Transformed microorganism, and method for producing polyhydroxyalkanoic acid

Abstract

Provided is a transformed microorganism which has polyhydroxyalkanoic acid productivity, which has a polyhydroxyalkanoic acid synthase gene, and to which a gene that encodes a chaperone belonging to the ClpB family is introduced or which reinforces the expression of said gene. The chaperone belonging to the ClpB family may be derived from the genus Cupriavidus, the genus Eschericia, or the genus Saccharomyces. Polyhydroxyalkanoic acid can be produced by culturing the transformed microorganism.

Description

Transformed microorganism and method for producing polyhydroxyalkanoic acid

[0001] The present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.

[0002] In recent years, growing awareness of environmental issues has led to the development of biodegradable plastics as environmentally friendly materials. One example of a biodegradable plastic is polyhydroxyalkanoic acid (hereinafter referred to as PHA), which is produced by microorganisms. PHA is not only biodegradable, but is also expected to be used industrially as a non-petroleum-derived material, since its raw materials are sugars and fats and oils. However, PHA production costs remain high, and there is a need to increase production volume and reduce costs.

[0003] Meanwhile, chaperones are known as proteins that help proteins form the correct three-dimensional structure. When proteins become misfolded due to stresses such as heat or oxidative stress, chaperones unfold the misfolded protein and assist in refolding. Low-molecular-weight chaperones such as IbpA and IbpB prevent aggregation by copolymerizing with denatured proteins to stabilize them. In addition, chaperones such as GroESL and DnaKJ contained in Hsp60 and Hsp70 not only prevent aggregation but also assist in protein refolding. Furthermore, the chaperone ClpB unfolds and disaggregates aggregated proteins.

[0004] Non-Patent Document 1 reports that overexpression of the chaperone GroESL in Cupriavidus necator resulted in an improvement in isopropanol productivity by 9 to 18%.

[0005] Metab. Eng. , 42, 74-84 (2017)

[0006] As mentioned above, it has been reported that overexpression of the chaperone GroESL in a microorganism improved isopropanol productivity, but the effect of the chaperone on polyhydroxyalkanoic acid productivity has not been reported.

[0007] In view of the above-mentioned current situation, an object of the present invention is to provide a transformed microorganism with improved productivity of polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid by culturing the microorganism.

[0008] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that the productivity of polyhydroxyalkanoic acid can be improved by introducing a gene encoding a chaperone belonging to the ClpB family into a microorganism capable of producing polyhydroxyalkanoic acid or by enhancing the expression of the gene, and thus completed the present invention.

[0009] Specifically, the present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, which has a polyhydroxyalkanoic acid synthase gene and into which a gene encoding a chaperone belonging to the ClpB family has been introduced or whose expression has been enhanced. The present invention also relates to a method for producing polyhydroxyalkanoic acid, which includes a step of culturing the transformed microorganism.

[0010] According to the present invention, it is possible to provide a transformed microorganism with improved productivity of polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid by culturing the microorganism.

[0011] The present disclosure relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid (hereinafter also referred to as PHA), and a method for producing PHA by culturing the transformed microorganism.

[0012] The transformed microorganism according to the present disclosure is a PHA-producing microorganism having a PHA synthase gene, into which a chaperone gene belonging to the ClpB family has been introduced or whose expression has been enhanced.

[0013] The host of the transformed microorganism according to the present disclosure may be a wild-type strain that inherently has a PHA synthase gene, 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.

[0014] Examples of hosts for transformed microorganisms according to the present disclosure include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, etc. From the viewpoints of safety and PHA productivity, bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, and Escherichia are preferred, microorganisms belonging to the genus Cupriavidus are more preferred, and Cupriavidus necator is particularly preferred.

[0015] (PHA) 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. However, preferred examples include 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.

[0016] Particularly preferred PHAs are homopolymers of 3-hydroxyalkanoic acids having 4 carbon atoms, or copolymers containing 3-hydroxyalkanoic acids having 4 carbon atoms. Examples include P(3HB), which is a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), P(3HB-co-3HH) (abbreviation: P3HB3HH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), P(3HB-co-4HB), and PHAs containing lactic acid (abbreviation: LA) as a constituent, such as P(LA-co-3HB), from the viewpoint of a wide range of applications as a polymer. However, these are not limited to these.

[0017] The type of PHA to be produced can be appropriately selected depending on the purpose, such as the type of PHA synthase gene possessed by the microorganism used or separately introduced, the type of metabolic gene involved in the synthesis, and the culture conditions.

[0018] (PHA synthase gene) The PHA synthase (PhaC) gene possessed by the transformed microorganism according to the present disclosure may be one inherent to the host or may be exogenous. Examples of PHA synthase genes include, but are not limited to, PHA synthase genes derived from Aeromonas charabiae, 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-mentioned 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. Furthermore, when multiple PHA synthase genes are possessed, they may be the same gene or different genes.

[0019] (ClpB family chaperone gene) The transformed microorganism according to the present disclosure has a gene encoding a ClpB family chaperone introduced therein or its expression enhanced. Introduction of the gene can increase PHA productivity 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.

[0020] While common chaperones GroESL and DnaKJ have the function of assisting protein refolding, chaperones belonging to the ClpB family according to the present disclosure do not have the function of assisting protein refolding, but rather have the function of unraveling aggregated proteins using ATP. In the present disclosure, the "ClpB family" also includes ClpB homologs. Examples of such homologs include HSP104 found in yeast.

[0021] The gene encoding a chaperone belonging to the ClpB family is not particularly limited, and examples thereof include a gene encoding ClpB derived from the genus Cupriavidus (particularly, Cupriavidus necator), a gene encoding ClpB derived from the genus Eschericia (particularly, Eschericia coli), and a gene encoding HSP104 derived from the genus Saccharomyces (Saccharomyces cerevisiae).

[0022] More specifically, the ClpB derived from Cupriavidus necator is preferably ClpB having the amino acid sequence set forth in SEQ ID NO: 1, or a protein having an amino acid sequence showing 65% or more sequence identity to said amino acid sequence and having ClpB activity. Furthermore, the ClpB derived from Escherichia coli is preferably ClpB having the amino acid sequence set forth in SEQ ID NO: 2, or a protein having an amino acid sequence showing 65% or more sequence identity to said amino acid sequence and having ClpB activity. The sequence identity between the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2 is 67%. Furthermore, the HSP104 derived from Saccharomyces cerevisiae is preferably HSP104 having the amino acid sequence set forth in SEQ ID NO: 3, or a protein having an amino acid sequence showing 90% or more sequence identity to said amino acid sequence and having HSP104 activity.

[0023] The sequence identity is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.

[0024] (Gene introduction) The method for introducing a target gene into a host is not particularly limited, and may include a method for directly inserting or substituting the target gene onto the chromosome of the host, a method for directly inserting or substituting the target gene onto a megaplasmid carried by the host, or a method for placing the target gene onto a vector such as a plasmid, phage, or phagemid and then introducing it, and two or more of these methods may be used in combination.

[0025] Considering the stability of the introduced gene, a method of directly inserting or substituting the gene of interest onto the host chromosome or onto a megaplasmid carried by the host is preferred, and a method of directly inserting or substituting the gene of interest onto the host chromosome is even more preferred.

[0026] To ensure the expression of the introduced gene, it is preferable to introduce the target gene so that it is located downstream of a "gene expression regulatory sequence" that the host originally has, or so that it is located downstream of an exogenous "gene expression regulatory sequence." In the present disclosure, a "gene expression regulatory sequence" is a DNA sequence that includes a base sequence (e.g., a promoter sequence) that controls the transcription level of the gene and / or a base sequence (e.g., a Shine-Dalgarno sequence) that controls the translation level of messenger RNA transcribed from the gene. As 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.

[0027] Examples of promoter sequences and Shine-Dalgarno sequences contained in the "gene expression regulatory sequence" include, but are not limited to, the base sequences shown in any of SEQ ID NOs: 10 to 15, or base sequences containing parts of these base sequences.

[0028] Substitution, deletion, insertion, and / or addition of at least a portion of genomic DNA can be performed by methods well known to those skilled in the art, such as a method utilizing transposons and the mechanism of 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 subsequent loss by a second step of homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)). Alternatively, a method can be used in which the sacB gene from Bacillus subtilis is coexisted, and a microbial strain from which the gene has been lost by second-stage homologous recombination is easily isolated as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195-1204 (1992), Lenz et al., J. Bacteriol., 176:4385-4393 (1994)). As another method, genome editing technology using the CRISPR / Cas9 system to modify target DNA (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732) can also be used. In the CRISPR / Cas9 system, the guide RNA (gRNA) has a sequence that can bind to a portion of the base sequence of the genomic DNA to be modified, and serves to transport Cas9 to the target.

[0029] The method for introducing a vector into a cell is not particularly limited, and examples thereof include the calcium chloride method, electroporation, polyethylene glycol method, and spheroplast method.

[0030] (Enhancing Gene Expression) Methods for enhancing the expression of a gene encoding a chaperone belonging to the ClpB family are not particularly limited, and include inserting a gene expression regulatory sequence upstream of the gene encoding the endogenous chaperone on a chromosome, or introducing a copy of the gene encoding the endogenous chaperone at a position different from the original position, thereby increasing the expression level, or introducing a mutation into the gene encoding the endogenous chaperone to increase the ClpB activity of the gene. Furthermore, these methods may be combined or used in combination.

[0031] When inserting a gene expression regulatory sequence upstream of a gene encoding the endogenous chaperone on a chromosome, a known method can be used, for example, homologous recombination, etc. Furthermore, the gene expression regulatory sequence described above can be used.

[0032] When a copy of a gene encoding an endogenous chaperone is introduced into a location different from its original location, the method of introduction is not particularly limited. It may be directly inserted or substituted into the host chromosome, introduced onto a megaplasmid possessed by the host, or introduced by arranging the copy on a vector such as a plasmid, phage, or phagemid. Two or more of these methods may be used in combination. However, since the plasmid may be lost during culture, it is preferable that a copy of the gene encoding the endogenous chaperone is inserted or substituted into the host chromosome. Known methods can be used for the introduction, insertion, replacement, or placement. For example, homologous recombination or the like can be used to replace or insert the gene encoding the endogenous chaperone into the host chromosome.

[0033] Furthermore, it is preferable that the copy of the gene encoding the endogenous chaperone to be introduced has, upstream thereof, a gene expression regulatory sequence involved in the expression of the endogenous chaperone. The gene expression regulatory sequence to be linked upstream of the gene encoding the endogenous chaperone may be a gene expression regulatory sequence originally possessed by the host, any gene expression regulatory sequence present in nature, or an artificially constructed or modified gene expression regulatory sequence.

[0034] The gene expression regulatory sequence used for the gene encoding the endogenous chaperone is not particularly limited, and the gene expression regulatory sequence located upstream of the gene encoding the endogenous chaperone may be introduced as is together with the gene, or an appropriate gene expression regulatory sequence may be selected and linked to the gene before being introduced into the host. Furthermore, when a copy of the gene encoding the endogenous chaperone is inserted into the chromosome of the host, the gene may be inserted so as to be linked to a gene expression regulatory sequence that is originally present on the chromosome of the host. The gene expression regulatory sequence selected here can be any of the gene expression regulatory sequences described above.

[0035] When a mutation is introduced into a gene encoding an endogenous chaperone, a known method can be used. For example, a gene having a mutation introduced therein can be obtained by using the gene encoding the chaperone as a template and performing error-prone PCR or PCR using primers containing a mutation.

[0036] (PhaA gene and phaB gene derived from thermophilic bacterium) The transformed microorganism according to the present disclosure preferably has the phaA gene and the phaB gene. The phaA gene and / or the phaB gene may be the phaA gene and / or the phaB gene originally possessed by the host, or a gene encoding PhaA derived from a thermophilic bacterium and / or a gene encoding PhaB derived from a thermophilic bacterium may be introduced.

[0037] The phaA gene and phaB gene encode β-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, the most common PHA biosynthetic substrate. Specifically, PhaA is involved in catalyzing the reaction that produces acetoacetyl-CoA by condensing two molecules of acetyl-CoA, and PhaB is involved in catalyzing the reaction that produces 3HB-CoA by reducing acetoacetyl-CoA.

[0038] Examples of genes encoding β-ketothiolase (PhaA) derived from thermophiles include, but are not limited to, a gene derived from Cupriavidus sp. strain S-6 that encodes PhaA having the amino acid sequence set forth in SEQ ID NO: 4, a gene derived from Caldimonas manganoxidans that encodes PhaA having the amino acid sequence set forth in SEQ ID NO: 5, a gene derived from Schlegelella thermodepolymerans that encodes PhaA having the amino acid sequence set forth in SEQ ID NO: 6, and genes having an amino acid sequence that shows 90% or more sequence identity to these amino acid sequences and having a nucleotide sequence that encodes a protein that exhibits β-ketothiolase activity.

[0039] The sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more. The sequence identity to the amino acid sequence set forth 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.

[0040] Examples of genes encoding acetoacetyl-CoA reductase (PhaB) derived from thermophiles include, but are not limited to, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 7 derived from Cupriavidus sp. strain S-6, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 8 derived from Caldimonas manganoxidans, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 9 derived from Schlegelella thermodepolymerans, and genes having an amino acid sequence showing 90% or more sequence identity to these amino acid sequences and having a nucleotide sequence encoding a protein exhibiting acetoacetyl-CoA reductase activity.

[0041] The sequence identity to the amino acid sequence set forth in SEQ ID NO: 7 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. The sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 9 is also preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0042] Introduction of the gene encoding PhaA and / or the gene encoding PhaB can be achieved by the methods described above.

[0043] (Production of PHA) PHA can be accumulated in the cells by culturing the transformed microorganism according to the present disclosure. The method for culturing the transformed microorganism according to the present disclosure can be a conventional microbial culture method, and the culture can be carried out in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, method of adding the carbon source, culture scale, aeration and agitation conditions, culture temperature, culture time, etc. The carbon source is preferably added to the medium continuously or intermittently.

[0044] The transformed microorganism according to the present disclosure can produce PHA with good productivity even at relatively high culture temperatures, for example, even when cultured at temperatures of 35°C or higher.

[0045] Any carbon source can be used as a carbon source during cultivation as long as it can be assimilated by the transformed microorganism of the present disclosure. Examples 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 their low-melting-point fractions such as palm olein, palm double olein, and palm kernel oil olein), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, as well as their fractionated oils and refined by-products; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, as well as their derivatives, and glycerol. Some or all of the oils and fats may be degraded oils. Degraded oils refer to oils and fats that have been thermally denatured or altered by reaction with oxygen and / or water under heat. The term "degraded oil" is not limited, and includes those commonly referred to as waste oil, discarded oil, waste cooking oil, waste vegetable oil, and used oil. Furthermore, when the transformed microorganism according to the present disclosure is capable of utilizing gases or alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources.

[0046] In the production of PHA according to the present disclosure, it is preferable to culture the microorganism using a medium containing the carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Examples of nitrogen sources include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, and yeast extract. Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. 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.

[0047] After culturing for an appropriate time to accumulate PHA in the cells, PHA can be recovered from the cells using a known method. The recovery method is not particularly limited, but for example, after the end of the culture, the cells are separated from the culture solution using a centrifuge or a separation membrane, etc., and dried, and then PHA is extracted from the dried cells using an organic solvent such as chloroform, and cellular components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA, and the supernatant is removed by filtration or centrifugation, and the PHA can be recovered by drying. Alternatively, cellular components other than PHA can be dissolved in water using a surfactant, alkali, enzyme, etc., and then PHA particles can be separated from the aqueous phase by filtration or centrifugation, dried, and recovered.

[0048] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to them. [Item 1] A transformed microorganism capable of producing polyhydroxyalkanoic acid, which has a polyhydroxyalkanoic acid synthase gene and into which a gene encoding a chaperone belonging to the ClpB family has been introduced or whose expression has been enhanced. [Item 2] The transformed microorganism according to Item 1, wherein the chaperone belonging to the ClpB family has an amino acid sequence that shows 65 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2. [Item 3] The transformed microorganism according to Item 1 or 2, wherein the chaperone belonging to the ClpB family is derived from the genus Cupriavidus or Eschericia. [Item 4] The transformed microorganism according to Item 3, wherein the chaperone belonging to the ClpB family is derived from Cupriavidus necator or Eschericia coli. [Item 5] The transformed microorganism according to Item 1 or 2, wherein the transformed microorganism belongs to the genus Cupriavidus. [Item 6] The transformed microorganism according to any one of Items 1 to 5, into which a gene encoding PhaA derived from a thermophilic bacterium and / or a gene encoding PhaB derived from a thermophilic bacterium has been introduced. [Item 7] The transformed microorganism according to Item 6, wherein the thermophilic bacterium is Cupriavidus sp. strain S-6. [Item 8] A method for producing polyhydroxyalkanoic acid, comprising a step of culturing the transformed microorganism according to any one of Items 1 to 7. [Item 9] A method for producing polyhydroxyalkanoic acid according to Item 8, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid. [Item 10] A method for producing polyhydroxyalkanoic acid according to Item 9, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

[0049] The present invention will be described 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 as described in, for example, Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, and the like used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. The enzymes used are not particularly limited as long as they can be used in genetic manipulation.

[0050] The KNK005 / dZ / trc-J4b strain used below is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, expression of the R-specific enoyl-CoA hydratase gene (phaJ4b gene) on the chromosome has been enhanced, and a gene encoding a PHA synthase mutant (N149S / D171G mutant (NSDG) gene) derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 19 has been introduced, and can be prepared in accordance with the method described in WO 2015 / 115619.

[0051] (Microorganism Preparation Example 1) First, a plasmid for expressing a chaperone (ClpB) gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 17) having the lacN17 promoter, a modified Escherichia coli lac promoter, was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with the restriction enzymes EcoRI and MunI, and the resulting DNA fragment was ligated to a plasmid vector pCUP2 described in WO 2007 / 049716 that had been cleaved with MunI. The DNA fragment ligated in an orientation such that the restriction enzyme SpeI recognition sequence of pCUP2 was located downstream of the lacN17 promoter was selected, yielding pCUP2-lacN17. Next, a DNA fragment (SEQ ID NO: 18) having the nucleotide sequence of a gene encoding ClpB, which has 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 the ClpB gene expression plasmid pCUP2-lacN17-ClpBre. Next, a ClpB expression-enhanced strain was constructed using the ClpB gene expression plasmid pCUP2-lacN17-ClpBre as follows. The ClpB gene expression plasmid pCUP2-lacN17-ClpBre was introduced into the KNK005 / dZ / trc-J4b strain, and the resulting strain was designated KNK005 / dZ / trc-J4b / pCUP2-lacN17-ClpBre strain (hereinafter sometimes referred to as "PHA-producing microbial strain (1)").

[0052] Introduction of the plasmid vector into cells was carried out by electroporation as follows. A Biorad Gene Pulser was used as the gene transfer device, and a Biorad Gap 0.2 cm cuvette was used. 400 μl of competent cells and 20 μl of expression vector were injected into the cuvette and set in the pulse device, and an electric pulse was applied under the conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance of 800 Ω. After pulsing, the bacterial solution in the cuvette was cultured with shaking 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 PHA-producing microbial strain (1).

[0053] The PHA-producing microbial strain (1) is a strain obtained by deleting the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Cupriavidus necator strain H16, enhancing the expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome, introducing a gene encoding a PHA synthase mutant derived from the genus Aeromonas and having the amino acid sequence set forth in SEQ ID NO: 19, and enhancing the expression of the gene encoding ClpB derived from the genus Cupriavidus and having the amino acid sequence set forth in SEQ ID NO: 1.

[0054] (Microorganism Preparation Example 2) First, a plasmid for expressing the chaperone (ClpB) gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 20) having the nucleotide sequence of a gene encoding ClpB having the amino acid sequence set forth in SEQ ID NO: 2 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 the ClpB gene expression plasmid pCUP2-lacN17-ClpBec.

[0055] Next, a ClpBec-introduced strain was prepared using the ClpB gene expression plasmid pCUP2-lacN17-ClpBec as follows: The ClpB gene expression plasmid pCUP2-lacN17-ClpBec was introduced into the KNK005 / dZ / trc-J4b strain by the same electroporation method as above, and the resulting strain was designated KNK005 / dZ / trc-J4b / pCUP2-lacN17-ClpBec strain (hereinafter, sometimes referred to as "PHA-producing microbial strain (2)").

[0056] The PHA-producing microbial strain (2) is a strain obtained by deleting the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain, enhancing the expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome, and introducing a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 19 and a gene encoding ClpB derived from the genus Escherichia having the amino acid sequence set forth in SEQ ID NO: 2.

[0057] (Microorganism Production Example 3) First, a plasmid for expressing a chaperone (GroESL) gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 23) having the nucleotide sequence of a gene encoding GroES having the amino acid sequence set forth in SEQ ID NO: 21 and GroEL having the amino acid sequence set forth in SEQ ID NO: 22 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 the GroESL gene expression plasmid pCUP2-lacN17-GroESL.

[0058] Next, a strain with enhanced GroESL expression was prepared using the GroESL gene expression plasmid pCUP2-lacN17-GroESL as follows: The GroESL gene expression plasmid pCUP2-lacN17-GroESL was introduced into the KNK005 / dZ / trc-J4b strain by the same electroporation method as above, and the resulting strain was designated KNK005 / dZ / trc-J4b / pCUP2-lacN17-GroESL strain (hereinafter, sometimes referred to as "PHA-producing microbial strain (3)").

[0059] The PHA-producing microbial strain (3) is a strain obtained by deleting the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Cupriavidus necator strain H16, enhancing the expression of the R-specific enoyl-CoA hydratase gene on the chromosome, and introducing a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 19 and a gene encoding GroESL derived from the genus Cupriavidus having the amino acid sequences set forth in SEQ ID NOs: 21 and 22.

[0060] (Microorganism Production Example 4) First, a plasmid for expressing the chaperone (DnaKJ) gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 26) having the nucleotide sequence of a gene encoding DnaK having the amino acid sequence set forth in SEQ ID NO: 24 and DnaJ having the amino acid sequence set forth in SEQ ID NO: 25 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 the DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ.

[0061] Next, a DnaKJ-introduced strain was prepared using the DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ as follows. The DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ was introduced into the KNK005 / dZ / trc-J4b strain by the same electroporation method as above, and the resulting strain was named KNK005 / dZ / trc-J4b / pCUP2-lacN17-DnaKJ strain (hereinafter, sometimes referred to as "PHA-producing microbial strain (4)").

[0062] The PHA-producing microbial strain (4) is a strain obtained by deleting the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain, enhancing expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome, and introducing a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 19 and a gene encoding DnaKJ derived from the genus Escherichia having the amino acid sequences set forth in SEQ ID NOs: 24 and 25.

[0063] (Microorganism Preparation Example 5) First, a plasmid for disrupting the phaA and phaB genes was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 27) having the nucleotide sequences upstream and downstream of the phaA and phaB structural genes of the Capriavidus necator H16 strain was obtained by PCR using synthetic oligo DNA. 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 DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), to prepare the plasmid vector pNS2X-sacB+phaABUD for disrupting the phaAB genes.

[0064] Next, a phaAB gene-disrupted strain was prepared using the phaAB gene-disruption plasmid vector pNS2X-sacB+phaABUD as follows: Escherichia coli S17-1 strain (ATCC 47055) was transformed with the phaAB gene-disruption plasmid vector pNS2X-sacB+phaABUD, and the resulting transformed microorganism was mixed and cultured with KNK005 / dZ / trc-J4b strain on Nutrient Agar medium (Difco) for conjugative transfer. The resulting culture medium was inoculated onto Simmons agar medium containing 250 mg / L of kanamycin (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8), and the strains that grew on the agar medium were selected to obtain a strain in which the plasmid was integrated onto the chromosome of the KNK005 / dZ / trc-J4b strain. This strain was cultured for two generations in Nutrient Broth medium (manufactured by Difco), and then diluted and spread onto Nutrient Agar medium containing 15% sucrose. The grown strain was obtained as a strain from which the plasmid had been lost. Furthermore, one strain in which the phaAB genes on the chromosome were deleted was isolated by PCR and DNA sequencer analysis. This gene-disrupted strain was designated KNK005 / dZ / trc-J4b / dphaAB strain.

[0065] Furthermore, a plasmid for introducing the phaA and phaB genes was prepared. This was done as follows. PCR using synthetic oligo DNA yielded a DNA fragment (SEQ ID NO: 28) containing the nucleotide sequences upstream and downstream of the phaA and phaB structural genes of the Capriavidus necator H16 strain, as well as the nucleotide sequences of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 4 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 7. 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 DNA ligase (Ligation High (Toyobo Co., Ltd.)) to prepare the plasmid vector pNS2X-sacB+phaAU-phaABsp-phaBD for introducing the phaA and phaB genes.

[0066] Next, the plasmid vector pNS2X-sacB+phaAU-phaABsp-phaBD for introducing the phaA and phaB genes was introduced into the KNK005 / dZ / trc-J4b / dphaAB strain by the same conjugal transfer method as above. Furthermore, by culturing in the same manner as above and selecting using Nutrient Agar medium containing 15% sucrose, one strain into which a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO:4 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO:7 had been introduced was isolated. The resulting strain was designated KNK005 / dZ / trc-J4b / dphaAB::phaABsp.

[0067] Furthermore, a ClpBre expression-enhanced strain was prepared using the ClpB gene expression plasmid pCUP2-lacN17-ClpBre as follows: The ClpB gene expression plasmid pCUP2-lacN17-ClpBre was introduced into the KNK005 / dZ / trc-J4b / dphaAB::phaABsp strain by the same electroporation method as above, and the resulting strain was designated KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-ClpBre strain (hereinafter, sometimes referred to as "PHA-producing microbial strain (5)").

[0068] The PHA-producing microbial strain (5) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene, and phaAB genes on the chromosome of the Cupriavidus necator strain H16 have been deleted, the expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas and having the amino acid sequence set forth in SEQ ID NO: 19, and the phaA gene having the amino acid sequence set forth in SEQ ID NO: 4 and the phaB gene having the amino acid sequence set forth in SEQ ID NO: 7 derived from Cupriavidus sp. strain S-6 have been introduced, and the expression of the gene encoding ClpB derived from the genus Cupriavidus and having the amino acid sequence set forth in SEQ ID NO: 1 has been enhanced.

[0069] (Microorganism Preparation Example 6) Furthermore, a ClpBec-introduced strain was prepared using the ClpB gene expression plasmid pCUP2-lacN17-ClpBec as follows. The ClpB gene expression plasmid pCUP2-lacN17-ClpBre was introduced into the KNK005 / dZ / trc-J4b / dphaAB::phaABsp strain by the same electroporation method as above, and the resulting strain was designated KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-ClpBec strain (hereinafter, sometimes referred to as "PHA-producing microorganism strain (6)").

[0070] The PHA-producing microbial strain (6) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene, and phaAB genes on the chromosome of the Cupriavidus necator strain H16 have been deleted, expression of the R-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas and having the amino acid sequence set forth in SEQ ID NO: 19, phaA genes having the amino acid sequence set forth in SEQ ID NO: 4 and phaB genes having the amino acid sequence set forth in SEQ ID NO: 7 derived from Cupriavidus sp. strain S-6, and a gene encoding ClpB derived from the genus Escherichia and having the amino acid sequence set forth in SEQ ID NO: 2 have been introduced.

[0071] (Microorganism Preparation Example 7) Furthermore, a GroESL-introduced strain was prepared using the GroESL gene expression plasmid pCUP2-lacN17-GroESL as follows: The GroESL gene expression plasmid pCUP2-lacN17-GroESL was introduced into the KNK005 / dZ / trc-J4b / dphaAB::phaABsp strain by the same electroporation method as above, and the resulting strain was designated KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-GroESL strain (hereinafter, sometimes referred to as "PHA-producing microorganism strain (7)").

[0072] The PHA-producing microbial strain (7) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene, and phaAB genes on the chromosome of the Cupriavidus necator strain H16 have been deleted, expression of the R-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas and having the amino acid sequence set forth in SEQ ID NO: 19, phaA genes having the amino acid sequence set forth in SEQ ID NO: 4 and phaB genes having the amino acid sequence set forth in SEQ ID NO: 7, derived from Cupriavidus sp. strain S-6, and a gene encoding GroESL derived from the genus Cupriavidus and having the amino acid sequences set forth in SEQ ID NOs: 21 and 22 have been introduced.

[0073] (Microorganism Preparation Example 8) Furthermore, a DnaKJ-introduced strain was prepared using the DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ as follows. The DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ was introduced into the KNK005 / dZ / trc-J4b / dphaAB::phaABsp strain by the same electroporation method as above, and the resulting strain was designated KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-DnaKJ strain (hereinafter, sometimes referred to as "PHA-producing microorganism strain (8)").

[0074] The PHA-producing microbial strain (8) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene, and phaAB genes on the chromosome of the Cupriavidus necator strain H16 have been deleted, expression of the R-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas and having the amino acid sequence set forth in SEQ ID NO: 19, phaA genes having the amino acid sequence set forth in SEQ ID NO: 4 and phaB genes having the amino acid sequence set forth in SEQ ID NO: 7, derived from Cupriavidus sp. strain S-6, and a gene encoding DnaKJ derived from the genus Escherichia and having the amino acid sequences set forth in SEQ ID NOs: 24 and 25 have been introduced.

[0075] (Example 1) PHA production by PHA-producing microbial strain (1) Cultivation studies were carried out using the PHA-producing microbial strain (1) under the following conditions.

[0076] (Method for measuring PHA accumulation amount) The ratio of PHA accumulation amount to dry bacterial cells was measured as follows. The bacterial cells were collected from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain dry bacterial cells, and their weight was measured. 100 ml of chloroform was added to 1 g of the obtained dry bacterial cells, and the mixture was stirred at room temperature for one day and one night to extract the PHA (PHA mixture) within the bacterial cells. The bacterial cell residue was filtered and then concentrated in an evaporator to a total volume of 30 ml, after which 90 ml of hexane was gradually added and the mixture was left to stand for 1 hour with slow stirring. The precipitated PHA was filtered and then vacuum-dried at 50°C for 3 hours. The weight of the dried PHA was measured, and the PHA production amount was calculated.

[0077] (PHA production culture in a flask) The composition of the seed medium was 10 g / L meat extract, 10 g / L bactotryptone, 2 g / L yeast extract, 9 g / L sodium dihydrogen phosphate dodecahydrate, 1.5 g / L dipotassium hydrogen phosphate, and 100 μg / L kanamycin sulfate.

[0078] The PHA production medium consisted of 11 g / L disodium hydrogen phosphate dodecahydrate, 1.9 g / L dipotassium hydrogen phosphate, 1.3 g / L ammonium sulfate, 5 mL / L magnesium solution, and 1 mL / L trace metal salt solution. The magnesium solution was prepared by dissolving 200 g / L magnesium sulfate heptahydrate in water. The trace metal salt solution was prepared by dissolving 0.218 g / L cobalt chloride hexahydrate, 16.2 g / L iron(III) chloride hexahydrate, 10.3 g / L calcium chloride dihydrate, 0.118 g / L nickel chloride hexahydrate, and 0.156 g / L copper sulfate pentahydrate in 0.1 N hydrochloric acid.

[0079] 50 μL of a glycerol stock solution of the KNK005 / dZ / trc-J4b / pCUP2-lacN17-ClpBre strain prepared in Microorganism Preparation Example 1 was inoculated into 10 mL of seed medium and cultured with shaking at 30° C. for 24 hours. The resulting culture solution was used as a preculture solution.

[0080] PHA production culture was carried out in a flask. 50 mL of PHA production medium was placed in a 500 mL shake flask. Immediately before inoculation, 250 μL of magnesium solution, 50 μL of trace metal solution, and 1 g of palm kernel oil were added. After medium preparation, 500 μL of preculture solution was inoculated into the shake flask, and shake culture was carried out at 36°C for 72 hours. After culture was completed, the PHA production amount was measured as described above. The PHA production amount in Comparative Example 1, in which the KNK005 / dZ / trc-J4b strain was cultured under the same conditions, was used as the reference value, and the PHA production amount is shown in Table 1 as a relative value to this reference value.

[0081] (Example 2) PHA production by PHA-producing microbial strain (2) Cultivation studies were carried out using the PHA-producing microbial strain (2) under the same conditions as in Example 1, and the PHA production amount was measured as described above. As in Example 1, the PHA production amount is shown in Table 1 as a relative value to the reference value.

[0082] (Comparative Examples 1 to 3) Cultivation studies were carried out using the KNK005 / dZ / trc-J4b strain, the PHA-producing microbial strain (3), or the PHA-producing microbial strain (4) under the same conditions as in Example 1, and the PHA production amount was measured as described above. As in Example 1, the PHA production amount is shown in Table 1 as a relative value to the reference value.

[0083]

[0084] The following can be seen from Table 1: Comparing Example 1 and Example 2 with Comparative Example 1, it can be seen that Examples 1 and 2 had improved PHA production amounts compared to Comparative Example 1. This suggests that PHA productivity was improved by introducing or enhancing expression of a gene encoding a chaperone belonging to the ClpB family.

[0085] Furthermore, when Examples 1 and 2 are compared with Comparative Examples 2 and 3, it is found that Examples 1 and 2 have higher PHA production amounts than Comparative Examples 2 and 3. This indicates that the introduction or enhanced expression of a gene encoding a chaperone belonging to the ClpB family is more effective in improving PHA productivity than the introduction or enhanced expression of a gene encoding other chaperones, GroESL or DnaKJ.

[0086] (Example 3) PHA production by PHA-producing microbial strain (5) Cultivation studies were carried out using the PHA-producing microbial strain (5) under the following conditions.

[0087] (PHA production culture in high-density culture) The composition of the seed 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, 0.15w / v% KH ２ P.O. ４ , (pH 6.8).

[0088] The composition of the pre-culture medium was 1.1 w / v% Na ２ HPO ４ ・12H ２ O, 0.19w / v%KH ２ P.O. ４ , 1.29w / v% (NH ４ ) ２ SO ４ , 0.1w / v% MgSO ４ ・7H ２ O, 2.5 w / v% palm olein oil, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid ３ ・6H ２ O, 1w / v% CaCl ２ ・2H ２ O, 0.02w / v% CoCl ２ ・6H ２ O, 0.016w / v% CuSO ４ ・5H ２ O, 0.012w / v% NiCl ２ ・6H ２ O was dissolved).

[0089] The composition of the PHA production medium was 0.385 w / v% Na ２ HPO ４ ・12H ２ O, 0.067w / v% KH ２ P.O. ４ , 0.291w / v% (NH ４ ) ２ SO ４ , 0.1w / v% MgSO４ ・7H ２ 0, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid) ３ ・6H ２ O, 1w / v% CaCl ２ ・2H ２ O, 0.02w / v% CoCl ２ ・6H ２ O, 0.016w / v% CuSO ４ ・5H ２ O, 0.012w / v% NiCl ２ ・6H ２ O was dissolved).

[0090] PHA production culture was carried out as follows. First, a glycerol stock (50 μl) of the PHA-producing microbial strain (5) was inoculated into a seed medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (Marubishi Bioengine MDL-300 model) 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 continued for 28 hours while controlling the pH between 6.7 and 6.8 to carry out preculture. A 14% aqueous ammonium hydroxide solution was used to control the pH.

[0091] Next, the preculture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (MDS-U50 model, manufactured by Marubishi Bioengineering) 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. The culture was continued for 48 hours. The PHA production amount was measured as described above. The PHA production amount in Comparative Example 4, in which the KNK005 / dZ / trc-J4b strain was cultured under the same conditions, was used as the reference value, and the PHA production amount relative to this reference value is shown in Table 2.

[0092] (Example 4) PHA production by PHA-producing microbial strain (6) Cultivation studies were carried out using the PHA-producing microbial strain (6) under the same conditions as in Example 3, and the PHA production amount was measured as described above. As in Example 3, the PHA production amount is shown in Table 2 as a relative value to the reference value.

[0093] (Comparative Examples 4 to 7) Cultivation studies were carried out using the KNK005 / dZ / trc-J4b strain, the KNK005 / dZ / trc-J4b / dphaAB::phaABsp strain, the PHA-producing microbial strain (7), or the PHA-producing microbial strain (8) under the same conditions as in Example 1, and the PHA production amount was measured as described above. As in Example 3, the PHA production amount is shown in Table 2 as a relative value to the reference value.

[0094]

[0095] The following can be seen from Table 2: Comparing Example 3 and Example 4 with Comparative Example 5, it can be seen that Examples 3 and 4 have higher PHA production amounts than Comparative Example 5. This indicates that PHA productivity was improved by introducing or enhancing expression of a gene encoding a chaperone belonging to the ClpB family.

[0096] Furthermore, when Examples 3 and 4 are compared with Comparative Examples 6 and 7, it is found that Examples 3 and 4 have higher PHA productivity than Comparative Examples 6 and 7. This indicates that the introduction or enhanced expression of a gene encoding a chaperone belonging to the ClpB family is more effective in improving PHA productivity than the introduction or enhanced expression of a gene encoding another chaperone, GroESL or DnaKJ.

Claims

1. A transformed microorganism capable of producing polyhydroxyalkanoic acid, Possessing the polyhydroxyalkanoate synthase gene, A gene encoding a chaperone belonging to the ClpB family is introduced or its expression is enhanced, A transformed microorganism into which a gene encoding PhaA derived from a thermophilic bacterium and / or a gene encoding PhaB derived from a thermophilic bacterium have been introduced.

2. 2. The transformed microorganism according to claim 1, wherein the chaperone belonging to the ClpB family has an amino acid sequence that shows 65 to 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 2.

3. The transformed microorganism according to claim 1 or 2, wherein the chaperone belonging to the ClpB family is derived from the genus Cupriavidus or Eschericia.

4. The transformed microorganism according to claim 3 , wherein the chaperone belonging to the ClpB family is derived from Cupriavidus necator or Escherichia coli.

5. The transformed microorganism according to claim 1 or 2, wherein the transformed microorganism belongs to the genus Cupriavidus.

6. 3. The transformed microorganism according to claim 1, wherein the thermophilic bacterium is Cupriavidus sp. strain S-6.

7. A method for producing a polyhydroxyalkanoic acid, comprising a step of culturing the transformed microorganism according to claim 1 or 2.

8. 8. The method for producing a polyhydroxyalkanoic acid according to claim 7, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids.

9. 9. The method for producing a polyhydroxyalkanoic acid according to claim 8, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.