivation” of genes refers to the state in which the genes were inserted, translocated or partially deleted, such that proteins encoded by the genes could not be produced.
  • the culture of the mutant according to the present invention can be carried out according to a widely known method, and conditions including the culture temperature and time, the pH of medium, and the like can be suitably controlled.
  • Collection of 1,3 -propanediol from the culture broth of the mutant can be carried out using conventional isolation techniques, for example, distillation, electrodialysis, evaporation, chromatography, solvent extraction, and reaction extraction, and these techniques may generally be used in combination to isolate highly pure substances.
  • a mutant having the ability to produce 1,3- propanediol without producing byproducts can be prepared by blocking only the oxidative pathway of glycerol in a microorganism having the ability to produce 1,3- propanediol from glycerol.
  • Example 1 Preparation of recombinant strains in which oxidative-reductive metabolic pathways of glycerol were blocked
  • a recombinant strain in which the glycerol metabolic pathway in Klebsiella pneumoniae MGH 78578 (ATCC 700721) was completely blocked was prepared as a base strain.
  • the antibiotic resistance characteristics of the Klebsiella pneumoniae MGH 78578 strain were analyzed.
  • apramycin 50 /zg/ml
  • plasmids originally present in the strain were removed by a curing method, and tetracycline was added as a usable selection marker.
  • a plasmid DNA-cured Klebsiella pneumoniae MGH 78578 strain (named "Cu") as a parent strain, the DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene of the dha regulon (FIG. 2) were substituted with apramycin-resistant genes by a homologous recombination method, thus preparing a recombinant strain AK with a deletion of both oxidative and reductive pathways.
  • a recombinant strain AR was prepared by substituting the DhaB enzyme reactivation factor, the DhaT gene and the DhaR regulator with apramycin-resistant gene.
  • the DhaR dependent promoter upstream of the DhaB gene was replaced with an artificially controllable lacZ promoter.
  • Klebsiella pneumoniae MGH78578 was inoculated into a liquid medium (LB) containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl and was cultured at 37 ° C at 180 rpm for 12 hours. Then, the cultured strain was subinoculated into a liquid medium containing 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2 mM Tris- Cl buffer and 0.1% EtBr and was cultured at 37 ° C at 180 rpm for 12 hours. The culture was repeated three times in the same manner as described above, and then the culture broth was plated onto a solid LB medium containing no antibiotic and was cultured at 37 "C for 12 hours, and single colonies were isolated from the culture broth.
  • LB liquid medium
  • the isolated colonies were inoculated into a medium supplemented with or without tetracycline, and colonies which no longer proliferated in the medium supplemented with tetracycline were selected. Plasmid DNA was isolated from the selected colonies and analyzed by agarose gel electrophoresis, and a strain lacking the plasmid DNA was finally selected. The selected strain was named "Klebsiella pneumoniae MGH78578 Cu” and used as a parent strain for preparing recombinant strains, after it was confirmed that the cellular proliferation and glycerol metabolic characteristics thereof did not significantly differ from those of the MGH78578 strain.
  • the strain was cultured in LB liquid medium (50 ml) for 12 hours, and then the cells were collected by centrifugation at 10 ° C at 14,000 ⁇ g for 10 minutes. The collected cells were washed with 50 mM Tris-Cl (pH 8.0), resuspended in a solution containing 3 mi of 25% sucrose, 175 ⁇ i of TES buffer, 20% SDS and 100 ⁇ i of 0.5 mM EDTA and allowed to stand at 37 ° C for 30 minutes. 40 ⁇ of RNase (10 mg/mt in TE buffer) was added to the cells which were then allowed at 37 ° C for 20 minutes.
  • DNA fragments for preparing a plasmid for homologous recombination were amplified by PCR using the extracted chromosomal DNA as a template and the following primer sets (FIG. 2).
  • the PCR reaction performed using 50 ⁇ i of a final reaction solution containing 2 ⁇ i of template DNA, 2 ⁇ i of each primer DNA, 0.2 mM dNTP mix, 2 mM MgC12, 5 ⁇ i of 1Ox buffer (Mg2+ free), 0.05 ⁇ MlA of Taq polymerase and 30.5 ⁇ i of triple-distilled water for one cycle of denaturation at 94 0 C for 30 sec, annealing at 52 ° C for 30 sec and extension at 72 ° C for 1 min.
  • SEQ ID NO: 1 5'-TCTAGAATGAAAAGATCAAAACGATTT-3'(dhaBI XbaI-480bpF)
  • SEQ ID NO: 2 S'-GGATCCGTCAGCGGCAATCTGCAC-S'CdhaBI BamHI-480bpR)
  • SEQ ID NO: 3 5'-AAGCTTCATGCTCTCCGGCGCCTGTC-3'(dhaK HindIII-200-700 bpF)
  • SEQ ID NO: 4 5'-AGATCTATTTGGTCCAGCGAGCTGAAGC-3'(dhaK BglII-200-700bpR)
  • SEQ ID NO: 5 5'-AGATCTCCTGGGATTTCGCGACGGCA-3'(dhaR bglII-200-700bpF)
  • SEQ ID NO: 6 5'-AAGCTTTCGACA ATCGGTTTT AAGGTG-3 '(dhaR HindIII-200-700bpR)
  • SEQ ID NO: 8 5'-AGATCTAAAAGCTTATGAGCTCAGCCAATCGA-S' Apr Hindlll-BglHR
  • the amplified DNA fragments were cloned using a pGEM TEasy vector, and the base sequence thereof was analyzed. Then, as shown in FIG. 3, plasmid DNAs were constructed.
  • a plasmid DNA for preparing an AK strain which was comprised of a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaK gene amino terminus (dhaK')
  • a plasmid DNA for preparing an AR strain which was comprised of a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaR gene amino end (dhaR') was constructed.
  • Each of the plasmids was treated with BamHI-BglU, and the collected DNA fragment was introduced into the Klebsiella pneumoniae Cu strain by electroporation. Then, recombinant strains forming colonies in a medium supplemented with apramycin were isolated. It was inferred that, in the obtained colonies, the insertion of the BamHl-BgHl DNA fragment containing the apramycin resistant gene into the chromosome occurred. It was finally confirmed by Southern blotting that homologous recombination occurred accurately in the dha regulon.
  • a recombinant strain AK with a deletion of the DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene of the dha regulon and an insertion of the lacZ promoter and the apramycin resistant gene was obtained, and a recombinant strain AR with a deletion of the DhaB enzyme reactivation factor, the DhaT gene and the DhaR regulator and an insertion of the lacZ promoter and the apramycin resistant gene was obtained.
  • Example 2 Analysis of characteristics of Klebsiella pneumoniae strains AK and AR
  • the proliferation and glycerol metabolic characteristics of the Klebsiella pneumoniae recombinant strains AK and AR in which the anaerobic metabolic pathway of glycerol was removed were analyzed.
  • Each of the AK and AR strains was inoculated into a medium supplemented with glycerol and was cultured at 37 ° C at 180 rpm for 12 hours.
  • the AK and AR strains showed cell proliferation rates which were about two-fold slower than that of the parent strain Cu used as a control, and the glycerol consumption and 1,3 -propanediol production thereof were shown to be about 40% and 20%, respectively (FIG. 5). It was shown that oxidation metabolites including 2,3-butanediol other than acetic acid were not produced, and this result was consistent with the genetic background in which the anaerobic metabolic pathway of glycerol in each of the AK and AR recombinant strains was blocked.
  • Example 3 Preparation of strains in which reductive pathway of glycerol was restored
  • a DhaB reactivation enzyme gene (or ⁇ V)-or ⁇ CDNA fragment and the dhaT, yqhD (derived from E. col ⁇ ) or yqhD homologous gene (derived from Klebsiella pneumoniae) having 1,3 -propanediol oxidoreductase activity were amplified using the following primer sequences.
  • the amplified genes were cloned into a pGEM TEasy vector, and the base sequences thereof were analyzed. Then, as shown in FIG. 5, plasmid DNAs were prepared.
  • SEQ ID NO: 9 S'-AGATCTATGAGCTATCGTATGTTTGA-S'fdhaT-Bglll F)
  • SEQ ID NO: 10 S'-CTCGAGAAGCTTCAGAATGCCTGGCGGAAAAT-3'fdhaT-HindIII/XhoI R)
  • SEO ID NO: 1 1 5'-AGATCTATGAACAACTTTAATCTGCAC-3'(yqhD-BglH F)
  • SEQ ID NO; 12 5'-AGATCTATGAATAATTTCGACCTGCA-3'(vqhD-HindIII/XhoI R)
  • SEQ ID NO: 14 5'-CTCGAGAAGCTTAGCGTGCAGCCTCGTAAAT-3'(yqhD KIe Hindlll, Xhol R)
  • FIG. 5 shows a process for constructing a plasmid DNA containing the DhaT gene and DhaB reactivation enzyme gene of Klebsiella pneumoniae downstream of the lacZ promoter or for constructing a plasmid DNA containing only the DhaT gene downstream of the lacZ promoter
  • FIG. 6 shows a process for constructing a plasmid DNA containing the 1,3 -propanediol oxidoreductase activity YqhD(E) gene (derived from E. coli) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(E) gene downstream of the lacZ promoter.
  • FIG. 5 shows a process for constructing a plasmid DNA containing the DhaT gene and DhaB reactivation enzyme gene of Klebsiella pneumoniae downstream of the lacZ promoter or for constructing a plasmid DNA containing only the DhaT gene
  • FIG. 7 shows a process for constructing a plasmid DNA containing the 1,3 -propanediol oxidoreductase activity YqhD(K) gene (derived from Klebsiella pneumoniae) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(K) gene downstream of the lacZ promoter.
  • Example 4 Analysis of characteristics of recombinant strains in which reductive pathway of glycerol was restored
  • Example 3 Each of the recombinant strains prepared in Example 3 was inoculated into a medium (50 ml) supplemented with glycerol (2%), tetracycline (10 ⁇ g/ml) and
  • IPTG (0.5 mM) and was cultured at 37 ° C at 120 rpm, while metabolites in the culture broth were analyzed.
  • the recombinant strains derived from Klebsiella pneumoniae Cu completely consumed the added glycerol after 14 hours of culture, whereas the recombinant strains derived from AK or AR showed glycerol consumption rates slightly slower that those of the Cu-derived recombinant strains, but their ability to produce 1,3 -propanediol was found to be restored (FIG. 8). It was shown that the two YqhD genes derived from E. coli or Klebsiella pneumoniae restored the reductive pathway of glycerol in the same manner as the DhaT gene.
  • the mutant according to the present invention when used, the production of byproducts which are inevitably obtained when producing 1,3 -propanediol from glycerol using prior strains can be minimized, and thus the cost of purification of 1,3-propandiol can be reduced.

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Abstract

La présente invention concerne un mutant microbien, dans lequel le gène codant pour un activateur de transcription ou le gène codant pour la dihydroxyacétone kinase dans un microorganisme étant capable de produire du 1,3-propanediol à l'aide de glycérol comme source de carbone, est supprimé ou inactivé, et un procédé de production de 1,2-propanediol à l'aide du mutant. Lorsque le mutant selon la présente invention est utilisé, la production de sous-produits qui sont inévitablement obtenus lors de la production du 1,3-propanediol à partir de glycérol à l'aide de souches antérieures peut être réduite à un minimum, et ainsi le coût de purification du 1,3-propanediol peut être réduit.
PCT/KR2008/007145 2008-12-03 2008-12-03 Mutant bloqué dans la voie d'oxydation du glycérol pour produire le 1,3-propanediol WO2010064744A1 (fr)

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CN200880132762.XA CN102395671B (zh) 2008-12-03 2008-12-03 用于制造1，3-丙二醇的丙三醇氧化途径中阻隔的突变体

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US20130095541A1 (en) * 2010-05-25 2013-04-18 Korea Research Institute Of Bioscience And Biotech Method for preparing 3-hydroxypropionic acid from glycerol in high yield

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US20120045808A1 (en) * 2009-03-12 2012-02-23 Korea Research Institute of Biosctechnology Production method for 1,3-propanediol using recombinant microbial strain having glycerol oxidation metabolic pathway blocked
US8338148B2 (en) * 2009-03-12 2012-12-25 Korea Research Institute Of Bioscience And Biotechnology Method of producing 1,3-propanediol using recombinant Klebsiella strain in which glycerol oxidative pathway has been blocked
US20130095541A1 (en) * 2010-05-25 2013-04-18 Korea Research Institute Of Bioscience And Biotech Method for preparing 3-hydroxypropionic acid from glycerol in high yield
US8951763B2 (en) * 2010-05-25 2015-02-10 Korea Research Institute Of Bioscience And Biotechnology Method for preparing 3-hydroxypropionic acid from glycerol in high yield

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