WO2007013695A1 - Méthode pour conférer la capacité d’assimilation du glycérol à des bactéries - Google Patents

Méthode pour conférer la capacité d’assimilation du glycérol à des bactéries Download PDF

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WO2007013695A1
WO2007013695A1 PCT/JP2006/315453 JP2006315453W WO2007013695A1 WO 2007013695 A1 WO2007013695 A1 WO 2007013695A1 JP 2006315453 W JP2006315453 W JP 2006315453W WO 2007013695 A1 WO2007013695 A1 WO 2007013695A1
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gene
glycerin
bacterium
corynebacterium
protein
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Japanese (ja)
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Masaharu Mukoyama
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Nippon Shokubai Co., Ltd.
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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • 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
    • 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
    • C12P7/56—Lactic acid

Definitions

  • the present invention relates to a method for imparting glycerin assimilation ability to a bacterium, a recombinant bacterium having a glycerin assimilation ability, and a fermented product or a reaction product using the recombinant bacterium. On the method.
  • glycerin In the surfactant industry and the biodiesel fuel industry, glycerin is discharged in large quantities as a by-product. Normally glycerin is used in the manufacture of nitroglycerin and other pharmaceuticals and cosmetics, but the glycerin discharged as a by-product is in the form of an aqueous solution containing impurities, so it can be used as it is for the above use! / The current situation is that it cannot be discarded. If glycerin, which is a waste material, can be used effectively in this way, it is highly desirable from the viewpoint of effective use of resources such as global environmental problems.
  • the glycerin aqueous solution containing impurities discharged as a by-product in the surfactant industry and the piodiesel fuel industry has no problem when used as a fermentation raw material.
  • the production of useful substances using glycerin as a raw material is desired not only from the viewpoint of developing fermentation raw materials other than sugar, but also from the viewpoint of effective use of waste. Disclosure of the invention
  • An object of the present invention is to provide means for imparting glycerol utilization ability to microorganisms.
  • the present inventors have found that the bacterium acquires glycerin assimilation ability by introducing a gene encoding a glycerin uptake protein into the bacterium.
  • the invention has been completed. '
  • the present invention includes the following inventions.
  • glycerin uptake protein gene is a g1pF gene derived from Corynebacterium diphtheria '.
  • Fermentation production comprising culturing the recombinant bacterium according to any one of (11) to (20) in a medium containing glycerin, and obtaining a fermentation product or a reaction product from the culture Product or reaction product production method.
  • FIG. 1 shows the result of extracting a plasmid from an E. coli transformant in Example 1 and confirming the insert.
  • FIG. 2 shows the result of confirming the introduction of the hybrid plasmid into the corynebacterium dartami force in Example 2.
  • FIG. 3 shows the results of PCR amplification of the g 1 p F K gene using KO D-gene polymerase in Example 3.
  • Fig. 4 shows the result of excising and collecting the band of g I pFK gene obtained by PCR amplification and immobilizing it on pCR4TOPO.
  • FIG. 5 shows the result of confirming the insert of g ip FK_pCG100-pHSG398 plasmid in Example 3.
  • Fig. 6 shows that the plasmid g 1 p.FK—pCGl OO—pHSG398, in which the Escherichia coli g l.pFK gene was introduced into the pCG100—pHSG398 shuttle vector, was electroporated into Corynebacterium dartamicum ATCC 13032 strain. Show the result.
  • the present invention will be described in detail.
  • a bacterium for introducing a glycerin uptake protein gene a bacterium that does not have glycerin assimilation ability is usually used.
  • a bacterium having a glycerol kinase gene is preferably used.
  • Coryneform bacteria are a group of microorganisms as defined in B argeys Manual of Det rm inative B acteriology, 8, 599 (1 974), aerobic, gram positive, non-acidic, spore-forming ability
  • the bacterium belonging to the genus Corynebacterium which has been previously classified as the genus Brevibacterium, has now been classified into the genus Corynebacterium.
  • Incorporated bacteria Int. J. Sys t. B acteriol., 41, 255 (1 98 1)
  • Brevibakyuterium bacterium belonging to the genus Corynepacteria are also closely related.
  • a bacterium belonging to the genus Ralstonia can also be used as a bacterium for introducing a gene.
  • Examples of the genus Ralstonia include Ralstonia eutropha, Ralstonia solanacearum, Ralstonia metal ldurans, Ralstonia pickettii, Ralstonia bxalatica., Ralstonia insidiosa, Ralstonia raannitolilytica ⁇ Ralstonia syzygii.
  • a bacterium for introducing a gene in the method of the present invention preferably Ralstonia eutropha is used.
  • Alcaligenes bacteria can also be used as a cell for gene introduction.
  • Alkagenes bacteria include, for example, Alcaligenes faecalis, Alcaligenes denitrif icans, Alcaligenes eutrophus, Alcaligenes xylosoxidans, Alcaligenes aestus, Alcaligenes aquamarinus N Alcaligenes aquatilis ⁇ Alcaligenes cupidus, Alcaligenes defragrantus, caligen Alcaligenes paradoxus, Alcaligenes piechaudii, Alcaligenes ruhlandii, Alcaligenes venustus, etc. are mentioned.
  • the bacterium into which the gene is introduced is preferably Alcaligenes faecalis.
  • the present inventors have clarified that Corynepacterium 'Glutamicum, Ralstonia' Eutropha and Alkaline Genus' fecalis in a medium using glycerin as a carbon source. I found that it did not show good growth.
  • Corynebacterium dartamicum, Ralstonia utropha and Arka ligenes faecalis have the ability to assimilate glycerin, despite the presence of the glycerol kinase gene, which phosphorylates glycerin and leads to the center of metabolism. It was amazing not to do it.
  • the present invention is based on the above findings.
  • Glycerin uptake protein is a hydrophobic protein, and is thought to act to promote uptake of glycerin into cells by forming a channel that connects the outside and inside by taking a state of being inserted into the cell membrane.
  • the Dari 'serine uptake protein gene to be introduced into bacteria is not particularly limited as long as it is a gene encoding a protein having a function of promoting the uptake of glycerin into cells, but the glycerin assimilation ability is not limited.
  • g 1 p F gene Derived from microorganisms, preferably having g 1 p F gene, for example, Salraone ⁇ 1 &, Yersinia genus,, Shigella, Erwiniaj3 ⁇ 4 s Haemophilus ⁇ Pasteureila 3 ⁇ 4, Mannheimia genus, Xylella ⁇ , Vibrio genus, Photobacterium genus, Pseudomonas, Francisella, Chromobacterium, Burkholderia, Bacillus ⁇ , Staphylococcus, Listeria, Lactococcus, Streptococcus, Lactobacillus, Enterococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Streptomyces j3 ⁇ 4 N Leifs , Rhodopirellula, Boirelia, Leptospira, Archaeoglobus, Escherichia Those that come, specifically, Salmonella
  • Microorganisms with glycerin assimilation ability such as E. coli corynebacterium 'Diphtheria' have a g 1 p FK gene that is thought to be involved in glycerin uptake.
  • Glycerin uptake protein also called facilitator
  • gl pK codes for glycerol kinase.
  • the glycerin-incorporating protein gene to be introduced into the bacterium is preferably a g 1 pF gene, particularly a g 1 p F gene derived from E. coli and a g 1 p F gene derived from Corynebacterium diphtheria.
  • the glycerin uptake protein gene include a gene comprising the nucleotide sequence represented by SEQ ID NO: 1 or 7.
  • the glycerin uptake protein gene of the present invention includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 7.
  • “functionally equivalent” means that the protein encoded by the gene of interest has the same biological function and biochemistry as the protein encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 1 or 7. It has a functional function.
  • Examples thereof include a gene that hybridizes with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence of No. 1 or 7 under stringent conditions.
  • the protein encoded by the gene has the activity of glycerin uptake protein, that is, the activity of promoting the uptake of glycerin into cells.
  • the darling uptake protein gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, to the nucleotide sequence of SEQ ID NO: 1 or 7.
  • a gene consisting of a base sequence having at least 99% identity is also included.
  • Examples of the glycerin uptake protein gene include a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8.
  • the gene includes a gene encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8.
  • “Functionally equivalent” means that the target protein has a biological function or biochemical function equivalent to that of the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 8.
  • the amino acid sequence of SEQ ID NO: 2 or 8 is deleted, added, inserted or substituted in one or several amino acids. And a protein consisting of the prepared amino acid sequence.
  • the protein has the activity of the above glycerin uptake protein, that is, glycerin into the cell. Has activity to promote uptake.
  • One or several amino acid deletions, additions, insertions or substitutions in the amino acid sequence of SEQ ID NO: 2 or 8 are commonly used techniques such as site-directed mutagenesis (Z o 1 1 er et al., Nu cleic Ac ids R es. 1 0 6478-6 500, 1 98 2).
  • the side chains of amino acids that constitute protein components differ in hydrophobicity, charge, size, etc., but they are essentially in the three-dimensional structure of the entire protein (also referred to as a three-dimensional structure).
  • Several relationships that are highly conserved in the sense that they have no effect are known from empirical and physicochemical measurements.
  • conservative substitutions between different amino acid residues include glycine (G 1 y) and proline (Pro), glycine and alanine (A la) or norin (V a 1), leucine (L eu) and isoleucine (I 1 e), glutamic acid (G 1 u) and glutamine (G in), aspartic acid (A sp) and asparagine (A sn), cysteine (Cy s) and threonine (Th r) ', Substitution between amino acids such as threonine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg) is known.
  • the mutant protein is composed of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 or 8, the mutation is If the mutation is highly conserved in the three-dimensional structure of the amino acid sequence shown in SEQ ID NO: 2 or 8, and the mutant protein has the activity of the glycerin uptake protein, these mutations
  • the gene encoding the type protein is also included in the glycerin uptake protein gene.
  • severe means usually 2 to 5, preferably 2 to 3.
  • the glycerin incorporation protein gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the amino acid sequence of SEQ ID NO: 2 or 8. More preferably, a gene encoding a protein consisting of an amino acid sequence having at least 99% identity is also included.
  • a glycerol kinase gene is further introduced into the bacterium.
  • Glycerol kinase is a protein having an activity of phosphorylating glycerin, and has an activity of catalyzing the following reaction in the metabolism of glycerin.
  • the glycerol kinase gene is not particularly limited as long as it is a gene encoding a protein having an activity to phosphorylate glycerin, but it is derived from a microorganism having glycerin assimilation ability, preferably has a g 1 p K gene.
  • the glycerol kinase gene to be introduced into bacteria in the present invention the g 1 p K gene in the above g 1 p F K gene, particularly the g 1 p K gene derived from Escherichia coli and the g 1 p K gene derived from Corynebacterium diphtheria are preferable.
  • the glycerol kinase gene is preferably derived from the same bacterium as the glycerin uptake protein gene to be introduced.
  • coli-derived g 1 p K gene present in the same operon is introduced into Escherichia coli, which has an excellent ability to assimilate glycerin. Replacement bacteria can be obtained.
  • the g1 p F gene derived from Corynebacterium diphtheria is introduced as a glycerin uptake protein gene, it is derived from Corynebacterium diphtheria present in the same operon in Corynebacterium diphtheria.
  • K gene By introducing the K gene, a recombinant bacterium excellent in glycerin assimilation ability can be obtained.
  • g 1 p F gene and the g 1 p K gene should be introduced as g 1 p FK genes contained in g 1 p (glycerol phosphate phosphate) in E. coli or corynebacterium diphtheria. Is more preferable.
  • glycerol kinase kinase gene examples include a gene consisting of the nucleotide sequence represented by SEQ ID NO: 3 or 9.
  • the glycerol kinase gene also includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 9.
  • “functionally equivalent” is the same as above.
  • 'A gene that is functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 9 includes a gene that is highly pre-hybridized under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 9 Is mentioned.
  • the protein encoded by the gene has glycerol phosphorylation activity.
  • the stringent conditions are as described above.
  • the entire length of the nucleotide sequence set forth in SEQ ID NO: 3 or 9 is subjected to various artificial treatments. Even if the sequence is partially changed, these mutant genes are hybridized under stringent conditions with a gene consisting of a base sequence complementary to the base sequence of SEQ ID NO: 3 or 9. Any gene that codes for a protein having glycerin phosphorylation activity is included in the glycerol kinase gene regardless of the difference from the nucleotide sequence shown in SEQ ID NO: 3 or 9.
  • the glycerol kinase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the nucleotide sequence of SEQ ID NO: 3 or 9. Furthermore, a gene consisting of a nucleotide sequence having at least 99% identity is preferably included.
  • the glycerol kinase gene also includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 or 10.
  • the gene includes a gene encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4 or 10.
  • the protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 4 or 10 is a deletion, addition, or insertion of ⁇ or several amino acids in the amino acid sequence of SEQ ID NO: 4 or 1.0.
  • a protein having a substituted amino acid sequence can be mentioned.
  • the protein has the above-mentioned glycerol kinase activity, that is, glycerin phosphorylation activity.
  • mutant protein consisting of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 4 or 10. If the mutation is highly conserved in the three-dimensional structure of the amino acid sequence described in No. 4 or 10, and the mutant protein has the activity of the glycerol kinase, these mutant types
  • the gene encoding the protein is also included in the glyceose mouth kinase gene.
  • severeal means usually 2 to 5, preferably 2 to 3.
  • the glycerol kinase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the amino acid sequence of SEQ ID NO: 4 or 10. Also included is a gene encoding a protein consisting of an amino acid sequence having a sex, more preferably at least 99% identity.
  • the target bacterium to be imparted with glycerin-utilizing ability does not have a glycerin kinase gene, it is necessary to introduce a glycerol kinase gene. However, if the target bacterium has a glycerol kinase gene, the glycerol kinase gene is not necessarily introduced. -In an embodiment, a glycerol triphosphate dehydrogenase gene is further introduced into the bacterium.
  • Glycerol triphosphate dehydrogenase (also referred to as glycerin triphosphate dehydrogenase) is a protein having an activity of dehydrating glycerol triphosphate (also referred to as glycerin triphosphate).
  • the glycerol triphosphate dehydrogenase gene is not particularly limited as long as it is a gene encoding a protein having an activity of dehydrating glycerol triphosphate, but it is derived from a microorganism having glycerin assimilation ability, preferably g Those having an I p A gene, for example, Escherichia coli fc, Salmonella ⁇ , Yersinia, Shigella, Haemophilus, Xanthomonas, Vibrio ⁇ , Pseudomonas, Methylococcus, Neisseria ⁇ , Ralstonia, Burkholderia, Bordetella , Nitrosomonas ⁇ , Helicobacter genus, Geobacter genus, 'Agrobacterium genus, Rhizobium genus, Brucella genus, Bradyrhizobium genus, Rhodopseudomonas genus, Rhodobacter
  • Escherichia coli K- 12 MG1655 Salmonella enterica serovar Typhi CT18, Salmonella typhi murium T2, Yersinia pest is C092, Shigella f lexneri 2457T, Shigella sonnei, Haemophilus influenzae (serotype d), Haemophilus ducreyi, estrisona p.
  • Xanthomonas oryzae KACC10331 Vibrio cholerae, Vibrio vulnificus CMCP6, Vibrio parahaeraolyticus, Vibrio f ischeri, Pseudomonas aeruginosa, Pseudomonas put i da, Pseudomonas -syringae pv.
  • Tomato DC3000 Pseudomonse lure solanacearum N Ralstonia eutropha ⁇ Burkholderia mallei, Burkholderia pseu domallei K96243, Burkholderia xenovorans ⁇ Burkholderia cenocepacia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Nitrosomonas europaea, Nitrosospira multiformis, Helicobacter pylori 26695, Helicobacter hepaticer sulfurreducens, Geobacter metallireducens, Agrobacterium tumefaciens C58 (UWash / Dupont), Rhizobiura etli, Brucella melitensis, Brucella abortus, Bradyrhizobium japonicum, Rhodopseudoraonas palustris CGA009, Nitrobacter winogradsky
  • the glycerol triphosphate dehydrogenase gene to be introduced into the bacterium in the present invention the g1 p A gene derived from Corynebacterium diphtheria is preferable.
  • the glycerol triphosphate dehydrogenase gene is preferably derived from a bacterium belonging to the same genus as the glycerin uptake protein gene to be introduced.
  • glycerol triphosphate dehydrogenase gene When introducing the glycerol triphosphate dehydrogenase gene, By introducing it as a g 1 p AFK gene contained in the same operon (g 1 p) in diphtheria and the like, a recombinant bacterium excellent in glycerin assimilation ability can be obtained.
  • a specific example of the glyce mouth triphosphate dehydrogenase gene is a gene consisting of the base sequence represented by SEQ ID NO: 11.
  • the glycerol kinase gene also includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 11.
  • “functionally equivalent” is the same as above.
  • a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 11 includes a gene that hybridizes with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 11 under stringent conditions. Can be mentioned.
  • the protein encoded by the gene has glycerin triphosphate dehydrogenase activity.
  • the stringent conditions are as described above.
  • the glycerin triphosphate dehydrogenase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: 11. More preferably, a gene comprising a base sequence having at least 99% identity is also included.
  • the glycerin triphosphate dehydrogenase gene also includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 12.
  • the gene includes a gene encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 12.
  • the protein has glycerin triphosphate dehydrogenase activity.
  • mutant protein consisting of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 2 If the difference is a highly conserved mutation in the three-dimensional structure of the amino acid sequence described in SEQ ID NO: 12, and the mutant protein has the activity of glycerin triphosphate dehydrogenase, Genes encoding these mutant proteins are also included in the glycerin triphosphate dehydrogenase gene.
  • severe means usually 2 to 5, preferably 2 to 3.
  • the glycerin triphosphate dehydrogenase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity with the amino acid sequence of SEQ ID NO: 12. More preferably, a gene encoding a protein consisting of an amino acid sequence having at least 99% identity is also included.
  • the target bacterium that is to be glycerinated does not have the glycerin triphosphate dehydrogenase gene, it is necessary to introduce the glycerin triphosphate dehydrogenase gene. However, if the target bacterium has a glycerin triphosphate dehydrogenase gene, the glycerin triphosphate dehydrogenase gene need not necessarily be introduced.
  • all of the genes described above are derived from bacteria belonging to the same genus as the bacterium to be introduced.
  • a gene when a gene is introduced into a bacterium belonging to the genus Corynebacterium, it is preferable to introduce a gene derived from a bacterium belonging to the genus Corynebacterium, preferably Corynebacterium diphtheria.
  • it is introduced as the g 1 p F gene derived from Corynebacterium diphtheria, particularly as the g 1 p A F K gene contained in the glycerol phosphate operon derived from Corynebacterium diphtheria.
  • Corynebacterium diphtheria has glycerin assimilation ability but was difficult to use because it is a pathogen.
  • a glycerin uptake protein gene derived from Corynebacterium diphtheria into another Corynebacterium genus 'bacteria', it is possible to obtain a bacterium having excellent glycerin utilization ability and having no pathogenicity.
  • Introducing a gene into a bacterium involves linking the above gene or a part thereof to an appropriate vector and introducing the resulting recombinant vector into a host so that the target gene can be expressed, or It can be carried out by inserting the gene of interest or a part thereof at any position on the genome by homologous recombination.
  • Part refers to a part of each gene capable of expressing the protein encoded by each gene when introduced into a host.
  • a method for obtaining a desired gene from a bacterial genome by cloning is well known in the field of molecular biology. For example, if the gene sequence is known, A more suitable genomic library can be created and screened using a probe complementary to the desired gene sequence. Once the sequence is isolated, the DNA is amplified using standard amplification techniques such as polymerase, chain reaction (PCR) (US Pat. No. 4,683,202) and is suitable for transformation. DNA can be obtained.
  • the glycerin uptake protein gene or glycerol kinase gene can be hybridized using a known synthetic glycerin uptake protein gene or a synthetic DNA primer appropriately designed based on the base sequence of the glycerol kinase gene as a cage. It can also be obtained by the PCR method or PCR method.
  • the vector to which the gene is linked is not particularly limited as long as it can be replicated in the host cell, and examples thereof include plasmids, phages and cosmids.
  • a shuttle vector that is movable between the bacterium from which the gene is introduced and the bacterium from which the gene is introduced is used.
  • a glycerin-incorporating protein gene derived from E. coli is introduced into a coryneform bacterium
  • a shuttle vector that can move between E. coli and the coryneform bacterium is preferable.
  • a glycerin uptake protein gene derived from E. coli is introduced into Ralstonia bacteria
  • a shuttle vector that can move between E. coli and Ralstonia bacteria is preferred.
  • a shuttle vector that can move between Escherichia coli and the genus Algigen genus is preferable.
  • pAM 3 30 derived from prebibataterium lactofamentum 2256 (JP 58-6 7699, Ag ric c. Biol. Chem. Vol. 48, 290 1-2903 ( 1 984) and Nu c 1 ei cAc ids S ymp Ser. Vol. 1 6, 26 5 -267 (1985)), Corynebacterium glutamicum ATCC 1 3 058 strain pHMl 5 1 9 (A gric. B iol. C he m. vol. 48, 29 0 1-2903 (1 984)), p CRY 30 (Ap pl. En viro n. Mi crobiol. Vo l.
  • an appropriate expression promoter is connected upstream of the gene.
  • the expression promoter to be used is not particularly limited as long as it is not repressed by glycerin, and may be appropriately selected by those skilled in the art depending on the host.
  • the host is Escherichia coli, T7 promoter, lac promoter, trp promoter, ⁇ -PL promoter, etc.
  • the host is a Bacillus genus, a SPO promoter or the like can be used.
  • coli g 1 p (glycerol 'phosphate operon) is used from the viewpoint of efficient expression of glycerin uptake protein. I like it.
  • the promoter contained in Corynebacterium diphtheria g 1 p (glycerol phosphate operon) (P glp FK) is preferably used. Also preferred are promoters that are induced by glycerin.
  • the method for introducing the above vector into the host is not particularly limited.
  • the electric pulse method (Y. K'u rusu, eta 1., Ag ri c. B io 1. C he m. 54: 443 -447 (19 90)) ⁇ Method using calcium ion (Proc. Natl. Acad. Sci. USA, 6 9, 21 10 (1 9 72)), Protoplast method (Japanese Patent Laid-Open No. 6-2483942) ), Electroporation (Nuclei cAcids Res., 16, 6, 1 27 (1 98 '8))) and the like.
  • the target gene is inserted into a sequence homologous to the sequence on the genome together with a promoter, and this DNA fragment is inserted into the cell by electroporation. It can be carried out by introducing and causing homologous recombination.
  • introducing into the genome use a DNA fragment linking the target gene and drug resistance gene. Then, strains that have undergone homologous recombination can be selected easily.
  • a gene linked to a drug-resistant gene and a gene that is lethal under specific conditions is inserted into the genome by homologous recombination as described above, and then the drug-resistant gene is lethal under specific conditions. It is also possible to introduce the target gene using homologous recombination in a form that replaces the gene.
  • a method for selecting a recombinant bacterium into which a target gene has been introduced is not particularly limited, but a method that allows easy selection of only a recombinant bacterium into which a target gene has been introduced is preferred.
  • the culture conditions for recombinant bacteria can be set based on the conditions normally used in the art depending on the type of bacteria used as the host. However, in this case, the culture medium for recombinant bacteria must be glycerin. It is desirable to contain it as the sole carbon source.
  • the glycerin assimilation test of recombinant bacteria can be carried out by culturing these recombinant bacteria in a medium containing glycerin and following changes in the glycerin uptake rate and bacterial cell amount.
  • Measurement of the glycerin uptake rate by the recombinant bacteria can be carried out by measuring the concentration of glycerin remaining in the medium using an HPLC analysis.
  • the change in the amount of bacterial cells can be measured by measuring the change in optical density at 60 nm with a spectrophotometer.
  • the recombinant bacterium of the present invention obtained as described above is cultured in a medium, a fermentation product or a reaction product is produced and accumulated in the medium or bacterial cell, and the product is collected from the medium or bacterial cell.
  • a fermentation product or a reaction product can be produced using glycerin.
  • Examples of fermentation products or reaction products to which the method of the present invention can be applied include substances produced by metabolism of glycerin and substances produced by utilizing energy produced by metabolism of glycerin. It is done. Specifically, for example, amino acids such as glutamic acid, lysine, threonine, phenylalanine, tryptophan, etc .; organic acids such as pyruvic acid, lactic acid, acetic acid surrounding organic acids such as pyruvic acid, lactic acid, acetic acid, kenic acid, fumaric acid Organic acids around the TCA cycle, such as malic acid, succinic acid, itaconic acid, glyceric acid, organic acids around glycerin such as 3-hydroxypropionic acid; alcohols such as ethanol, propanol, 1 3-Propanediol; vitamins such as vitamin C; and polymer substances such as various enzymes.
  • amino acids such as glutamic acid, lysine, threonine, phenylalanine
  • the production of amino acids using bacteria can be carried out by methods known in the art except that a medium containing glycerin as a carbon source is used (for example, amino acid fermentation, Hiroshi Aida et al., Academic Publishing Center).
  • the culture medium, culture medium and culture conditions can be set appropriately according to the type of bacteria used, but a normal medium containing a nitrogen source, inorganic ions and other organic micronutrients as necessary. Can be used.
  • Glycerin is used as the carbon source.
  • sugars such as glucose, lactose, galactose, fructose or starch hydrolysate, alcohols such as sorbitol, or organic acids such as fumaric acid, succinic acid or succinic acid may be used in combination.
  • glycerin as carbon can be used in the form of an aqueous solution containing impurities that is discharged in large quantities as a by-product in the surfactant and biodiesel fuel industries. Therefore, the present invention is also advantageous from the viewpoint that the by-products that have been discarded can be used effectively.
  • inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate
  • organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like
  • inorganic ions potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added.
  • organic micronutrients it is desirable to contain required amounts of L-homoserine, vitamin B1, etc. or yeast extract as required.
  • Culturing is performed under conditions suitable for the growth of the bacteria to be used.
  • the culture temperature is 20 ° C to 45 ° C, and the pH is 5 to 8.5 during the culture.
  • an inorganic or organic acidic or alkaline substance as well as ammonia gas.
  • the culture * temperature can be cultured at 42 ° C. to 60 ° C.
  • Culture can be performed by aerobic cell growth followed by anaerobic conditions. This method is particularly effective when producing organic acids such as lactic acid and succinic acid.
  • succinic acid it is preferable to add carbon dioxide and carbonate ions to the medium because the yield increases.
  • the collection of the fermentation product or reaction product from the culture medium after completion of the culture does not require a special method in the present invention. That is, it can be carried out by combining conventionally known ion exchange resin method, precipitation method and other methods.
  • glycerin is used as a carbon source, it may be possible to simplify the purification of the target substance and the waste liquid treatment process, compared to the case where saccharides derived from agricultural products are used.
  • hybrid plasmids For the preparation of hybrid plasmids, use the plasmid ⁇ CG 100 (3 kbp) derived from Corynepacterum 'Dartamicam ATCC 1 3058 strain and the plasmid pHSG398 (Takara Bio Inc.) derived from Escherichia coli. It was.
  • PCG100 plasmid derived from Corynebacterium dartamicum ATCC 13058 strain was obtained and ligated with pHSG398 to prepare a hybrid plasmid.
  • Corynebacterium 'glutamicum ATCC 13058 strain was cultured with shaking in JCM26 medium (5 m' 1) at 30 ° C.
  • the cultured Corynebacterium 'Dartamicam A TCC 13058 strain was seeded on a JCM26 plate and cultured at 30 ° C with shaking.
  • JC M26 plate was inoculated with 1 loop in a loop, and cultured with shaking in JCM26 medium (5 ml) at 30 ° C for 6 hours.
  • the preculture solution lm 1 was inoculated into 5 ml of JCM26 medium (isonicotinic acid hydrazide 200 mg / 50 ml) and cultured at 30 ° C with shaking. After completion of the culture, it was cooled on ice. The culture was centrifuged at 15000 rpm for 10 minutes to recover the cells.
  • JCM26 medium isonicotinic acid hydrazide 200 mg / 50 ml
  • a plasmid was extracted from the collected cell pellet using Ml] 3 I Kit (Qiagen). At the time of recovery, the bacterial cell pellet was suspended in 1 ml of P 1 buffer containing 6 mg of lysozyme and shaken for 1 hour in a 37 ° C water bath. ,
  • Transformation was performed by applying 2.5 kvZ 200 ⁇ / 25 ⁇ F to the TOP 10 electrified cells.
  • the obtained E. coli transformant was inoculated with 0.5 ni U of SOC medium and placed in a 15 ml centrifuge tube. The culture was shaken at 37 ° C for 1 hour. 37 100 mu 1 spread LBCm culture destinations in ° C, at 30 ° C, and stationary culture was performed ⁇
  • the Escherichia coli containing the hybrid plasmid obtained in Example 1 was cultured again to prepare a large amount of the plasmid, which was introduced into the Corynebacterium dartamicam ATCC 13032 strain.
  • a colony of Corynebacterium 'glutamicum ATCC 13032 strain was inoculated into one loop. Culturing was carried out for 16 hours at 300 rpm and 30 ° C. in 5 ml of LB medium containing 0.2 ml of 50% glucose. At the end of the culture, OD 660 was measured.
  • the OD 66Q was measured, and the culture was sufficiently cooled on ice.
  • the culture solution was centrifuged at 15000 rpm for 10 minutes to recover the cells.
  • the obtained cells were suspended in 5 ml of 10% glycerin and cooled on ice. Centrifuge at 15000 rpm for 10 minutes and discard the supernatant. Pipetting with 10% glycerin in lm 1 And transferred to a 1.5 ml sterilized Eppen. Centrifugation was performed at 1 500 rpm for 1 minute, and the supernatant was discarded. Pipetted again with 1 ml of 10% glycerin. Centrifugation was carried out at 1 500 rpm for 1 minute, and the supernatant was discarded (4 times).
  • a voltage was applied at 2.5 k.v / 600 ⁇ / 25 ⁇ F to the competent cell obtained in 2-1. It was transferred to BH I S medium l rn 1 and placed in a 15 m 1 centrifuge tube. 4 Restoration culture was performed by allowing to stand in a 6 ° C water bath for 6 minutes, ice-cooling for 3 minutes, and then shaking culture at 30 ° C for 1 hour. L BHI IS medium Cm 30 and 60 were each plated with 5 plates in 2 sets for a total of 20 plates and statically cultured at 30 ° C.
  • JCM 26 medium, Epo medium, BH I S medium and L BH I S plate were prepared as follows.
  • JCM 26 medium composition JCM 26 medium composition:
  • BH IS medium The following A and B were mixed immediately before use.
  • the constructed hybrid plasmid pCG100-pHSG 398 was confirmed to be stably retained in both E. coli and Corynebacterium dartamicam as a shuttle vector.
  • Escherichia coli has (a) glycerol uptake protein (glycerol facilitator) and (b) glycerol kinase as operon (g 1 p FK operon) as glycerin utilization gene, and in the order of promoter, facilitator, kinase. Arranged.
  • a PCR was prepared from the fragment of promoter + (a) + (b) using the following primers, inserted into the pCG100_pHSG398 shuttle vector, and introduced into Corynebata glutamicum.
  • the pHSG398 plasmid was confirmed to contain 5 fragments of 1, 6, 8, 9 and 10 (Fig. 5). .
  • a large number of plasmid No. 10 was prepared and introduced into Corynepacteria d'artamicam ATCC 13032 strain.
  • composition of the growth test medium was as follows. Urea 4 g
  • the wild strains of les were also vigorously growing when dulcose was used as the carbon source, but when Dali serine was used as the carbon source, there was a difference from the condition without carbon source. There wasn't. Therefore, it was confirmed that the wild strain classified as Corynebacterium dartamicam has no glycerin assimilation ability.
  • the following medium was placed in a 2 L jar fermenter, autoclaved, and cultured in 50 ml of the same composition.
  • Escherichia coli g 1 p FK gene introduced Corynepactum glutamicum ATC C 1 3 0 3 2
  • the strain was inoculated and cultured at 30 ° C. for 48 hours while adjusting the pH to 7.2 with 25% aqueous ammonia while aeration was performed at 0.5 vvm.
  • Example 7 Production of organic acid Culture medium 10 Om 1 is sterilized in a 500 ml 1 flask, sterilized 50% glycerol solution 4 ml 1 is added, and E. coli g 1 p FK gene introduced Corynebacterium glutamicum AT CC 13032 strain is inoculated, 30 ° Incubated with C for 24 hours (1 Sakaguchi). After completion of the culture, the cells were centrifuged. Wet cells obtained from 100 ml of culture broth were 3.7'3 g. This was used for the reaction.
  • reaction medium 100 ml of the reaction medium to a 100 ml medium bottle, add the bacterial cells and 12 ml of 50% glycerin solution (work in the glove box), and keep the reaction sealed at 30 ° C for 24 hours with stirring with a stirrer. I let you. After the reaction, the culture solution was centrifuged and analyzed.
  • the culture broth contained malic acid 0.4%, succinic acid 1.4%, acetic acid 0.2%, and lactic acid '1.9%.
  • Examples 6 and 7 indicate that amino acids and organic acids can be produced by culturing a recombinant bacterium introduced with a gene encoding a glycerin uptake protein in a medium containing glycerin:! It was.
  • Ralstonia eutropha ATCC 17697 cannot grow using glycerin as a carbon source. Glycerol-utilizing ability was conferred by introducing the glycerol-utilizing genes g 1 pF and g 1 pK derived from Escherichia coli into this Ralstonia eutropha ATCC 17697 strain.
  • the g 1 p FK gene fragment was amplified by PCR using the following primer set using the g 1 p FK / p CG 100-pHSG 398 plasmid introduced into Corynebata d'artamicam as a saddle type.
  • the broad host vector pBBR122 plasmid contains resistance genes for two antibiotics, chloramphenicol and kanamycin.
  • N co I site existing on this chloramfu unicol resistance gene it was cleaved with the restriction enzyme Nco I, then treated with alkaline phosphatase and dephosphorylated. did.
  • This vector DNA fragment was separated by electrophoresis, cut out and recovered. This fragment and the previous g 1 p FK gene fragment were ligated and introduced into E. coli to obtain g 1 p FKZp BBR122 plasmid.
  • the KpBBR122 plasmid was introduced into the Lanorestonia utropha ATC C 17697 strain by electroporation.
  • a voltage was applied to a solution in which 2 ⁇ l of pBBR 12 2 plasmid solution without insert was added instead of gl pFK / pBBR122 plasmid solution.
  • the suspension was suspended in 1 ml of SOC medium, and cultured with shaking at 30 for 1 hour. After ⁇ time, the entire amount was inoculated into 50 ml of medium supplemented with 50 ppm of kanamycin in NB medium and cultured overnight at 3 CL ° C. After one night, the culture solution was diluted to 10 3 to 10 6 , spread on a medium obtained by adding 300 ppm of kanamycin to NB medium, and statically cultured at 30 ° C. to form colonies.
  • the grown colonies were inoculated into a medium obtained by adding 50 ppm of kanamycin to NB medium, and cultured at 30 for 10 hours.
  • the cells were collected from 1.5 ml of the culture solution by centrifugation, and the plasmid was extracted.
  • the restriction enzyme NcoI When confirmed by electrophoresis, it was confirmed that the transformant with gl pFKZpBBR122 plasmid introduced was found to have bands derived from g 1 p FK and p BBR 122 plasmid at positions 5300 bp and 3300 bp. In the transformant into which BBR 122 plasmid was introduced, only a band of a size considered to be derived from the pBBR 122 plasmid could be confirmed at the 5300 bp position.
  • the Ralstonia eutropha transformants were named Ralstonia eutropha / g 1 p FK / p B BR 122 and Ralstonia eutropha ZpBBRl 22, respectively. 8-5. Growth test of Ralstonia eutropha 'transformant
  • Ralstonia 'Utropha ATCC 17697 strain can be grown by using 3 strains of Ralstonia eutropha ATCC 17697 wild strain, the previous Lars small nya eutrophano g 1 p FK / p B BR 122 and ralstonia eutropha ZpBBRl 2 2 Growth was confirmed using a minimal medium containing L-malic acid as a single carbon source and a minimal medium containing glycerin as a single carbon source.
  • Ralstonia eutropha ATCC 17697 wild strain is NB medium 5 ml, ralstonia 'eutropha / g 1 p FK / p B BR 122 strain and Ralstonia eutropha / B BR 122 strain kanamycin 300 pm on NB medium
  • composition of the medium used in this example is as follows.
  • Alkaline Genes faecalis ATCC 18750 cannot grow T using glycerin as a carbon source.
  • the glycerin utilization genes g 1 pF and g 1 pK derived from E. coli were introduced into the Alkaligenes' Faecaris ATCC 18750 strain to impart the ability to assimilate dalyserin.
  • Alkaline Genes' Faekaris ATCC 18750 wild strain is 5 ml in NB medium, Al Carigenes' faecaris Zg 1 p FK / p BBR 122 strain and Al force ligenes faye squirrel / p BBR 122 strain were inoculated into 5 ml of medium with 300 kpm of kanamycin added to NB medium, 30 ° C. overnight culture.
  • 50 1 of a medium supplemented with L-malic acid or glycerin as a carbon source in the following minimal medium was inoculated with 50 1 of the culture medium cultured overnight and cultured at 30 ° C. for 24 hours.
  • the wild-type strain of Alcaligenes' Faecaris does not have glycerin assimilation ability, whereas the recombinant bacterium of the present invention has glycerin assimilation ability. Indicated. It can also be seen that the recombinant bacterium of the present invention has glycerin assimilation ability equivalent to L-malate assimilation ability in the wild strain.
  • the composition of the minimal medium used for the growth test is as follows.
  • This vector was ligated with the g 1 p AFK gene fragment excised from pCR40PO, and transformed into large intestine.
  • the plasmid was recovered from the obtained transformant, cut with restriction enzymes XbaI and PstI, and the insert was confirmed.
  • a 4600 bp band of the same size as the introduced g1p AF K gene was obtained.
  • This plasmid was named Cd_g 1 p AFK-pCG100-pHSG39 &.
  • a combinatorial cell of Corynebatarum-Glutamicum was prepared and this plasmid was introduced by the electroporation method.
  • Plasmid-only strain 15.8 0.74 0.80 The above results show that the wild-type strain of Corynebacterium dartamicam does not have glycerin-utilizing ability, whereas the recombinant bacterium of the present invention has glycerin-utilizing ability. It was done. In addition, it can be seen that the recombinant bacterium of the present invention has a glycerin assimilation ability equivalent to that in the wild strain.
  • glycerol-assimilating ability can be imparted to bacteria.
  • means for effectively utilizing glycerin is provided.

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Abstract

L’invention a pour objet un moyen pour conférer une capacité d'assimilation du glycérol à un micro-organisme, à savoir, une méthode qui comprend le transfert d’un gène de la protéine d’incorporation du glycérol dans une bactérie afin de lui conférer une capacité d'assimilation du glycérol.
PCT/JP2006/315453 2005-07-29 2006-07-28 Méthode pour conférer la capacité d’assimilation du glycérol à des bactéries WO2007013695A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008126667A2 (fr) 2007-03-19 2008-10-23 Sumitomo Chemical Company, Limited Procédé destiné à produire de l'acide lactique
WO2008126669A3 (fr) * 2007-03-19 2008-12-24 Sumitomo Chemical Co Procédé de production d'acide pyruvique
WO2010084995A2 (fr) 2009-01-23 2010-07-29 Ajinomoto Co.,Inc. Procédé de production d'un acide l-aminé
US7833761B2 (en) 2007-09-04 2010-11-16 Ajinomoto Co., Inc. Amino acid producing microorganism and a method for producing an amino acid
EP2260104A1 (fr) * 2007-11-20 2010-12-15 CJ Cheiljedang Corporation Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries
JP2011511643A (ja) * 2008-02-13 2011-04-14 バイオコンバージョン テクノロジーズ リミテッド 細菌細胞によるエタノール産生の増加
JP2012179003A (ja) * 2011-03-01 2012-09-20 Institute Of National Colleges Of Technology Japan バイオディーゼル燃料の製造方法
JP2013539368A (ja) * 2010-08-30 2013-10-24 コリア アドバンスド インスティチュート オブ サイエンス アンド テクノロジィ スクロースとグリセロールとを同時に利用する新規コハク酸生成変異微生物及びこれを利用したコハク酸製造方法
US8679798B2 (en) 2007-12-21 2014-03-25 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family
JP2014060974A (ja) * 2012-09-21 2014-04-10 Kao Corp バチルス（Ｂａｃｉｌｌｕｓ）属細菌のグリセリン資化能を向上させる方法
JP2014060975A (ja) * 2012-09-21 2014-04-10 Kao Corp 微生物のグリセリン資化能を向上させる方法
US9150827B2 (en) * 2005-10-05 2015-10-06 Evonik Degussa Gmbh Method for the fermentative production of L-amino acids with the aid of coryneform bacteria capable of using glycerin as the only carbon source
WO2019123324A1 (fr) * 2017-12-21 2019-06-27 Glycom A/S Construction d'acide nucléique permettant l'expression d'un gène in vitro et in vivo
WO2020255054A1 (fr) * 2019-06-21 2020-12-24 Glycom A/S Construction d'acide nucléique comprenant une boucle-tige 5'utr pour l'expression génique in vitro et in vivo
CN114149954A (zh) * 2021-12-01 2022-03-08 上海交通大学 利用谷氨酸棒状杆菌高效分泌生产类蛛丝、类弹性蛋白并快速纯化的方法

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US9150827B2 (en) * 2005-10-05 2015-10-06 Evonik Degussa Gmbh Method for the fermentative production of L-amino acids with the aid of coryneform bacteria capable of using glycerin as the only carbon source
WO2008126667A3 (fr) * 2007-03-19 2008-12-24 Sumitomo Chemical Co Procédé destiné à produire de l'acide lactique
WO2008126669A3 (fr) * 2007-03-19 2008-12-24 Sumitomo Chemical Co Procédé de production d'acide pyruvique
WO2008126667A2 (fr) 2007-03-19 2008-10-23 Sumitomo Chemical Company, Limited Procédé destiné à produire de l'acide lactique
US7833761B2 (en) 2007-09-04 2010-11-16 Ajinomoto Co., Inc. Amino acid producing microorganism and a method for producing an amino acid
EP2260104A4 (fr) * 2007-11-20 2012-03-14 Cj Cheiljedang Corp Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries
EP2260104A1 (fr) * 2007-11-20 2010-12-15 CJ Cheiljedang Corporation Corynébactéries utilisant des sources de carbone contenant du glycérol et procédé de fabrication d'un produit de fermentation à l'aide de ces corynébactéries
US8679798B2 (en) 2007-12-21 2014-03-25 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family
JP2011511643A (ja) * 2008-02-13 2011-04-14 バイオコンバージョン テクノロジーズ リミテッド 細菌細胞によるエタノール産生の増加
WO2010084995A2 (fr) 2009-01-23 2010-07-29 Ajinomoto Co.,Inc. Procédé de production d'un acide l-aminé
JP2013539368A (ja) * 2010-08-30 2013-10-24 コリア アドバンスド インスティチュート オブ サイエンス アンド テクノロジィ スクロースとグリセロールとを同時に利用する新規コハク酸生成変異微生物及びこれを利用したコハク酸製造方法
JP2012179003A (ja) * 2011-03-01 2012-09-20 Institute Of National Colleges Of Technology Japan バイオディーゼル燃料の製造方法
JP2014060974A (ja) * 2012-09-21 2014-04-10 Kao Corp バチルス（Ｂａｃｉｌｌｕｓ）属細菌のグリセリン資化能を向上させる方法
JP2014060975A (ja) * 2012-09-21 2014-04-10 Kao Corp 微生物のグリセリン資化能を向上させる方法
WO2019123324A1 (fr) * 2017-12-21 2019-06-27 Glycom A/S Construction d'acide nucléique permettant l'expression d'un gène in vitro et in vivo
CN111727253A (zh) * 2017-12-21 2020-09-29 格礼卡姆股份公司 用于体外和体内基因表达的核酸构建体
US11608504B2 (en) 2017-12-21 2023-03-21 Glycom A/S Nucleic acid construct for in vitro and in vivo gene expression
WO2020255054A1 (fr) * 2019-06-21 2020-12-24 Glycom A/S Construction d'acide nucléique comprenant une boucle-tige 5'utr pour l'expression génique in vitro et in vivo
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