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WO2024166830A1 - Micro-organisme transformé et procédé de production d'acide polyhydroxyalcanoïque - Google Patents

Micro-organisme transformé et procédé de production d'acide polyhydroxyalcanoïque Download PDF

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WO2024166830A1
WO2024166830A1 PCT/JP2024/003569 JP2024003569W WO2024166830A1 WO 2024166830 A1 WO2024166830 A1 WO 2024166830A1 JP 2024003569 W JP2024003569 W JP 2024003569W WO 2024166830 A1 WO2024166830 A1 WO 2024166830A1
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gene
microorganism
transformed
strain
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尚志 有川
俊輔 佐藤
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Kaneka Corp
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Kaneka Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • 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

Definitions

  • the present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.
  • PHA polyhydroxyalkanoic acid
  • microorganisms see Non-Patent Document 1.
  • PHA polyhydroxyalkanoic acid
  • PHA is a thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species, and is biodegradable.
  • non-petroleum-based plastic alternatives are attracting attention due to increased environmental awareness, and it is expected that PHA produced and accumulated within the cells of microorganisms will have a small adverse effect on the ecosystem, as it is incorporated into the carbon cycle process in nature.
  • PHA production using microorganisms for example, it is known that PHA is produced by feeding sugar, vegetable oils and fatty acids as carbon sources to Capriavidus bacteria and allowing them to accumulate PHA within the cells (see Non-Patent Documents 2 and 3).
  • the present invention aims to provide a transformed microorganism with improved productivity of polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid by culturing the microorganism.
  • thermophilic microorganism a gene encoding ⁇ -ketothiolase (PhaA) derived from a thermophilic microorganism and/or a gene encoding acetoacetyl-CoA reductase (PhaB) derived from a thermophilic microorganism into a mesophilic microorganism capable of producing polyhydroxyalkanoic acid, and thus completed the present invention.
  • PhaA ⁇ -ketothiolase
  • PhaB acetoacetyl-CoA reductase
  • the present invention relates to a method for producing a polyhydroxyalkanoic acid synthase gene
  • the present invention relates to a transformed mesophilic microorganism into which a phaA gene derived from a thermophilic microorganism and/or a phaB gene derived from a thermophilic microorganism has been introduced.
  • the present invention also relates to a method for producing polyhydroxyalkanoic acid, which comprises the step of culturing the transformed mesophilic microorganism in the presence of a carbon source.
  • the present invention it is possible to provide a transformed microorganism having improved productivity of polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid by culturing the microorganism. According to the present invention, the efficiency of polyhydroxyalkanoic acid production by a microorganism can be improved, and the production cost of polyhydroxyalkanoic acid can be reduced.
  • the present disclosure relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid (hereinafter also referred to as PHA), and to a method for producing PHA by culturing the transformed microorganism.
  • PHA polyhydroxyalkanoic acid
  • the transformed microorganism according to the present disclosure may be a transformed microorganism in which the host is a mesophilic microorganism, has a PHA synthase gene, and has a gene encoding ⁇ -ketothiolase (PhaA) derived from a thermophilic microorganism introduced therein.
  • the transformed microorganism may be a transformed microorganism in which the host is a mesophilic microorganism, has a PHA synthase gene, and has a gene encoding acetoacetyl-CoA reductase (PhaB) derived from a thermophilic microorganism introduced therein.
  • the transformed microorganism may be a transformed microorganism in which the host is a mesophilic microorganism, has a PHA synthase gene, and has a gene encoding PhaA derived from a thermophilic microorganism and a gene encoding PhaB derived from a thermophilic microorganism introduced therein.
  • mesophilic microorganisms refer to microorganisms whose optimum growth temperature is within the range of 25-40°C
  • thermophilic microorganisms refer to microorganisms that can grow at temperatures of 50°C or higher.
  • the host of the transformed microorganism according to the present disclosure may be a wild-type strain that inherently has a PHA synthase gene, or a mutant strain obtained by artificially mutating such a wild-type strain, or a transformed strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques.
  • the method for introducing a foreign gene is not particularly limited, and may be selected from a method for directly inserting or replacing a gene on a chromosome of the host, a method for directly inserting or replacing a gene on a megaplasmid possessed by the host, or a method for arranging a gene on a vector such as a plasmid, phage, or phagemid and introducing the gene, or two or more of these methods may be used in combination.
  • a method for directly inserting or replacing a gene on a chromosome of the host or a megaplasmid possessed by the host is preferred, and a method for directly inserting or replacing a gene on a chromosome of the host is more preferred.
  • the host of the transformed microorganism according to the present disclosure is not particularly limited as long as it is a mesophilic microorganism.
  • the mesophilic microorganism may be a microorganism that originally has a PHA synthase gene, a gene encoding ⁇ -ketothiolase (PhaA), and a gene encoding acetoacetyl-CoA reductase (PhaB).
  • hosts for the transformed microorganisms disclosed herein include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, etc., and having an optimal growth temperature within the range of 25 to 40°C.
  • the bacteria are preferably those belonging to the genera Ralstonia, Cupriavidus, and Wautersia, and have an optimum growth temperature within the range of 25 to 40°C, more preferably those belonging to the Cupriavidus, and have an optimum growth temperature within the range of 25 to 40°C, and particularly preferably Cupriavidus necator.
  • PHAs The type of PHA produced by the transformed microorganism according to the present disclosure is not particularly limited as long as it is a PHA that can be produced by a microorganism, but preferred are a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (e.g., 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid.
  • 2-hydroxyalkanoic acids 4-hydroxyalkanoic acids, 5-hydroxyalkano
  • PHAs are homopolymers of 3-hydroxyalkanoic acid having 4 carbon atoms, or copolymers containing 3-hydroxyalkanoic acid having 4 carbon atoms.
  • PHAs include homopolymers of 3-hydroxybutyric acid (abbreviation: 3HB), copolymers of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), copolymers of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), copolymers of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), copolymers of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), PHAs containing lactic acid (abbreviation: LA) as a component, such as copolymers of 3HB and LA, such as copolymers of 3HB and LA, but are not limited thereto.
  • P3HB3HH is preferred from the viewpoint of a wide range of applications as a polymer.
  • the type of PHA produced can be appropriately selected depending on the purpose, such as the type of PHA synthesis enzyme gene possessed by the microorganism used or introduced separately, the type of metabolic gene involved in the synthesis, and the culture conditions.
  • the PHA synthase (PhaC) gene possessed by the transformed microorganism according to the present disclosure may be one that is inherent to the host or may be an exogenous one.
  • the PHA synthase gene is not particularly limited, and examples thereof include PHA synthase genes derived from Aeromonas kiyaviei, Aeromonas hydrophila, Pseudomonas SP 61-3, or Capriavidus necator, chimeric PHA synthase genes combining two or more of the above PHA synthase genes, and genes encoding proteins consisting of amino acid sequences showing 90% or more sequence identity to the amino acid sequences of the above PHA synthases.
  • the sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.
  • the number of PHA synthase genes possessed by the transformed microorganism according to the present disclosure may be one or more.
  • the genes may be the same or different.
  • the phaA gene and the phaB gene are genes encoding ⁇ -ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB), respectively. These genes are possessed by many microorganisms that can naturally accumulate PHA, and are involved in the biosynthesis of 3HB-CoA, which is the most common biosynthetic substrate for PHA. Specifically, PhaA is involved in catalysis of a reaction in which two molecules of acetyl-CoA are condensed to produce acetoacetyl-CoA, and PhaB is involved in catalysis of a reaction in which acetoacetyl-CoA is reduced to produce 3HB-CoA.
  • PhaA ⁇ -ketothiolase
  • PhaB acetoacetyl-CoA reductase
  • the transformed microorganism according to the present disclosure has a mesophilic host, and the phaA gene derived from a thermophilic microorganism and/or the phaB gene derived from a thermophilic microorganism has been introduced into it. By introducing these genes, it is possible to increase the productivity of PHA by the transformed microorganism.
  • the transformed microorganism can produce PHA with good productivity even under high stress, for example, at a relatively high culture temperature.
  • phaA gene derived from a high-temperature microorganism may be introduced, only the phaB gene derived from a high-temperature microorganism may be introduced, or both genes may be introduced.
  • the transformed microorganism according to the present disclosure is one in which only the phaA gene derived from a high-temperature microorganism has been introduced, it is preferable that the transformed microorganism has the phaB gene that the host originally has. In this case, the transformed microorganism may or may not further have the phaA gene that the host originally has.
  • the transformed microorganism according to the present disclosure is one in which only the phaB gene derived from a high-temperature microorganism has been introduced, it is preferable that the host of the transformed microorganism has the phaA gene that it originally possesses. In this case, the transformed microorganism may or may not further have the phaB gene that the host originally possesses.
  • the transformed microorganism according to the present disclosure has both the phaA gene derived from a high-temperature microorganism and the phaB gene derived from a high-temperature microorganism, the transformed microorganism does not need to have the phaA gene originally possessed by the host and the phaB gene originally possessed by the host. However, since this further improves PHA productivity, it is preferable that the transformed microorganism also has the phaA gene originally possessed by the host and the phaB gene originally possessed by the host.
  • the high-temperature microorganism is not particularly limited as long as it is a microorganism that can grow at 50°C or higher and has the phaA gene and/or the phaB gene.
  • microorganisms include the genera Cupriavidus, Caldimonas, Schlegelella, Thermus, Chelatococcus, Bacillus, Aneurinibacillus, Pseudomonas, Synechococcus, and Spirulina.
  • the genera Cupriavidus, Caldimonas, and Schlegelella are preferred.
  • genes encoding ⁇ -ketothiolase (PhaA) derived from thermophilic microorganisms include, but are not limited to, a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO:1 derived from Cupriavidus sp.
  • strain S-6 a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO:2 derived from Caldimonas manganoxidans, a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO:3 derived from Schlegelella thermodepolymerans, or a gene having an amino acid sequence that shows 90% or more sequence identity to these amino acid sequences and a base sequence that encodes a protein that exhibits ⁇ -ketothiolase activity.
  • sequence identity to the amino acid sequence described in SEQ ID NO:1 is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.
  • sequence identity to the amino acid sequence described in SEQ ID NO:2 or 3 is also preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.
  • thermophilic microorganisms examples include, but are not limited to, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 4 derived from Cupriavidus sp.
  • strain S-6 a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 5 derived from Caldimonas manganoxidans, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 6 derived from Schlegelella thermodepolymerans, or a gene having an amino acid sequence that shows 90% or more sequence identity to these amino acid sequences and a base sequence that encodes a protein that exhibits acetoacetyl-CoA reductase activity.
  • sequence identity to the amino acid sequence described in SEQ ID NO:4 is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.
  • sequence identity to the amino acid sequence described in SEQ ID NO:5 or 6 is also preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.
  • the method for introducing the target gene into the host is not particularly limited, but may include directly inserting or replacing the target gene on the host chromosome, directly inserting or replacing the target gene on a megaplasmid carried by the host, or locating and introducing the target gene on a vector such as a plasmid, phage, or phagemid, and two or more of these methods may be used in combination.
  • a method in which the target gene is directly inserted or replaced onto the host chromosome or onto a megaplasmid carried by the host is preferred, and a method in which the target gene is directly inserted or replaced onto the host chromosome is even more preferred.
  • a “gene expression regulatory sequence” is a DNA sequence that includes a base sequence that controls the transcription amount of the gene (e.g., a promoter sequence) and/or a base sequence that controls the translation amount of messenger RNA transcribed from the gene (e.g., a Shine-Dalgarno sequence).
  • a “gene expression regulatory sequence” any base sequence that exists in nature can be used, or an artificially constructed or modified base sequence can be used.
  • the promoter sequence or Shine-Dalgarno sequence contained in the "gene expression regulatory sequence” includes, but is not limited to, the base sequences shown in any of SEQ ID NOs: 7 to 14, or base sequences containing parts of these base sequences.
  • the substitution, deletion, insertion and/or addition of at least a portion of the genomic DNA can be performed by methods well known to those skilled in the art.
  • Representative methods include a method that utilizes the mechanism of transposon and homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (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., 154:197-217 (1987)).
  • the sacB gene from Bacillus subtilis can be coexisted, and the microbial strain from which the gene has been lost by second-stage homologous recombination can be easily isolated as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195-1204 (1992); Lenz et al., J. Bacteriol., 176:4385-4393 (1994)).
  • genome editing technology using the CRISPR/Cas9 system to modify target DNA Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732
  • gRNA guide RNA
  • gRNA guide RNA
  • the method for introducing the vector into the cells is not particularly limited, but examples include the calcium chloride method, electroporation method, polyethylene glycol method, and spheroplast method.
  • PHA can be accumulated in the cells.
  • the method for culturing the transformed microorganism according to the present disclosure can be a conventional microbial culture method, and the culture can be performed in a medium containing an appropriate carbon source.
  • the medium composition, the method for adding the carbon source, the culture scale, the aeration and agitation conditions, the culture temperature, the culture time, etc. are not particularly limited.
  • the carbon source is preferably added to the medium continuously or intermittently.
  • the transformed microorganisms disclosed herein can produce PHA with good productivity even at relatively high culture temperatures. For example, good productivity can be achieved even when cultured at temperatures of 35°C or higher.
  • carbon sources can be used as a carbon source during cultivation as long as it can be assimilated by the transformed microorganism according to the present disclosure.
  • carbon sources include, but are not limited to, sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil and palm kernel oil (including palm olein, palm double olein, palm kernel oil olein, etc., which are low-melting point fractions obtained by fractionating these oils), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and fractionated oils thereof, or by-products of their refinement; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, and derivatives thereof, and glycerol.
  • sugars such as glucose, fructose, and sucrose
  • oils and fats such as palm oil and palm kernel oil (including palm olein, palm double olein, palm kernel oil olein
  • oils and fats may be partially or entirely degraded oils.
  • Degraded oils refer to oils and fats that have been thermally denatured or that have been altered by reacting with oxygen and/or water under heating.
  • the name of the oils and fats is not limited, and includes those called waste oil, discarded oil, waste cooking oil, waste edible oil, waste vegetable oil, used oil, etc.
  • gases or alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources.
  • a nitrogen source which is a nutrient source other than the carbon source
  • inorganic salts and other organic nutrient sources.
  • nitrogen sources 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.
  • PHA can be recovered from the cells using known methods. There are no particular limitations on the recovery method, but for example, after culturing is completed, the cells are separated from the culture liquid using a centrifuge or separation membrane, etc., and dried, and then PHA is extracted from the dried cells using an organic solvent such as chloroform, and cell components are removed from the organic solvent solution containing PHA by filtration, etc., a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA, the supernatant is removed by filtration or centrifugation, and the PHA can be recovered by drying.
  • cell components other than PHA can be dissolved in water using a surfactant, alkali, enzyme, etc., and then the PHA particles can be separated from the aqueous phase by filtration or centrifugation, dried, and recovered.
  • [Item 1] Possessing the polyhydroxyalkanoate synthase gene, A transformed mesophilic microorganism into which a phaA gene derived from a thermophilic microorganism and/or a phaB gene derived from a thermophilic microorganism has been introduced.
  • [Item 2] Item 1. The transformed mesophilic microorganism according to item 1 or 2, wherein the phaA gene derived from the thermophilic microorganism is a gene encoding an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
  • a method for producing a polyhydroxyalkanoic acid comprising culturing the transformed mesophilic microorganism according to any one of items 1 to 5 in the presence of a carbon source.
  • Item 7 Item 7. The method for producing a polyhydroxyalkanoic acid according to item 6, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids.
  • Item 8 8. The method for producing a polyhydroxyalkanoic acid according to Item 7, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyric acid as a monomer unit.
  • Item 9 Item 9. The method for producing a polyhydroxyalkanoic acid according to Item 7 or 8, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.
  • the present invention will be described in more detail below with reference to examples, although 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, and the like used in the genetic manipulation can be purchased from commercial suppliers and used according to their instructions. There are no particular limitations on the enzymes used, so long as they can be used in genetic manipulation.
  • KNK005 trc-phaJ4b/ ⁇ phaZ1,2,6 strain used in this example is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, the PHA synthase gene on the chromosome has been replaced with a modified version of the PHA synthase gene derived from the genus Aeromonas (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 15, i.e., the N149S/D171G mutant (NSDG) gene), and the expression of the R-specific enoyl-CoA hydratase gene on the chromosome (phaJ4b gene) has been enhanced, and can be prepared in accordance with the method described in PCT Publication WO 2015/115619.
  • This DNA fragment was digested with restriction enzymes EcoRI and MunI, and the resulting DNA fragment was ligated to a plasmid vector pCUP2 described in International Publication WO 2007/049716 that was cleaved with MunI, and the DNA fragment ligated in such an orientation that the restriction enzyme SpeI recognition sequence of pCUP2 was located downstream of the lacN17 promoter was selected to obtain pCUP2-lacN17.
  • a DNA fragment (SEQ ID NO: 17) having the base sequence of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 1 was obtained by PCR using synthetic oligo DNA.
  • This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain a plasmid for expressing phaAsp, pCUP2-lacN17-phaAsp.
  • the phaAsp expression plasmid pCUP2-lacN17-phaAsp was introduced into the KNK005dZ/trc-J4b strain, and the resulting strain was named KNK005dZ/trc-J4b/pCUP2-lacN17-phaAsp (hereinafter, also referred to as the "high-temperature microorganism-derived phaA gene-introduced strain").
  • the plasmid vector was introduced into the cells by electroporation as follows.
  • a Biorad Gene Pulser was used as the gene introduction device, and a Biorad gap 0.2 cm cuvette was used. 400 ⁇ l of competent cells and 20 ⁇ l of expression vector were poured into the cuvette and set in the pulse device, and an electric pulse was applied under the conditions of capacitance 25 ⁇ F, voltage 1.5 kV, and resistance value 800 ⁇ .
  • the bacterial solution in the cuvette was shake-cultured in Nutrient Broth medium (DIFCO) at 30°C for 3 hours, and then cultured on a selection plate (Nutrient Agar medium (DIFCO), kanamycin 100 mg/L) at 30°C for 2 days to obtain the grown high-temperature microorganism-derived phaA gene-introduced strain.
  • DIFCO Nutrient Broth medium
  • DIFCO Nutrient Agar medium
  • kanamycin 100 mg/L kanamycin 100 mg/L
  • This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain a plasmid for expressing phaBsp, pCUP2-lacN17-phaBsp.
  • KNK005dZ/trc-J4b/pCUP2-lacN17-phaBsp strain hereinafter, sometimes referred to as the "high-temperature microorganism-derived phaB gene-introduced strain").
  • This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain a plasmid pCUP2-lacN17-phaABsp for expressing phaAsp and phaBsp.
  • KNK005dZ/trc-J4b/pCUP2-lacN17-phaABsp strain hereinafter, sometimes referred to as the "high-temperature microorganism-derived phaAB gene-introduced strain"
  • This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using a DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare a plasmid vector pNS2X-sacB+phaABUD for disrupting the host phaAB genes.
  • a DNA ligase Ligation High (manufactured by Toyobo Co., Ltd.)
  • a host phaAB gene-disrupted strain was prepared as follows using the plasmid vector pNS2X-sacB+phaABUD for disrupting the host phaAB genes.
  • the host phaAB gene disruption plasmid vector pNS2X-sacB+phaABUD was used to transform E. coli S17-1 strain (ATCC47055), and the resulting transformed microorganism was mixed and cultured with KNK005dZ/trc-J4b strain on Nutrient Agar medium (Difco) to perform conjugation transfer.
  • the resulting culture solution was inoculated onto Son agar medium (sodium citrate 2g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, ammonium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, agar 15g/L, pH 6.8) containing 250mg/L kanamycin, and the strains that had grown on the agar medium were selected to obtain strains in which the plasmid was integrated onto the chromosome of KNK005dZ/trc-J4b strain.
  • Son agar medium sodium citrate 2g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, ammonium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, agar 15g/L, pH 6.8
  • a plasmid for introducing the phaA gene (phaAsp) and the phaB gene (phaBsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6 was constructed as follows.
  • a DNA fragment (SEQ ID NO: 21) was obtained by PCR using synthetic oligo DNA, which has the nucleotide sequences of the upstream of the phaA structural gene and the downstream of the phaB structural gene of the KNK005dZ/trc-J4b strain, and the nucleotide sequences of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 1 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 4.
  • This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A, which had also been digested with SwaI, using a DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to transform the thermophilic microorganism Cupriavidus sp.
  • a plasmid vector pNS2X-sacB+phaABsp for introducing the phaA gene (phaAsp) and the phaB gene (phaBsp) derived from strain S-6 was prepared.
  • strain S-6 was introduced into the KNK005dZ/trc-J4b/dphaAB strain by the same conjugation transfer method as above. Furthermore, by culturing as above and selecting with Nutrient Agar medium containing 15% sucrose, one strain in which phaAsp and phaBsp were introduced at the position on the chromosome where the original host had the phaA and phaB genes was isolated.
  • the resulting strain was named KNK005dZ/trc-J4b/phaAB::phaABsp (hereinafter, also referred to as "high-temperature microorganism-derived phaAB gene chromosomal substitution strain 1").
  • thermophilic microorganism a plasmid for introducing the phaA gene (phaAcm) and the phaB gene (phaBcm) derived from a thermophilic microorganism Caldimonas manganoxidans was prepared as follows.
  • a DNA fragment (SEQ ID NO: 22) was obtained by PCR using synthetic oligo DNA, which has the base sequences of the upstream phaA structural gene and downstream phaB structural gene of the KNK005dZ/trc-J4b strain, a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 2, and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 5.
  • This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using a DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare a plasmid vector pNS2X-sacB+phaABcm for introducing the phaA gene (phaAcm) and phaB gene (phaBcm) derived from the thermophilic microorganism Caldimonas manganoxidans.
  • a DNA ligase Ligation High (manufactured by Toyobo Co., Ltd.)
  • the plasmid vector pNS2X-sacB+phaABcm for introducing the phaA gene (phaAcm) and phaB gene (phaBcm) derived from the thermophilic microorganism Caldimonas manganoxidans was introduced into the KNK005dZ/trc-J4b/dphaAB strain by the same conjugation transfer method as above. Furthermore, by culturing as above and selecting with Nutrient Agar medium containing 15% sucrose, one strain in which phaAcm and phaBcm were introduced at the position on the chromosome where the original host had the phaA and phaB genes was isolated. The obtained strain was named KNK005dZ/trc-J4b/phaAB::phaABcm strain (hereinafter, sometimes referred to as "thermophilic microorganism-derived phaAB gene chromosomal replacement strain 2").
  • thermophilic microorganism a plasmid for introducing the phaA gene (phaAst) and the phaB gene (phaBst) derived from the thermophilic microorganism Schlegelella thermodepolymerans was prepared as follows.
  • a DNA fragment (SEQ ID NO: 23) was obtained by PCR using synthetic oligo DNA, which has the base sequences of the upstream phaA structural gene and downstream phaB structural gene of the KNK005dZ/trc-J4b strain, a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 3, and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 6.
  • This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare a plasmid vector pNS2X-sacB+phaABst for introducing the phaA gene (phaAst) and phaB gene (phaBst) derived from the high-temperature microorganism Schlegelella thermodepolymerans.
  • DNA ligase Ligation High (manufactured by Toyobo Co., Ltd.)
  • the plasmid vector pNS2X-sacB+phaABst for introducing the phaA gene (phaAst) and phaB gene (phaBst) derived from the high-temperature microorganism Schlegelella thermodepolymerans was introduced into the KNK005dZ/trc-J4b/dphaAB strain by the same conjugation transfer method as above. Furthermore, by culturing in the same manner as above and selecting with Nutrient Agar medium containing 15% sucrose, one strain in which phaAst and phaBst were introduced at the position on the chromosome where the original host had the phaA and phaB genes was isolated.
  • the resulting strain was named KNK005dZ/trc-J4b/phaAB::phaABst (hereinafter, also referred to as "high-temperature microorganism-derived phaAB gene chromosomal substitution strain 3").
  • composition of the seed mother medium was 1 w/v % meat extract, 1 w/v % Bacto-Tryptone, 0.2 w/v % yeast extract, 0.9 w/v % Na 2 HPO 4 ⁇ 12H 2 O, and 0.15 w/v % KH 2 PO 4 (pH 6.8).
  • the composition of the preculture medium was 1.1 w/v% Na2HPO4.12H2O , 0.19 w /v % KH2PO4 , 1.29 w /v% ( NH4 ) 2SO4 , 0.1 w /v% MgSO4.7H2O, 2.5 w/v% palm olein oil, and 0.5 v/v% trace metal salt solution (1.6 w/v% FeCl3.6H2O, 1 w/v % CaCl2.2H2O, 0.02 w/v% CoCl2.6H2O , 0.016 w /v% CuSO4.5H2O , and 0.012 w/v% NiCl2.6H2O dissolved in 0.1 N hydrochloric acid ) .
  • the composition of the PHA production medium was 0.385 w/v% Na2HPO4.12H2O , 0.067 w /v % KH2PO4 , 0.291 w /v % ( NH4 ) 2SO4 , 0.1 w/v % MgSO4.7H2O , and 0.5 v/v% trace metal salt solution (1.6 w/v% FeCl3.6H2O, 1 w / v % CaCl2.2H2O, 0.02 w/v % CoCl2.6H2O, 0.016 w / v% CuSO4.5H2O , and 0.012 w/v% NiCl2.6H2O dissolved in 0.1 N hydrochloric acid).
  • the amount of PHA produced was measured as follows. The cells were collected from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain the dried cells, and the weight was measured to calculate the dry cell concentration in the culture medium. Furthermore, 100 ml of chloroform was added to 1 g of the obtained dried cells, and the mixture was stirred at room temperature for a day and night to extract the PHA in the cells. After filtering the cell residue, the mixture was concentrated in an evaporator until the total volume was 30 ml, and then 90 ml of hexane was gradually added and left for 1 hour while slowly stirring. The precipitated PHA was filtered and then vacuum dried at 50° C. for 3 hours. The weight of the dried PHA obtained from 1 g of dried cells was measured, and the amount of PHA produced per culture medium was calculated.
  • PHA production culture PHA production culture was carried out as follows. First, a glycerol stock (50 ⁇ l) of the KNK005dZ/trc-J4b strain was inoculated into a seed medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture liquid was inoculated at 1.0 v/v% into a 3 L jar fermenter (Marubishi Bioengineering MDL-300 type) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 30° C., a stirring speed of 500 rpm, and an aeration rate of 1.8 L/min, and the culture was carried out for 28 hours while controlling the pH between 6.7 and 6.8 to carry out preculture. A 14% aqueous solution of ammonium hydroxide was used for pH control.
  • the preculture solution was inoculated at 5.0 v/v% into a 5 L jar fermenter (Marubishi Bioengineering MDS-U50 type) containing 2.5 L of PHA production medium.
  • the operating conditions were a culture temperature of 36°C, an agitation speed of 420 rpm, and an aeration rate of 2.1 L/min, and the pH was controlled between 6.7 and 6.8.
  • a 25% aqueous solution of ammonium hydroxide was used for pH control.
  • the carbon source was added intermittently. Palm olein oil was used as the carbon source. After 48 hours of cultivation, the amount of PHA produced per culture solution was measured using the method described above.
  • Examples 1 to 6 Under the same conditions as in Comparative Example 1, a culture study was carried out using each of the strains obtained in Microorganism Preparation Examples 1 to 6, and the amount of PHA produced was measured.
  • Table 1 the PHA production amount measured in Comparative Example 1 is set as a reference value, and the PHA production amount of each strain is shown as a relative value to the reference value.
  • thermophilic microorganism From Table 1, it was confirmed that the PHA production of microorganisms was improved by introducing the phaA gene derived from a thermophilic microorganism and/or the phaB gene derived from a thermophilic microorganism.

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Abstract

L'invention concerne un micro-organisme mésophile transformé qui a un gène de synthase d'acide polyhydroxyalcanoïque et dans lequel un gène phaA dérivé d'un micro-organisme thermophile et/ou un gène phaB dérivé d'un micro-organisme thermophile ont été introduits. Le micro-organisme mésophile transformé peut appartenir au genre Cupriavidus. Un acide polyhydroxyalcanoïque peut être produit par culture du micro-organisme mésophile transformé en présence d'une source de carbone.
PCT/JP2024/003569 2023-02-06 2024-02-02 Micro-organisme transformé et procédé de production d'acide polyhydroxyalcanoïque Ceased WO2024166830A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120398255A (zh) * 2025-04-30 2025-08-01 天津大学 一种利用二价锰离子自养反硝化的废水脱氮工艺

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