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WO2024185708A1 - Micro-organisme transformé et procédé de production d'acide lactique ou de co-polyester - Google Patents

Micro-organisme transformé et procédé de production d'acide lactique ou de co-polyester Download PDF

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Publication number
WO2024185708A1
WO2024185708A1 PCT/JP2024/007858 JP2024007858W WO2024185708A1 WO 2024185708 A1 WO2024185708 A1 WO 2024185708A1 JP 2024007858 W JP2024007858 W JP 2024007858W WO 2024185708 A1 WO2024185708 A1 WO 2024185708A1
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gene
lactic acid
acid
transformed microorganism
amino acid
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Japanese (ja)
Inventor
相昊 高
精一 田口
祥 古舘
俊輔 佐藤
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Kaneka Corp
Kobe University NUC
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Kaneka Corp
Kobe University NUC
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/78—Preparation processes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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.)
    • 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
    • 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
    • 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
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates to a transformed microorganism capable of producing lactic acid or a copolymer polyester, and a method for producing lactic acid or a copolymer polyester using the microorganism.
  • Lactic acid (hereafter abbreviated as LA) is one of the basic chemicals with high demand among the organic acids obtained by fermentation production. It is used as a raw material for food, medicines, and cosmetics, and as a precursor for plastics, additives, medicines, etc., and has a major role in industry.
  • the fermentative production of LA has been achieved using lactic acid bacteria or yeast as a host, but these microorganisms have high nutritional requirements, necessitating the use of expensive culture media. Therefore, there is a demand for fermentative production of LA using microorganisms that can use synthetic media with low nutritional requirements.
  • LA is in demand as a monomer raw material for plastic materials, especially as a raw material for polylactic acid.
  • Poly(3-hydroxybutyrate-co-lactic acid) hereinafter abbreviated as LAHB
  • LAHB Poly(3-hydroxybutyrate-co-lactic acid)
  • PHA polyhydroxyalkanoic acid
  • 3HB 3-hydroxybutyric acid
  • Such LAHB can be produced by microbial fermentation, and in particular, there are known examples of fermentative production using transformants of hydrogen-oxidizing bacteria, Capriavidus genus microorganisms. Capriavidus genus microorganisms are known to grow well in synthetic media and produce LAHB through fermentation, and are expected to be useful as hosts for the fermentative production of organic acids using inexpensive synthetic media.
  • Non-Patent Document 1 reports that LAHB was successfully produced by introducing PCT Cp V193A, a single amino acid mutant of propionyl CoA transferase derived from Clostridium propionicum, into the microorganism Cupriavidus necator.
  • the aim of the study was to suppress the supply of 3HB-CoA for copolymerization of 3HB with LA by deleting the genes encoding PhaA and PhaB1, enzymes that supply 3HB-CoA, in Cupriavidus necator.
  • Non-Patent Document 1 shows that the productivity of LAHB was only 0.14 g/L when glucose was used as a carbon source.
  • Non-Patent Document 1 in order to produce LAHB with a high LA ratio, the ability to supply 3HB-CoA was suppressed by deleting the genes encoding PhaA and PhaB1, which are enzymes that supply 3HB-CoA. As a result, although the LA ratio could be increased, there was a problem in that the productivity of LAHB was reduced.
  • the present invention aims to provide a transformed microorganism of the genus Capriavidus that is capable of producing lactic acid or a copolymer polyester of lactic acid and another hydroxyalkanoic acid with high productivity.
  • Capriavidus microorganisms tend to grow well using lactic acid as a carbon source. This suggests the existence of a metabolic pathway that consumes lactic acid. With the aim of blocking such a metabolic pathway, the inventors attempted to disrupt various genes present in the genome of Capriavidus microorganisms that may be involved in lactic acid metabolism.
  • the present invention relates to a transformed microorganism belonging to the genus Capriavidus having the ability to produce lactic acid
  • the present invention relates to a transformed microorganism in which at least one gene selected from the group consisting of the following genes (A) and (B) has been disrupted: Gene (A): a gene encoding a 2-hydroxyacid dehydrogenase having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, or a gene encoding a protein having an amino acid sequence with 90% or more sequence identity to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and having 2-hydroxyacid dehydrogenase activity.
  • Gene (B) a gene encoding a regulator protein that controls the expression of at least one of the genes (A).
  • the present invention also relates to a method for producing lactic acid, which includes a step of culturing the transformed microorganism.
  • the present invention further relates to a method for producing a copolymer polyester of lactic acid and another hydroxyalkanoic acid, which comprises a step of culturing the transformed microorganism.
  • the present invention provides a transformed microorganism of the genus Capriavidus that is capable of producing lactic acid or a copolymer polyester of lactic acid and another hydroxyalkanoic acid with high productivity.
  • the transformed microorganism can produce a copolyester having a high LA ratio with good productivity.
  • the transformed microorganism according to the present invention does not need to be cultured in a natural medium, and can produce lactic acid or a copolymer polyester containing lactic acid by culturing in a synthetic medium.
  • the present embodiment relates to a transformed microorganism belonging to the genus Capriavidus, which has the ability to produce lactic acid and/or the ability to produce a copolymer polyester of lactic acid and another hydroxyalkanoic acid.
  • the transformed microorganism according to this embodiment uses a microorganism belonging to the genus Cupriavidus as a host.
  • Microorganisms belonging to the genus Cupriavidus have a proven track record in the industrial production of polyesters such as PHBH, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, and can synthesize the polyester with high efficiency using an inexpensive synthetic medium and carbon source.
  • polyesters such as PHBH, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid
  • the transformed microorganism according to this embodiment has the ability to produce lactic acid.
  • lactic acid is produced and released outside the cell, so that lactic acid can be easily obtained from the culture solution.
  • the transformed microorganism according to this embodiment may have the ability to produce 2-hydroxyalkanoic acids other than lactic acid, in addition to lactic acid.
  • 2-hydroxyalkanoic acids include glycolic acid, 2-hydroxybutyric acid, 2-hydroxy-4-methylvaleric acid, and 2-hydroxy-3-methylvaleric acid. Only one of these may be produced, or two or more may be produced simultaneously.
  • a transformed microorganism according to a preferred embodiment can have the ability to produce a copolymer polyester of lactic acid and a hydroxyalkanoic acid other than lactic acid.
  • the hydroxyalkanoic acid other than lactic acid that may be contained in the copolymer polyester is not particularly limited, and may be, for example, a hydroxyalkanoic acid having 2 to 15 carbon atoms that can be copolymerized with lactic acid.
  • 3-hydroxybutyric acid (sometimes abbreviated as 3HB), glycolic acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, 5-hydroxyvaleric acid, 6-hydroxyhexanoic acid, 4-hydroxyvaleric acid, 4-hydroxyhexanoic acid, 2-hydroxybutyric acid, 3-hydroxy-2-methylbutyric acid, 3-hydroxy-2-methylvaleric acid, 2-hydroxy-4-methylvaleric acid, and 2-hydroxy-3-methylvaleric acid. Only one of these may be contained in the copolymer polyester, or two or more may be contained. Among them, it is preferable that at least 3HB is contained. In particular, LAHB composed of lactic acid and 3HB is preferable as the
  • the ratio of lactic acid in the copolymer polyester is not particularly limited. By adjusting the configuration of the transformed microorganism according to the present disclosure, the carbon source, the culture conditions, etc., it is possible to produce copolymer polyesters with various ratios. Specifically, the ratio of lactic acid units to all monomer units constituting the copolymer polyester may be about 0.1 to 70 mol%, about 0.3 to 50 mol%, about 0.5 to 30 mol%, or about 1 to 10 mol%.
  • the transformed microorganism according to the present disclosure is one in which at least one gene selected from the group consisting of the following genes (A) and (B) inherently possessed by microorganisms of the genus Capriavidus has been disrupted. By disrupting these genes, it is possible to improve the productivity of lactic acid by the transformed microorganism and/or the ratio of lactic acid in the copolymer polyester produced by the transformed microorganism.
  • Gene (A) a gene encoding a 2-hydroxyacid dehydrogenase having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, or a gene encoding a protein having an amino acid sequence with 90% or more sequence identity to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and having 2-hydroxyacid dehydrogenase activity.
  • Gene (B) a gene encoding a regulator protein that controls the expression of at least one of the genes (A).
  • the amino acid sequence represented by SEQ ID NO: 1 is that of lactate dehydrogenase Dld (H16_A3091) possessed by Capriavidus necator strain H16.
  • the amino acid sequence represented by SEQ ID NO: 2, 3, or 4 is that of glycolate dehydrogenase GlcD1 (H16_A3094), GlcE (H16_A3096), or GlcF (H16_A3097) possessed by Capriavidus necator strain H16.
  • sequence identity for gene (A) may be 95% or more, 97% or more, 99% or more, or 99.5% or more.
  • only one of the genes (A) may be disrupted, or two or more of the genes may be disrupted. Of these, it is preferable that three or more genes are disrupted, since this is effective in increasing the ratio of lactic acid in the copolymer polyester produced.
  • Methods for disrupting genes (A) and/or (B) include, but are not limited to, methods for deleting all or part of the base sequence of the target gene, inserting extra bases into all or part of the base sequence of the target gene, replacing all or part of the base sequence of the target gene with a different base sequence, and introducing a stop codon into the middle of the base sequence of the target gene.
  • Gene (B) is a gene that codes for a regulator protein that controls the expression of at least one of the above-mentioned genes (A).
  • Control of expression here refers to positively controlling the expression of gene (A) and promoting the transcription of gene (A).
  • the regulator protein encoded by gene (B) is not particularly limited, but examples thereof include a regulator protein having an amino acid sequence represented by SEQ ID NO:5, and a regulator protein having an amino acid sequence with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:5.
  • the amino acid sequence represented by SEQ ID NO:5 is that of the regulator protein LysR (H16_A3092) of Capriavidus necator strain H16.
  • sequence identity for gene (B) may be 95% or more, 97% or more, 99% or more, or 99.5% or more.
  • the transformed microorganism according to this embodiment preferably has at least one gene selected from a gene encoding a copolymer polyester synthase, a gene encoding propionyl CoA transferase (PCT) or butyl CoA transferase (BCT), and a gene encoding an enzyme that supplies 2-hydroxy acid.
  • PCT propionyl CoA transferase
  • BCT butyl CoA transferase
  • polyester copolymer synthase is an enzyme that has the activity of synthesizing the polyester copolymer by copolymerizing lactic acid with another hydroxyalkanoic acid using the CoA form of lactic acid and the CoA form of the other hydroxyalkanoic acid as substrates.
  • Polyester synthase is also called polyester synthase.
  • the copolyester synthase is not particularly limited as long as it has the above activity, but may be a known enzyme having activity to synthesize a copolyester containing a lactic acid unit.
  • the gene encoding such an enzyme may be the phaC gene inherent to the host of the transformed microorganism according to this embodiment, a gene in which a mutation has been introduced into the phaC gene, or an exogenous gene.
  • the copolymer polyester synthase is not particularly limited, but a polyhydroxyalkanoic acid synthase derived from a microorganism of the genus Pseudomonas or a mutant thereof can be suitably used.
  • the mutant refers to a protein that has 90% or more sequence identity to the amino acid sequence of the polyhydroxyalkanoic acid synthase and exhibits polyhydroxyalkanoic acid synthesis activity.
  • the sequence identity is preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and most preferably 99.5% or more.
  • an STQK mutant in which the 325th serine of the enzyme PhaC1 Ps derived from Pseudomonas sp. 61-3 is converted to threonine and the 481st glutamine is converted to lysine can be preferably used. It has been reported that LAHB can be produced by culturing a transformant obtained by introducing a gene encoding the STQK mutant into Escherichia coli in a natural medium (Proc. Natl. Acad. Sci. 105, 17323-17327 (2008)).
  • the propionyl CoA transferase (PCT) or butyl CoA transferase (BCT) has the activity of adding CoA to a hydroxyalkanoic acid such as lactic acid to synthesize the CoA form of the hydroxyalkanoic acid.
  • the transformed microorganism according to this embodiment is preferably one into which a foreign gene encoding such a CoA transferase has been introduced.
  • the number of foreign genes to be introduced may be one or more.
  • the PCT and BCT are not particularly limited as long as they are enzymes having the above-mentioned activities.
  • propionyl-CoA transferase PCT Es derived from Epulopiscium sp. having the amino acid sequence shown in SEQ ID NO: 6, or a homolog thereof, can be preferably used.
  • the homologue is a gene that has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO:6 and encodes a protein having propionyl-CoA transferase activity or butyl-CoA transferase activity.
  • the sequence identity is preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and most preferably 99.5% or more.
  • the homologue may be a protein having propionyl-CoA transferase activity or butyl-CoA transferase activity, and may have an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO:6.
  • substitution, deletion, insertion, or addition of one or more amino acids is preferably a conservative mutation that maintains the normal function of the protein, and conservative substitution is more preferable.
  • conservative substitutions include substitutions between aromatic amino acids, between hydrophobic amino acids, between polar amino acids, between basic amino acids, and between amino acids having a hydroxyl group.
  • the substitution, deletion, insertion, or addition of amino acids may be an artificial mutation, or may be a natural mutation due to individual differences in the organism from which the gene is derived, differences in species, etc.
  • CoA transferase is not an enzyme that catalyzes only the CoA transfer reaction to lactate, but is also known to have the activity of transferring CoA to hydroxyalkanoic acids other than lactate. For example, it has been reported that CoA transferase can transfer CoA to hydroxyalkanoic acids such as 2-hydroxybutyric acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, and 3-hydroxy-2-methylbutyric acid (Microbial. Cell Factories, 21, 84 (2020), JP 2019-119806 A).
  • the transformed microorganism according to this embodiment can produce a copolymer polyester of lactic acid and a hydroxyalkanoic acid other than lactic acid.
  • the enzymes that supply the 2-hydroxy acid are not particularly limited, but examples include lactate dehydrogenase (LDH), which reduces pyruvic acid and supplies lactate; lactate dehydratase, which adds water to methylglyoxal and supplies lactate; glyoxylate reductase, which reduces glyoxylic acid and supplies glycolic acid; and phosphoglycolate phosphatase, which dephosphorylates 2-phosphoglycolic acid to obtain glycolic acid.
  • LDH lactate dehydrogenase
  • lactate dehydratase which adds water to methylglyoxal and supplies lactate
  • glyoxylate reductase which reduces glyoxylic acid and supplies glycolic acid
  • phosphoglycolate phosphatase which dephosphorylates 2-phosphoglycolic acid to obtain glycolic acid.
  • a pathway is established that can convert pyruvate to D-lactic acid, making it possible to efficiently produce lactic acid or copolymer polyesters containing lactic acid using sugars such as glucose instead of lactic acid as a carbon source.
  • the LDH is not particularly limited as long as it is an enzyme that has the activity of converting pyruvic acid to D-lactic acid, but examples include lactate dehydrogenase derived from Escherichia coli, lactate dehydrogenase derived from the genus Lactobacillus, lactate dehydrogenase derived from the genus Leuconostoc, and mutants thereof.
  • the introduced gene When introducing a foreign gene into a Capriavidus microorganism, the introduced gene may be present on a chromosome possessed by the host microorganism, or on DNA such as a plasmid or megaplasmid. From the viewpoint of maintaining the introduced gene, it is preferable for the introduced gene to be present on a chromosome or megaplasmid possessed by the microorganism, and it is even more preferable for the introduced gene to be present on a chromosome possessed by the microorganism.
  • Methods for site-specifically replacing or inserting any DNA into DNA possessed by a microorganism, or methods for deleting any site in DNA possessed by a microorganism are well known to those skilled in the art and can be used when producing the transformed microorganism of the present disclosure.
  • representative methods include a method that utilizes the mechanism of transposon and homologous recombination (Ohman et al., J. Bacteriol., vol. 162: p. 1068 (1985)), and a method based on the principle of site-specific integration caused by the mechanism of homologous recombination and loss by second-stage homologous recombination (Noti et al., Methods Enzymol., vol.
  • Methods for introducing vectors into cells are not particularly limited, and examples include the calcium chloride method, electroporation method, polyethylene glycol method, and spheroplast method.
  • the base sequence of the gene to be introduced is not particularly limited, and may be appropriately optimized for the host microorganism of the genus Capriavidus.
  • the promoter for expressing the introduced gene is not particularly limited, but examples that can be used include the trp promoter, lac promoter, lacUV5 promoter, trc promoter, tic promoter, tac promoter, lacN17 promoter, lacN19 promoter, and modified versions of these promoters derived from Escherichia coli. It is also possible to use a promoter sequence that is naturally present near the position where the target gene is to be inserted on the genomic DNA without using an exogenous promoter. Furthermore, it is also possible to use a combination of the naturally present promoter sequence and an exogenous promoter.
  • lactic acid can be produced or the copolymer polyester can be accumulated in the microbial cells.
  • the transformed microorganism can be cultured according to a conventional microbial culture method, and the culture may be performed in a medium containing an appropriate carbon source.
  • the medium composition, the method of adding the carbon source, the culture scale, the aeration and agitation conditions, the culture temperature, the culture time, and the like are not particularly limited.
  • the carbon source is preferably added to the medium continuously or intermittently.
  • Any carbon source can be used as a carbon source during cultivation, as long as the transformed microorganism can assimilate it.
  • Examples include, but are not limited to, sugars such as glucose, fructose, sucrose, and xylose; oils and fats such as palm oil and palm kernel oil (including palm olein, palm double olein, and palm kernel oil olein, which are low-melting point fractions obtained by fractionating these), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and fractionated oils thereof, or refined by-products thereof; other hydroxyalkanoic acids such as D-lactic acid; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, and derivatives thereof, or glycerol.
  • the copolymerized PHA mixture-producing microorganism can utilize gases and alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and
  • a nitrogen source which is a nutrient source other than the carbon source
  • inorganic salts and other organic nutrient sources.
  • the nitrogen source include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like.
  • inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride, and the like.
  • examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C, and the like.
  • the transformed microorganism In producing lactic acid, the transformed microorganism is cultured for an appropriate period of time to produce lactic acid, and then the lactic acid is recovered from the culture supernatant by a known method.
  • the transformed microorganism in the production of the copolymer polyester, is cultured for an appropriate time to accumulate the copolymer polyester in the microbial cells, and then the copolymer polyester is recovered using a known method.
  • the recovery method is not particularly limited, but industrially, recovery by separation and purification in an aqueous system with low environmental impact is preferable.
  • a cell lysate in which cell components other than the copolymer polyester are dissolved in water can be obtained by applying mechanical shearing force or disrupting the cells using a surfactant, alkali, enzyme, or the like.
  • the copolymer polyester can be recovered by separating the copolymer polyester from the aqueous phase by filtration or centrifugation of the cell lysate and then drying.
  • [Item 1] A transformed microorganism belonging to the genus Capriavidus having the ability to produce lactic acid, A transformed microorganism in which at least one gene selected from the group consisting of the following genes (A) and (B) has been disrupted: Gene (A): a gene encoding a 2-hydroxyacid dehydrogenase having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, or a gene encoding a protein having an amino acid sequence with 90% or more sequence identity to an amino acid sequence represented by any one of SEQ ID NOs: 1 to 4 and having 2-hydroxyacid dehydrogenase activity; Gene (B): a gene encoding a regulator protein that controls the expression of at least one of the genes (A) [Item 2] 2.
  • the transformed microorganism according to item 1 wherein the regulator protein has an amino acid sequence represented by SEQ ID NO:5 or has an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO:5.
  • the transformed microorganism according to item 1 or 2 further comprising a gene encoding a copolymer polyester synthase and having the ability to produce a copolymer polyester of lactic acid and another hydroxyalkanoic acid.
  • a method for producing lactic acid comprising a step of culturing the transformed microorganism according to any one of items 1 to 4.
  • a method for producing a copolymer polyester of lactic acid and another hydroxyalkanoic acid comprising a step of culturing the transformed microorganism according to item 3 or 4.
  • the present invention will be explained in more detail below with reference to examples. However, the present invention is not limited to these examples.
  • the overall genetic manipulation can be carried out, for example, as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, etc. used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. The enzymes are not particularly limited as long as they can be used in genetic manipulation.
  • the Cupriavidus necator H16 strain was genetically modified to replace the PHA polymerase gene phaC 1Re on the genome with another PHA polymerase gene, destroy the PHA decomposition enzyme gene phaZ 1,2,6 , and to enhance glucose assimilation ability, the 793rd base G of the N-acetylglucosamine uptake gene nagE was replaced with C, and the gene encoding the transcriptional regulator nagR was destroyed to prepare the KNK005 ⁇ phaZ 1,2,6 /nagE G793C.dR strain (see International Publication No. 2017/104722).
  • the PHA synthase gene phaC 1Re on the genome of the KNK005 ⁇ phaZ 1,2,6 /nagE G793C.dR strain was replaced with a gene encoding an STQK mutant (a PHA synthase in which the 325th serine of the polymerase PhaC1 Ps derived from Pseudomonas sp. 61-3 was converted to threonine and the 481st glutamine was converted to lysine), to prepare H16 phaC 1Re ::STQK ⁇ phaZ 1,2,6 /nagE G793C.dR. This strain is used as the host (1).
  • PCR was performed using the genomic DNA of C. necator H16 strain as a template and oligo DNAs shown in SEQ ID NO: 7 and SEQ ID NO: 8 as primers.
  • Prime STAR GXL DNA polymerase (manufactured by Takara Bio) was used as the DNA polymerase.
  • PCR was performed using DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 10 as primers.
  • Overlap PCR was performed using the two DNA fragments obtained by the above PCR as templates and DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 10 as primers.
  • the obtained DNA fragment is a fragment in which about 500 base pairs upstream and about 500 base pairs downstream of the ORF of D-lactate dehydrogenase (Locus tag: H16_A3091) are linked.
  • This DNA fragment was treated with the restriction enzyme SmiI and ligated with the vector pNS2X-sacB (described in JP 2007-259708 A) which had also been treated with SmiI, using DNA ligase.
  • the obtained gene disruption plasmid (1) containing the base sequence shown in SEQ ID NO:11 was named pNS2X-sacB- ⁇ dld.
  • This gene disruption plasmid (1) is a plasmid used to disrupt the gene dld:A3091 encoding D-lactate dehydrogenase.
  • plasmid for gene introduction In order to introduce the gene group necessary for LAHB production into the genome of C. necator, a plasmid for gene introduction was prepared.
  • pNS2X-sacB-phaJ4b::REP-LDH Lm was prepared, which can introduce a gene sequence for expressing lactate dehydrogenase LDH Lm derived from Leuconostoc mesenteroides with the REP promoter by replacing the ORF of phaJ4b (Locus tag: H16_B0397) on the genome of the host (1).
  • This plasmid is a plasmid in which a DNA fragment represented by SEQ ID NO: 42 is inserted into the pNS2X-sacB vector by ligation.
  • This plasmid it is possible to impart the ability to produce D-lactic acid from glucose to C. necator.
  • This plasmid is designated as the plasmid for gene introduction (1).
  • pNS2X-sacB-phaJ4a::lacN17-PCT Es was prepared, which can introduce a gene sequence for expressing propionyl CoA transferase PCT Es derived from Epulopiscium sp. with the lacN17 promoter by replacing the ORF of phaJ4a (Locus tag: H16_A1070) on the genome of the host (1).
  • This plasmid is a plasmid in which a DNA fragment represented by SEQ ID NO: 43 is inserted into the pNS2X-sacB vector by ligation. By using this plasmid, it is possible to add CoA to lactic acid produced from glucose to supply a substrate for the copolymer polyester synthase.
  • This plasmid is designated as the gene introduction plasmid (2).
  • pNS2X-sacB-phaZ1::lacUV5-XylAB which can introduce gene sequences that express xylose isomerase (XylA) and xylulokinase (XylB) derived from E. coli using the lacUV5 promoter by replacing the ORF of phaZ1 on the genome of the host (1).
  • This plasmid is a plasmid in which the DNA fragment represented by SEQ ID NO: 44 is inserted into the pNS2X-sacB vector by ligation. By using this plasmid, it is possible to confer the ability to assimilate xylose to C. necator, which does not originally have a metabolic pathway for xylose.
  • This plasmid is designated as the gene introduction plasmid (3).
  • the gene disruption plasmid or the gene introduction plasmid was introduced into Escherichia coli S17-1 strain (ATCC47055) by electroporation, and the resulting strain was mixed and cultured with the target C. necator recombinant strain on Nutrient Agar medium (Difco) to perform conjugative transfer.
  • the strains in which the plasmid was inserted on the genome were selected on Simmons agar medium (sodium citrate 2g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, diammonium hydrogen phosphate 1g/L, agar 15g/L, pH 6.8) containing 250 mg/L kanamycin sulfate, and isolated.
  • the strains isolated in Nutrient Agar medium containing 250 mg/L kanamycin sulfate were further purified, and then inoculated into Nutrient Agar medium containing 15% sucrose to obtain strains from which the plasmid had been removed.
  • strains Two types of strains were generated at the stage of plasmid removal by homologous recombination: a strain that returns to the original genome sequence and a strain in which the desired gene modification had been performed, and the latter was isolated and obtained by colony PCR. The obtained genetically modified strain was purified again in Nutrient Agar medium containing sucrose to obtain a homologous recombinant strain.
  • the host (1) was genetically modified by the above-mentioned method to produce H16 phaC 1Re ::STQK ⁇ phaZ 1,2,6 /nagE G793C.dR phaJ4a::lacN17-PCT Es phaJ4b::REP-LDH Lm .
  • This strain was used as the base strain (Comparative Example 1) to evaluate the effect of gene disruption.
  • This base strain is capable of producing LAHB using glucose as a carbon source, and does not require the addition of lactic acid as a carbon source.
  • the genes were disrupted by the above-mentioned method using the gene disruption plasmids shown in Table 1 to obtain the gene-disrupted strains of Examples 1 to 10 and Comparative Examples 2 and 3. These gene-disrupted strains are also capable of producing LAHB using glucose as a carbon source, and do not require the addition of lactic acid as a carbon source.
  • the basic strain and each gene-disrupted strain were cultured under the conditions described below, and the production of lactic acid and LAHB were evaluated.
  • the bacteria were inoculated into a Sakaguchi flask containing a synthetic medium (composition: 1.1% (w/v) disodium hydrogen phosphate dodecahydrate, 0.19% (w/v) potassium dihydrogen phosphate, 0.129% (w/v) ammonium sulfate, 0.15% (w/v) magnesium sulfate heptahydrate, 50 ⁇ g/L kanamycin, 0.75% (v/v) trace metal salt solution (1.6% (w/v) iron (II) hydrochloride hexahydrate, 1% (w/v) calcium chloride dihydrate, 0.02% (w/v) cobalt chloride hexahydrate, 0.016% (w/v) copper sulfate pentahydrate, 0.012% (w/v) nickel chloride hexahydrate in 0.1 N hydrochloric acid)) containing 20 g/L glucose as a carbon source, and cultured at 30 ° C. and 130 rpm for 72
  • the LAHB biosynthesized by each strain was extracted with chloroform from the dried cells washed with ethanol and hexane, and the chloroform was removed by volatilization to recover the LAHB.
  • the composition of the obtained LAHB was analyzed by 1 H NMR, and the LA fraction in the LAHB was calculated. The results are shown in Table 2.
  • the lactic acid concentration in the culture supernatant was calculated based on a calibration curve prepared using a D-lactic acid standard solution of known concentration.
  • the results of the LA fraction in LAHB and the lactate concentration in the supernatant in flask culture are shown in Table 2.
  • Example 1 the LA fraction in the produced LAHB was improved compared to Comparative Examples 1 to 3.
  • the LA fraction in Examples 1 to 9 was several times higher than in Comparative Examples 1 to 3, and in particular, the glcD1 disruption strain (Example 2), the glcF disruption strain (Example 4), the double disruption strain (Examples 5 to 7), and the triple disruption strain (Examples 8 and 9) were able to achieve the highest LA fraction among past cases of LAHB production in Capriavidus microorganisms using glucose as a carbon source.
  • glcD1, glcE, and glcF are known as genes encoding glycolic acid reductase, but this is the first time that it has been revealed that these genes are involved in lactic acid metabolism in Capriavidus microorganisms.
  • the effects achieved by disrupting glcD1, glcE, or glcF were greater than those achieved by disrupting dld, ldhA1, and ldhA2, which are known to be genes encoding enzymes that reduce D-lactate.
  • Example 11 and Comparative Example 4 Using the plasmid for gene introduction (3), the basic strain (Comparative Example 1) was genetically modified by the above-mentioned method to obtain a xylose-utilizing strain (Comparative Example 4).
  • This xylose-utilizing strain is capable of producing LAHB using xylose as a carbon source.
  • dld, glcD1, and glcF were disrupted in the same manner as in Example 9 using the gene disruption plasmid shown in Table 1, and a gene-disrupted strain having the ability to utilize xylose (Example 11) was obtained by the above-mentioned method.
  • the above-mentioned flask culture was carried out using the strains of Comparative Example 4 and Example 11. However, xylose was used instead of glucose as the carbon source in the flask culture. The culture time was 168 hours.
  • the LA fraction in the LAHB biosynthesized by each strain was calculated in the same manner as above. As a result, it was 3.4 mol% in Comparative Example 4, while it was 7.2 mol% in Example 11. This confirmed that the LA fraction was also improved by gene disruption in the xylose-utilizing strain.
  • the aeration rate was set to 100 mL/min (1 vvm), the stirring speed was set to 500 rpm, the culture temperature was set to 30 ° C, and the pH was maintained at 6.5 for 72 hours.
  • the amount (g/L) of biosynthesized LAHB was quantified by gas chromatography. Furthermore, the LA fraction in the LAHB and the lactic acid concentration in the supernatant were quantified in the same manner as in the flask culture. The results are shown in Table 3.
  • pH stud culture produced results showing the same tendency as flask culture in Table 2.
  • the amount of lactic acid accumulated in the culture supernatant was greatly improved by culturing while maintaining a constant pH by adding alkali.
  • a maximum of 4.0 g/L of lactic acid was produced in the culture supernatant. This is the highest value in past cases of lactic acid production in synthetic medium using Capriavidus microorganisms.
  • This preculture solution was added to a 500 ml Sakaguchi flask containing 100 ml of meat medium and cultured with shaking at 30°C for 6 hours.
  • the above culture solution was inoculated into a 5 L jar fermenter (Bioneer Neo type, manufactured by Marubishi Bioengineering) containing 1.8 L of PHA production medium.
  • the operating conditions were a culture temperature of 30°C, an agitation speed of 500 rpm, and an aeration rate of 1.8 L/min.
  • the culture was continued for 48 hours while controlling the pH between 6.7 and 6.8.
  • a 7% aqueous solution of ammonium hydroxide was used for pH control.
  • the composition of the PHA production medium was 0.578% (w/v) disodium hydrogen phosphate dodecahydrate, 0.101% (w/v) potassium dihydrogen phosphate, 0.437% (w/v) ammonium sulfate, 0.15% (w/v) magnesium sulfate heptahydrate, 0.75% (v/v) trace metal salt solution (1.6% (w/v) iron chloride (II) hexahydrate, 1% (w/v) calcium chloride dihydrate, 0.02% (w/v) cobalt chloride hexahydrate, 0.016% (w/v) copper sulfate pentahydrate, and 0.012% (w/v) nickel chloride hexahydrate dissolved in 0.1N hydrochloric acid).
  • the carbon source was glucose, with an initial concentration of 20 g/L, and after glucose was consumed to 10 g/L, it was maintained at 10 g/L.
  • LAHB When the basic strain (Comparative Example 1) was cultured at high density under the above conditions, LAHB was obtained at a yield of 59 g/L dry cell weight and 61 wt% LAHB content, with an LA fraction of 5.2 mol% and a weight-average molecular weight of 1,040,000.
  • the gene-disrupted strain of Example 9 was similarly cultured at high density, LAHB with an LA fraction of 10.2 mol% and a weight-average molecular weight of 1,100,000 was obtained at a yield of 45 g/L dry cell weight and 60 wt% LAHB content. From the above, it can be seen that the LA fraction is improved and high molecular weight LAHB can be obtained in the high-density culture of the gene-disrupted strain.
  • Example 12 In order to incorporate lactic acid released into the supernatant during culture into the polymer, a strain was prepared in which the expression level of PHA polymerase was enhanced using a plasmid.
  • a DNA fragment encoding the STQK mutant was amplified by PCR, and the DNA fragment was ligated to a pCUP2 vector (see International Publication No. 2007/049716) treated with MunI and SpeI using DNA ligase to obtain a plasmid expressing the STQK mutant under the strong lacUV5 promoter (sequence number 45).
  • This plasmid was introduced into the basic strain (Comparative Example 1) and the gene-disrupted strain of Example 9 by electroporation to obtain the strain of Comparative Example 5 and the gene-disrupted strain of Example 12. High-density cultivation was carried out as described above, except that a sugar mixture with a glucose:xylose concentration ratio of 4:1 was used as the carbon source.
  • the strain of Comparative Example 5 produced a yield of 75 g/L dry cell weight, 64 wt% LAHB content, and 3.2 mol% LAHB, whereas the gene-disrupted strain of Example 12 produced a yield of 90 g/L dry cell weight, 57 wt% LAHB content, and 18.2 mol% LAHB.

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Abstract

Dans la présente invention, au moins l'un des gènes (A) et (B) mentionnés ci-dessous est interrompu dans un micro-organisme transformé appartenant au genre Cupriavidus.　Gène (A) : un gène qui code pour la 2-hydroxy-acide déshydrogénase comprenant une séquence d'acides aminés représentée par l'une quelconque des SEQ ID NO : 1 à 4, ou un gène qui code pour une protéine qui comprend une séquence d'acides aminés ayant une identité de séquence de 90 % ou plus avec une séquence d'acides aminés représentée par l'une quelconque des SEQ ID NO : 1 à 4 et qui présente une activité 2-hydroxy-acide déshydrogénase. Gène (B) : gène codant pour une protéine régulatrice qui régule l'expression d'au moins un des gènes inclus dans le gène (A).
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WO2016168681A1 (fr) * 2015-04-15 2016-10-20 William Marsh Rice University Plate-forme itérative pour la synthèse de produits fonctionnalisés en alpha

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WO2016168681A1 (fr) * 2015-04-15 2016-10-20 William Marsh Rice University Plate-forme itérative pour la synthèse de produits fonctionnalisés en alpha

Non-Patent Citations (9)

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Title
DATABASE GenPept 7 March 2015 (2015-03-07), ANONYMOUS: "D-Lactate dehydrogenase (Cytochrome) [Cupriavidus necator H16] - Protein - NCBI", XP093208206, Database accession no. CAJ94166.1 *
DATABASE Protein 7 March 2015 (2015-03-07), ANONYMOUS: "2-Hydroxy-acid oxidase, FAD-binding subunit [Cupriavidus necator H16", XP093208209, Database accession no. CAJ94171.1 *
DATABASE Protein 7 March 2015 (2015-03-07), ANONYMOUS: "2-Hydroxy-acid oxidase, Fe-S subunit [Cupriavidus necator H16] ", XP093208216, Database accession no. CAJ94172.1 *
DATABASE Protein 7 March 2015 (2015-03-07), ANONYMOUS: "glycolate oxidase subunit GlcD [Cupriavidus necator H16] ", XP093208208, Database accession no. CAJ94169.1 *
DATABASE Protein 7 March 2015 (2015-03-07), ANONYMOUS: "transcriptional regulator, LysR-family [Cupriavidus necator H16] ", XP093208219, Database accession no. CAJ94167.1 *
GUO PENGYE, LUO YUANCHAN, WU JU, WU HUI: "Recent advances in the microbial synthesis of lactate-based copolymer", BIORESOURCES AND BIOPROCESSING, SPRINGER SINGAPORE, vol. 8, no. 1, 1 December 2021 (2021-12-01), XP093208201, ISSN: 2197-4365, DOI: 10.1186/s40643-021-00458-3 *
HERNÁNDEZ-HERREROS NATALIA; RIVERO-BUCETA VIRGINIA; PARDO ISABEL; PRIETO M. AUXILIADORA: "Production of poly(3-hydroxybutyrate)/poly(lactic acid) from industrial wastewater by wild-type Cupriavidus necator H16", WATER RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 249, 18 November 2023 (2023-11-18), AMSTERDAM, NL, XP087444588, ISSN: 0043-1354, DOI: 10.1016/j.watres.2023.120892 *
ISHIHARA SHIZURU, ORITA IZUMI, MATSUMOTO KEN’ICHIRO, FUKUI TOSHIAKI: "(R/S)-lactate/2-hydroxybutyrate dehydrogenases in and biosynthesis of block copolyesters by Ralstonia eutropha", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 107, no. 24, 1 December 2023 (2023-12-01), Berlin/Heidelberg, pages 7557 - 7569, XP093208223, ISSN: 0175-7598, DOI: 10.1007/s00253-023-12797-6 *
SALINAS ALEJANDRO, MCGREGOR CALLUM, IRORERE VICTOR, ARENAS-LÓPEZ CHRISTIAN, BOMMAREDDY RAJESH REDDY, WINZER KLAUS, MINTON NIGEL P.: "Metabolic engineering of Cupriavidus necator H16 for heterotrophic and autotrophic production of 3-hydroxypropionic acid", METABOLIC ENGINEERING, ACADEMIC PRESS, AMSTERDAM, NL, vol. 74, 1 November 2022 (2022-11-01), AMSTERDAM, NL, pages 178 - 190, XP093208220, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2022.10.014 *

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