Transforming microorganism and method for producing polyhydroxyalkanoate

Abstract

A transformed microorganism having the ability to produce a polyhydroxyalkanoate, the transformed microorganism having a polyhydroxyalkanoate synthase gene into which a gene encoding a molecular chaperone belonging to the ClpB family is introduced, or the expression thereof is enhanced. The molecular chaperone belonging to the ClpB family is optionally derived from the genus Cupriavidus, the genus Escherichia, or the genus Saccaromyces, and the molecular chaperone is selected from the genus Cupriavidus, the genus Escherichia, or the genus Saccaromyces. By culturing the transformed microorganism, a polyhydroxyalkanoate can be produced.

Description

Technical Field

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

[0002] In recent years, due to the increasing awareness of environmental issues, biodegradable plastics are attracting attention as materials with low environmental burden. As an example of biodegradable plastics, polyhydroxyalkanoates (hereinafter referred to as PHAs) produced by microorganisms can be cited. PHAs are biodegradable and, because their raw materials are sugars and oils, they are non-petroleum-based and are expected to be used in industry. However, since the production cost of PHAs is still relatively high, there is a need to increase production volume and reduce costs.

[0003] On the other hand, as proteins that assist proteins in forming the correct three-dimensional structure, there are known molecular chaperones. When proteins are misfolded due to stresses such as heat stress and oxidative stress, molecular chaperones untie the misfolding of the protein and refold it. Low-molecular molecular chaperones such as IbpA and IbpB copolymerize with denatured proteins to stabilize them, thereby preventing aggregation. In addition, molecular chaperones such as GroESL and DnaKJ contained in Hsp60 and Hsp70 can not only prevent aggregation but also assist in the refolding of proteins. In addition, molecular chaperone ClpB has a deaggregation function that unties aggregated proteins.

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

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent literature 1: Metab. Eng., 42, 74-84 (2017) Summary of the Invention

[0008] Problems to be solved by the invention

[0009] As described above, there have been reports that the productivity of isopropanol is improved by overexpressing the molecular chaperone GroESL in microorganisms, but there have been no reports that molecular chaperones affect the productivity of polyhydroxyalkanoates.

[0010] In view of the above-mentioned current situation, an object of the present invention is to provide a transformed microorganism having improved productivity of polyhydroxyalkanoate, and a method for producing polyhydroxyalkanoate by culturing the microorganism.

[0011] Solutions to the Problem

[0012] To solve the above problems, the present inventors conducted intensive studies and found that the productivity of polyhydroxyalkanoate can be improved by introducing a gene encoding a molecular chaperone belonging to the ClpB family or enhancing its expression into a microorganism capable of producing polyhydroxyalkanoate, thereby completing the present invention.

[0013] That is, the present invention relates to a transformed microorganism having the ability to produce polyhydroxyalkanoate.

[0014] The transformed microorganism has a polyhydroxyalkanoate synthase gene,

[0015] The transformed microorganism has a gene introduced therein encoding a molecular chaperone belonging to the ClpB family, or the expression of the gene is enhanced.

[0016] The present invention also relates to a method for producing polyhydroxyalkanoate, which comprises the step of culturing the above-mentioned transformation microorganism.

[0017] Effects of the Invention

[0018] According to the present invention, there can be provided a transformed microorganism having improved productivity of polyhydroxyalkanoate and a method for producing polyhydroxyalkanoate by culturing the microorganism. DETAILED DESCRIPTION

[0019] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments.

[0020] The present disclosure relates to a transformed microorganism capable of producing polyhydroxyalkanoate (hereinafter also referred to as PHA), and also relates to a method for producing PHA by culturing the transformed microorganism.

[0021] The transformed microorganism disclosed herein is a PHA-producing microorganism having a PHA synthase gene, and a molecular chaperone gene belonging to the ClpB family has been introduced into the transformed microorganism, or its expression has been enhanced.

[0022] The host of the transformed microorganism of the present disclosure may be a wild strain originally possessing a PHA synthase gene, a mutant obtained by artificially mutating such a wild strain, or a transformant into which an exogenous PHA synthase gene has been introduced by genetic engineering methods.

[0023] Examples of hosts for the transformation microorganisms of the present disclosure include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Aeromonas, Escherichia, Alcaligenes, and Pseudomonas. From the perspectives of safety and PHA productivity, bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, and Escherichia are preferred, microorganisms belonging to Cupriavidus are more preferred, and Cupriavidus necator is particularly preferred.

[0024] (PHA)

[0025] The type of PHA produced by the transformed microorganism of the present disclosure is not particularly limited as long as it is a PHA that can be produced by the microorganism, but preferably includes 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 other hydroxyalkanoic acids (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 other hydroxyalkanoic acids.

[0026] 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, but are not limited to, P(3HB), a homopolymer of 3-hydroxybutyric acid (abbreviated as 3HB), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviated as 3HV), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviated as 3HH), P(3HB-co-3HH) (abbreviated as P3HB3HH), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviated as 4HB), and PHAs containing lactic acid (abbreviated as LA) as a component, such as P(LA-co-3HB), a copolymer of 3HB and LA. Among these, P3HB3HH is preferred due to its wide range of applications as a polymer.

[0027] It should be noted that, depending on the purpose, the type of PHA produced can be appropriately selected based on the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of genes of the metabolic system involved in the synthesis, culture conditions, and the like.

[0028] (PHA synthase gene)

[0029] The PHA synthase (PhaC) gene possessed by the transformed microorganism of the present disclosure may be a gene originally possessed by the host or an exogenous gene. Examples of PHA synthase genes are not particularly limited and include PHA synthase genes derived from Aeromonas caviae, Aeromonas hydrophila, Pseudomonas sp 61-3, or Cupriavidus necrotizingus, chimeric PHA synthase genes combining two or more of the aforementioned PHA synthase genes, or genes encoding proteins comprising an amino acid sequence that exhibits 90% or greater sequence identity with the amino acid sequences of the aforementioned PHA synthases.

[0030] The sequence identity is preferably 95% or higher, more preferably 97% or higher, and even more preferably 99% or higher.

[0031] The number of PHA synthase genes possessed by the transformed microorganism of the present disclosure may be one or more. Furthermore, when the transformed microorganism possesses multiple PHA synthase genes, they may be the same gene or different genes.

[0032] (Molecular chaperone gene belonging to the ClpB family)

[0033] The transformed microorganisms disclosed herein have been introduced with a gene encoding a molecular chaperone belonging to the ClpB family, or their expression has been enhanced. By introducing this gene, the productivity of PHAs in the transformed microorganisms can be improved. These transformed microorganisms are capable of producing PHAs with good productivity under high stress conditions, such as at elevated culture temperatures.

[0034] Conventional molecular chaperones such as GroESL and DnaKJ have a function of assisting protein refolding. In contrast, the molecular chaperone belonging to the ClpB family according to the present disclosure does not have a function of assisting protein refolding, but rather has a function of disentangling aggregated proteins using ATP.

[0035] In the present disclosure, the term "ClpB family" also includes homologs of ClpB. Examples of such homologs include HSP104 possessed by yeast.

[0036] Genes encoding molecular chaperones belonging to the ClpB family are not particularly limited, and examples thereof include genes encoding ClpB derived from the genus Cupriavidus (particularly Cupriavidus necator), genes encoding ClpB derived from the genus Eschericia (particularly Eschericia coli), and genes encoding HSP104 derived from the genus Saccaromyse (Saccaromyse cerevisiae).

[0037] More specifically, ClpB 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 greater sequence identity with respect to the amino acid sequence and having ClpB activity. Furthermore, ClpB from Eschericia 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 greater sequence identity with respect to the amino acid sequence and having ClpB activity. It should be noted that 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%.

[0038] Furthermore, HSP104 derived from Saccaromyse cerevisiae is preferably HSP104 having the amino acid sequence shown in SEQ ID NO: 3, or a protein having an amino acid sequence showing 90% or greater sequence identity with the amino acid sequence and having HSP104 activity.

[0039] The sequence identity is preferably 70% or higher, more preferably 80% or higher, further preferably 90% or higher, even more preferably 95% or higher, particularly preferably 97% or higher, and most preferably 99% or higher.

[0040] (Gene introduction)

[0041] The method for introducing the target gene into the host is not particularly limited, and may include a method of directly inserting or replacing the target gene into the host chromosome, a method of directly inserting or replacing the target gene into a giant plasmid possessed by the host, or a method of introducing the target gene by configuring it on a vector such as a plasmid, phage, or phagemid. Two or more of these methods may be used in combination.

[0042] Considering the stability of the introduced gene, a method of directly inserting or replacing the target gene on the host chromosome or on a giant plasmid harbored by the host is preferred, and a method of directly inserting or replacing the target gene on the host chromosome is more preferred.

[0043] In order to ensure the expression of the introduced gene, it is preferably introduced in a manner such that the target gene is located downstream of a "gene expression regulatory sequence" originally possessed by the host, or in a manner such that the target gene is located downstream of an external "gene expression regulatory sequence." A "gene expression regulatory sequence" as used herein refers to a DNA sequence comprising a base sequence that controls the transcription amount of the gene (e.g., a promoter sequence) and / or a base sequence that regulates the translation amount of messenger RNA transcribed from the gene (e.g., a Shine-Dalgarno sequence). As a "gene expression regulatory sequence," any base sequence existing in nature can be used, as well as an artificially constructed or modified base sequence.

[0044] Examples of promoter sequences and Shine-Dalgarno sequences included in the “gene expression regulatory sequence” include, but are not particularly limited to, the base sequences represented by any one of SEQ ID NOs: 10 to 15, or base sequences comprising a portion of these base sequences.

[0045] The displacement, deletion, insertion and / or addition of at least a portion of genomic DNA can be carried out by methods well known to those skilled in the art. As representative methods, following methods are arranged: the method (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)) utilizing the mechanism of homologous recombination with transposon; the method (Noti et al., Methods Enzymol., 154:197-217 (1987)) etc. using the site-specific introduction caused by the mechanism of homologous recombination and the shedding caused by the homologous recombination in the second phase as principle. In addition, a method can be used to coexist the sacB gene from Bacillus subtilis and to easily separate the microbial strain from which the gene is removed as a sucrose-resistant strain by homologous recombination in the second stage (Schweizer, Mol. Microbiol., 6:1195-1204 (1992), Lenz et al., J. Bacteriol., 176:4385-4393 (1994)). In addition, as another method, the genome editing technology of the CRISPR / Cas9 system for changing the target DNA can also be used (Y. Wang et al., ACS Synth Biol. 2016, 5 (7):721-732). 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 has the effect of targeted delivery of Cas9.

[0046] The method for introducing a vector into cells is not particularly limited, and examples thereof include a calcium chloride method, an electroporation method, a polyethylene glycol method, and a spheroplast method.

[0047] (Enhanced gene expression)

[0048] Methods for enhancing expression of genes encoding molecular chaperones belonging to the ClpB family are not particularly limited. Expression levels can be increased by inserting a gene expression regulatory sequence upstream of the gene encoding the endogenous molecular chaperone on the chromosome, or by introducing a copy of the gene encoding the endogenous molecular chaperone at a different location than the original location. Alternatively, the ClpB activity of the gene encoding the endogenous molecular chaperone can be enhanced by introducing a mutation into the gene encoding the endogenous molecular chaperone. These methods can also be combined and used in combination.

[0049] When inserting a gene expression regulatory sequence upstream of the gene encoding the endogenous molecular chaperone on the chromosome, a known method can be used, for example, homologous recombination, etc. In addition, the gene expression regulatory sequence described above can be used as the gene expression regulatory sequence.

[0050] When a copy of the gene encoding the endogenous molecular chaperone is introduced into a position different from the original position, the method of introduction is not particularly limited, and any of the following methods can be arbitrarily selected: a method of directly inserting or replacing the gene on the host's chromosome; a form of importing the gene onto a giant plasmid held by the host, or a form of importing the gene by configuring the copy on a plasmid, phage, phagemid or other vector. Two or more of these methods can also be used in combination. However, there is a possibility that the plasmid will fall off during cultivation, so it is preferred to insert or replace a copy of the gene encoding the endogenous molecular chaperone on the host's chromosome. As the method of the aforementioned introduction, insertion, replacement or configuration, a known method can be used. For example, the gene encoding the endogenous molecular chaperone can be replaced or inserted on the host's chromosome using homologous recombination methods or the like.

[0051] Furthermore, the introduced copy of the gene encoding the endogenous molecular chaperone preferably has a gene expression regulatory sequence upstream thereof that regulates its expression. The gene expression regulatory sequence linked upstream of the gene encoding the endogenous molecular chaperone may be a gene expression regulatory sequence inherent to the host, any gene expression regulatory sequence existing in nature, or an artificially constructed or modified gene expression regulatory sequence.

[0052] The gene expression regulatory sequence used relative to the gene encoding the endogenous aforementioned molecular chaperone is not particularly limited. The gene expression regulatory sequence located upstream of the gene encoding the endogenous aforementioned molecular chaperone can be imported together in its original form. Alternatively, a suitable gene expression regulatory sequence can be selected and connected to the aforementioned gene and introduced into the host. Additionally, when a copy of the gene encoding the endogenous aforementioned molecular chaperone is inserted into the host's chromosome, the gene expression regulatory sequence originally present on the host's chromosome can also be connected to the aforementioned gene and inserted. As the gene expression regulatory sequence selected here, the gene expression regulatory sequence described above can also be utilized.

[0053] When introducing a mutation into the gene encoding the endogenous molecular chaperone, a known method can be used. For example, using the gene encoding the molecular chaperone as a template, error-prone PCR and PCR using primers that introduce the mutation can obtain the gene into which the mutation has been introduced.

[0054] (Derived from the phaA and phaB genes of thermophilic bacteria)

[0055] The transformed microorganism of the present disclosure preferably has the phaA gene and the phaB gene. The phaA gene and / or the phaB gene can be the phaA gene and / or the phaB gene originally possessed by the host, and a gene encoding PhaA and / or a gene encoding PhaB derived from a thermophilic bacterium can be introduced.

[0056] The phaA and phaB genes encode β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB), respectively. These genes are present in many microorganisms that can naturally accumulate PHA and are involved in the biosynthesis of 3HB-CoA, the most common substrate for PHA biosynthesis. Specifically, PhaA catalyzes the reaction that condenses two molecules of acetyl-CoA to produce acetoacetyl-CoA, while PhaB catalyzes the reaction that reduces acetoacetyl-CoA to produce 3HB-CoA.

[0057] Examples of genes encoding β-ketothiolase (PhaA) derived from thermophilic bacteria include, but are not limited to, a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 4 derived from Cupriavidus sp. strain S-6; a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 5 derived from Caldimonas manganoxidans; a gene encoding PhaA 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 exhibits 90% or greater sequence identity with these amino acid sequences and having a base sequence encoding a protein exhibiting β-ketothiolase activity.

[0058] The sequence identity with respect to the amino acid sequence described in SEQ ID NO: 4 is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more. In addition, the sequence identity with respect to the amino acid sequence described in SEQ ID NO: 5 or 6 is also preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more.

[0059] Examples of genes encoding acetoacetyl-CoA reductase (PhaB) derived from thermophilic bacteria 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; or a gene having an amino acid sequence that exhibits 90% or greater sequence identity with these amino acid sequences and having a base sequence encoding a protein exhibiting acetoacetyl-CoA reductase activity.

[0060] The sequence identity with respect to the amino acid sequence described in SEQ ID NO: 7 is preferably 90% or more, more preferably 95% or more, further preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. In addition, the sequence identity with respect to the amino acid sequence described in SEQ ID NO: 8 or 9 is also preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more.

[0061] Introduction of the gene encoding PhaA and / or the gene encoding PhaB can be achieved by the above-mentioned method.

[0062] (Production of PHA)

[0063] By culturing the transformed microorganisms disclosed herein, PHA can be accumulated within the microorganisms. The transformed microorganisms disclosed herein can be cultured in a culture medium containing an appropriate carbon source according to conventional microbial culture methods. The composition of the culture medium, the method for adding the carbon source, the culture scale, the aeration and stirring conditions, the culture temperature, and the culture time are not particularly limited. The carbon source is preferably added to the culture medium continuously or intermittently.

[0064] 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.

[0065] As a carbon source during cultivation, any carbon source can be used as long as the transformed microorganism of the present invention can assimilate it. There are no special restrictions, and examples include: sugars such as glucose, fructose, and sucrose; palm oil, palm kernel oil (including palm olein, palm diolein, palm kernel olein, etc. as low-melting-point fractions obtained by separating them), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, Jatropha oil, and other oils and their separated oils, or their refined by-products; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, myristic acid, their derivatives, or glycerol, etc. Part or all of the above-mentioned oils and fats may be deteriorated oils. Deteriorated oils refer to oils and fats that have been thermally denatured or have deteriorated by reacting with oxygen and / or water under heating. Its name is not limited, and includes oils such as waste oil, discarded oil, waste cooking oil, waste edible oil, waste vegetable oil, and used oil. Furthermore, when the above-mentioned transforming microorganism can utilize gases such as carbon dioxide, carbon monoxide, methane, methanol, ethanol, and alcohols, these can be used as carbon sources.

[0066] In the production of PHA disclosed herein, the microorganisms are preferably cultured using a culture medium containing the aforementioned 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, although not limited to the following, include 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.

[0067] After culturing for an appropriate period of time to allow PHA to accumulate within the cells, PHA can be recovered from the cells using known methods. The recovery method is not particularly limited. For example, after the culture is completed, the cells are separated from the culture medium using a centrifuge, a separation membrane, or the like, and dried. PHA is then extracted from the dried cells using an organic solvent such as chloroform. Cellular components are removed from the organic solvent solution containing the PHA by filtration, for example. A poor solvent such as methanol or hexane is added to the filtrate to precipitate the PHA. The supernatant is removed by filtration or centrifugation and then dried to recover PHA. Alternatively, cellular components other than PHA can be dissolved in water using a surfactant, an alkali, an enzyme, or the like, followed by filtration or centrifugation to separate the PHA particles from the aqueous phase and drying for recovery.

[0068] In the following items, preferred aspects of the present disclosure are listed, but the present invention is not limited to the following items.

[0069] [Project 1]

[0070] A transformed microorganism having the ability to produce polyhydroxyalkanoate,

[0071] The transformed microorganism has a polyhydroxyalkanoate synthase gene,

[0072] The transformed microorganism has a gene encoding a molecular chaperone belonging to the ClpB family introduced therein, or its expression is enhanced.

[0073] [Project 2]

[0074] The transformed microorganism according to item 1, wherein

[0075] The molecular 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.

[0076] [Item 3]

[0077] The transformed microorganism according to item 1 or 2, wherein

[0078] The molecular chaperone belonging to the ClpB family is derived from the genus Cupriavidus or the genus Escherichia.

[0079] [Item 4]

[0080] The transformed microorganism according to item 3, wherein

[0081] The molecular chaperone belonging to the ClpB family is derived from Cupriavidus necator or Eschericia coli.

[0082] [Item 5]

[0083] The transformed microorganism according to item 1 or 2, wherein

[0084] The transformed microorganism belongs to the genus Cupriavidus.

[0085] [Item 6]

[0086] The transformed microorganism according to any one of items 1 to 5, wherein a gene encoding PhaA derived from a thermophilic bacterium and / or a gene encoding PhaB derived from a thermophilic bacterium is introduced.

[0087] [Item 7]

[0088] The transformed microorganism according to item 6, wherein

[0089] The thermophilic bacteria is Cupriavidus sp. strain S-6.

[0090] [Item 8]

[0091] A method for producing a polyhydroxyalkanoate, comprising the step of culturing the transformed microorganism according to any one of items 1 to 7.

[0092] [Item 9]

[0093] The method for producing a polyhydroxyalkanoate according to item 8, wherein

[0094] Polyhydroxyalkanoate is a copolymer of two or more hydroxyalkanoic acids.

[0095] [Item 10] The method for producing a polyhydroxyalkanoate according to Item 9, wherein

[0096] The polyhydroxyalkanoate is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

[0097] Example

[0098] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0099] It should be noted that overall genetic manipulation can be performed, for example, as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Furthermore, enzymes and cloning hosts used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. It should be noted that any enzyme is not particularly limited as long as it can be used in genetic manipulation.

[0100] The KNK005 / dZ / trc-J4b strain used below is a strain that lacks the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Cupria necrotica strain H16; enhances the expression of the R-body-specific enoyl-CoA hydratase gene (phaJ4b gene) on the chromosome; and introduces a gene encoding a PHA synthase mutant from Aeromonas (N149S / D171G mutant (NSDG) gene) having the amino acid sequence set forth in SEQ ID NO: 19. This strain can be produced according to the method described in International Publication No. 2015 / 115619.

[0101] (Microorganism Preparation Example 1)

[0102] First, a plasmid for expressing the molecular chaperone (ClpB) gene was prepared as follows.

[0103] A DNA fragment (SEQ ID NO: 17) containing the lacN17 promoter, a variant of the Escherichia coli lac promoter, was obtained by PCR using synthetic oligoDNA. This DNA fragment was digested with restriction endonucleases EcoRI and MunI, and the resulting DNA fragment was ligated with the plasmid vector pCUP2 described in International Publication No. 2007 / 049716 cleaved with MunI. The product in which the digested DNA fragment and the cleaved pCUP2 were ligated in such a way that the pCUP2 restriction endonuclease SpeI recognition sequence was located downstream of the lacN17 promoter was screened, thereby obtaining pCUP2-lacN17.

[0104] Next, PCR using synthetic oligoDNA yielded a DNA fragment (SEQ ID NO: 18) having the base sequence encoding the ClpB gene having the amino acid sequence described in SEQ ID NO: 1. This DNA fragment was digested with restriction endonucleases MunI and SpeI, and the resulting DNA fragment was ligated with a product obtained by cleaving pCUP2-lacN17 with MunI and SpeI, thereby obtaining the ClpB gene expression plasmid pCUP2-lacN17-ClpBre.

[0105] Next, a ClpB 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 strain, and the resulting strain was named KNK005 / dZ / trc-J4b / pCUP2-lacN17-ClpBre strain (hereinafter sometimes referred to as "PHA-producing microbial strain (1)").

[0106] The plasmid vector was introduced into the cells by electroporation as described below. The gene introduction device used was a Gene Pulser manufactured by Biorad, and the cuvette used was a gap 0.2 cm also manufactured by Biorad. 400 μl of competent cells and 20 μl of expression vector were injected into the cuvette, and placed in a pulse device. Electric pulses were applied under the conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance of 800 Ω. After the pulse, the bacterial liquid in the cuvette was shaken and cultured at 30°C for 3 hours in Nutrient Broth medium (manufactured by DIFCO), and cultured at 30°C for 2 days in a selection plate (Nutrient Agar medium (manufactured by DIFCO), kanamycin 100 mg / L), thereby obtaining a growing PHA-producing microbial strain (1).

[0107] The PHA-producing microbial strain (1) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19 is introduced; and the expression of the gene encoding ClpB derived from the genus Cupriavidus having the amino acid sequence recorded in sequence number 1 is enhanced.

[0108] (Microorganism Preparation Example 2)

[0109] First, a plasmid for expressing the molecular chaperone (ClpB) gene was prepared. Preparation was performed as follows. PCR using synthetic oligoDNA yielded a DNA fragment (SEQ ID NO: 20) containing the base sequence encoding the ClpB gene having the amino acid sequence described in SEQ ID NO: 2. This DNA fragment was digested with the restriction endonucleases MunI and SpeI, and the resulting DNA fragment was ligated with the product of cleavage of pCUP2-lacN17 with MunI and SpeI, yielding the ClpB gene expression plasmid pCUP2-lacN17-ClpBec.

[0110] 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 described above. The resulting strain was named KNK005 / dZ / trc-J4b / pCUP2-lacN17-ClpBec strain (hereinafter sometimes referred to as "PHA-producing microbial strain (2)").

[0111] The PHA-producing microbial strain (2) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19 and a gene encoding ClpB derived from the genus Eschrichia having the amino acid sequence recorded in sequence number 2 are introduced.

[0112] (Microorganism Preparation Example 3)

[0113] First, a plasmid for expressing a molecular chaperone (GroESL) gene was prepared. Preparation was performed as follows. Using PCR with synthetic oligoDNA, a DNA fragment (SEQ ID NO: 23) containing the base sequence encoding the genes for GroES having the amino acid sequence described in SEQ ID NO: 21 and GroEL having the amino acid sequence described in SEQ ID NO: 22 was obtained. This DNA fragment was digested with the restriction endonucleases MunI and SpeI, and the resulting DNA fragment was ligated with the product of cleaving pCUP2-lacN17 with MunI and SpeI to obtain the GroESL gene expression plasmid pCUP2-lacN17-GroESL.

[0114] 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 described above. The resulting strain was named KNK005 / dZ / trc-J4b / pCUP2-lacN17-GroESL strain (hereinafter sometimes referred to as "PHA-producing microbial strain (3)").

[0115] The PHA-producing microbial strain (3) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19 and a gene encoding GroESL derived from the genus Cupriavidus having the amino acid sequence recorded in sequence numbers 21 and 22 are introduced.

[0116] (Microorganism Preparation Example 4)

[0117] First, a molecular chaperone (DnaKJ) gene expression plasmid was produced. Production was performed as described below. By using PCR with synthetic oligo DNA, a DNA fragment (SEQ ID NO: 26) containing the base sequence of genes encoding DnaK with the amino acid sequence recorded in SEQ ID NO: 24 and DnaJ with the amino acid sequence recorded in SEQ ID NO: 25 was obtained. This DNA fragment was digested with restriction endonucleases MunI and SpeI, and the resulting DNA fragment was ligated to the product of pCUP2-lacN17 after MunI and SpeI had cut off, thereby obtaining the DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ.

[0118] 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 described 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)").

[0119] The PHA-producing microbial strain (4) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19 and a gene encoding DnaKJ derived from the genus Eschrichia having the amino acid sequence recorded in sequence numbers 24 and 25 are introduced.

[0120] (Microorganism Preparation Example 5)

[0121] First, plasmids for phaA and phaB gene disruption were prepared as follows.

[0122] PCR using synthetic oligoDNA yielded a DNA fragment (SEQ ID NO: 27) containing the upstream and downstream base sequences of the phaA and phaB structural genes of C. necrotica strain H16. This DNA fragment was digested with the restriction endonuclease SwaI and ligated with DNA ligase (Ligation High, manufactured by Toyobo Co., Ltd.) to the vector pNS2X-sacB described in Japanese Patent Application Publication No. 2007-259708, which had also been digested with SwaI, to create the phaAB gene disruption plasmid vector pNS2X-sacB+phaABUD.

[0123] Next, using the phaAB gene disruption plasmid vector pNS2X-sacB+phaABUD, a phaAB gene-disrupted strain was prepared as follows.

[0124] Escherichia coli S17-1 strain (ATCC47055) was transformed with the phaAB gene disruption plasmid vector pNS2X-sacB+phaABUD, and the resulting transformed microorganism was co-cultured with the KNK005 / dZ / trc-J4b strain on NutrientAgar medium (Difco) for conjugation transfer.

[0125] The culture solution obtained is inoculated in Simmons ' 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 is 6.8) of the kanamycin that comprises 250mg / L, be selected in the bacterial strain that grows on agar medium, obtained the bacterial strain that has imported plasmid on the chromosome of KNK005 / dZ / trc-J4b strain.After this bacterial strain is carried out two generations of cultivation with Nutrient Broth medium (Difco company system), diluted and coated on the Nutrient Agar medium that comprises 15% sucrose, the bacterial strain of growth is obtained as the bacterial strain after plasmid comes off.Further by the analysis based on PCR and DNA sequencer, separated 1 strain and lacked the bacterial strain of the phaAB gene on the chromosome.This gene disruption strain is called after as KNK005 / dZ / trc-J4b / dphaAB strain.

[0126] Furthermore, plasmids for introducing the phaA and phaB genes were prepared as follows.

[0127] PCR using synthetic oligoDNA yielded a DNA fragment (SEQ ID NO: 28) containing the following base sequences: the upstream and downstream base sequences of the phaA and phaB structural genes of the Cupria necrotica strain H16, the gene encoding PhaA having the amino acid sequence described in SEQ ID NO: 4, and the gene encoding PhaB having the amino acid sequence described in SEQ ID NO: 7. This DNA fragment was digested with the restriction endonuclease SwaI, and the resulting DNA fragment was ligated with DNA ligase (Ligation High, manufactured by Toyobo Co., Ltd.) to the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708, which had also been digested with SwaI, to create the plasmid vector pNS2X-sacB+phaAU-phaABsp-phaBD for introducing the phaA and phaB genes.

[0128] Next, the phaA and phaB genes were introduced into the KNK005 / dZ / trc-J4b / dphaAB strain using the same conjugative transfer method as described above, using the plasmid vector pNS2X-sacB+phaAU-phaABsp-phaBD. Further, through the same culture and selection using Nutrient Agar medium containing 15% sucrose, a strain harboring the genes encoding PhaA with the amino acid sequence set forth in SEQ ID NO: 4 and PhaB with the amino acid sequence set forth in SEQ ID NO: 7 was isolated. The resulting strain was designated KNK005 / dZ / trc-J4b / dphaAB::phaABsp.

[0129] Furthermore, using the ClpB gene expression plasmid pCUP2-lacN17-ClpBre, a ClpBre expression-enhanced strain was prepared as follows.

[0130] 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 described above, and the resulting strain was named KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-ClpBre strain (hereinafter sometimes also described as "PHA-producing microbial strain (5)").

[0131] The PHA-producing microbial strain (5) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene and phaAB gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19, and a phaA gene having the amino acid sequence recorded in sequence number 4 derived from Cupriavidus sp. strain S-6 and a phaB gene having the amino acid sequence recorded in sequence number 7 are introduced; and the expression of the gene encoding ClpB derived from the genus Cupriavidus having the amino acid sequence recorded in sequence number 1 is enhanced.

[0132] (Microorganism Preparation Example 6)

[0133] Furthermore, using the ClpB gene expression plasmid pCUP2-lacN17-ClpBec, a ClpBec-introduced strain was prepared as follows.

[0134] 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 described above, and the resulting strain was named KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-ClpBec strain (hereinafter sometimes referred to as "PHA-producing microbial strain (6)").

[0135] The PHA-producing microbial strain (6) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene and phaAB gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19, a phaA gene having the amino acid sequence recorded in sequence number 4 derived from Cupriavidus sp. strain S-6 and a phaB gene having the amino acid sequence recorded in sequence number 7, and a gene encoding ClpB derived from the genus Escherichia having the amino acid sequence recorded in sequence number 2 are introduced.

[0136] (Microorganism Preparation Example 7)

[0137] Furthermore, using the GroESL gene expression plasmid pCUP2-lacN17-GroESL, a GroESL-transfected strain was prepared as follows.

[0138] 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 described above, and the resulting strain was named KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-GroESL strain (hereinafter sometimes also referred to as "PHA-producing microbial strain (7)").

[0139] The PHA-producing microbial strain (7) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene and phaAB gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19, the phaA gene having the amino acid sequence recorded in sequence number 4 and the phaB gene having the amino acid sequence recorded in sequence number 7 derived from Cupriavidus sp. strain S-6, and a gene encoding GroESL derived from the genus Cupriavidus having the amino acid sequence recorded in sequence numbers 21 and 22 are introduced.

[0140] (Microorganism Preparation Example 8)

[0141] Furthermore, using the DnaKJ gene expression plasmid pCUP2-lacN17-DnaKJ, a DnaKJ-transfected strain was prepared as follows.

[0142] 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 described above, and the resulting strain was named KNK005 / dZ / trc-J4b / dphaAB::phaABsp / pCUP2-lacN17-DnaKJ strain (hereinafter sometimes also described as "PHA-producing microbial strain (8)").

[0143] The PHA-producing microbial strain (8) is a strain as described below: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene, phaZ6 gene and phaAB gene on the chromosome of the H16 strain of Coptis nebulosa are deleted; the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced; a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 19, the phaA gene having the amino acid sequence recorded in sequence number 4 and the phaB gene having the amino acid sequence recorded in sequence number 7 derived from Cupriavidus sp. strain S-6, and a gene encoding DnaKJ derived from the genus Escherichia having the amino acid sequence recorded in sequence numbers 24 and 25 are introduced.

[0144] (Example 1) PHA production based on PHA-producing microbial strain (1)

[0145] Cultivation studies using the PHA-producing microbial strain (1) were conducted under the following conditions.

[0146] (Measurement method of PHA accumulation)

[0147] The ratio of PHA accumulation to dry cells was determined as follows. The cells were recovered from the culture medium by centrifugation, washed with ethanol, freeze-dried, and dried cells were obtained, and the weight was measured. 100 ml of chloroform was added to 1 g of the obtained dry cells, and the cells were stirred at room temperature for a day and a night to extract the PHA (PHA mixture) in the cells. After filtering and separating the cell residue, the cells were concentrated to a total volume of 30 ml using a rotary evaporator, and then 90 ml of hexane was slowly added and allowed to stand for 1 hour while slowly stirring. The precipitated PHA was separated by filtration and vacuum dried at 50°C for 3 hours. The weight of the dry PHA was measured to calculate the PHA production.

[0148] (PHA production culture in flasks)

[0149] The composition of the seed culture medium was set to 10 g / L meat extract, 10 g / L Bacto-Trypton, 2 g / L yeast extract, 9 g / L sodium dihydrogen phosphate dodecahydrate, 1.5 g / L potassium dihydrogen phosphate, and 100 μg / L kanamycin sulfate.

[0150] The composition of the PHA production medium was 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 0.1N hydrochloric acid.

[0151] 50 μL of the 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 the seed culture medium and cultured with shaking at 30° C. for 24 hours. The resulting culture solution was used as a pre-culture solution.

[0152] PHA production culture was carried out using flasks. 50 mL of PHA production medium was added to a 500 mL shaking 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 the culture medium was prepared, 500 μL of the pre-culture solution was inoculated into the shaking flask and cultured with shaking at 36°C for 72 hours. After the culture was completed, PHA production was measured as described above. The PHA production of Comparative Example 1, in which the KNK005 / dZ / trc-J4b strain was cultured under the same conditions, was used as a baseline value, and the PHA production was shown in Table 1 as a relative value relative to this baseline value.

[0153] (Example 2) PHA production by PHA-producing microbial strain (2)

[0154] Cultivation studies using the PHA-producing microbial strain (2) were conducted under the same conditions as in Example 1, and PHA production was measured as described above. Similarly to Example 1, PHA production was shown in Table 1 as a relative value to the reference value.

[0155]

[0156] (Comparative Examples 1 to 3)

[0157] Cultivation studies were conducted 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 PHA production was measured as described above. PHA production was performed as in Example 1, and is shown in Table 1 as a relative value relative to the baseline value.

[0158] The following can be seen from Table 1. When comparing Examples 1 and 2 with Comparative Example 1, it can be seen that the PHA production in Examples 1 and 2 was improved compared to Comparative Example 1. Therefore, it is believed that PHA productivity can be improved by introducing a gene encoding a molecular chaperone belonging to the ClpB family or enhancing its expression.

[0159] Furthermore, when comparing Examples 1 and 2 with Comparative Examples 2 and 3, it was found that Examples 1 and 2 had higher PHA production than Comparative Examples 2 and 3. Therefore, it is believed that the introduction of a gene encoding a molecular chaperone belonging to the ClpB family or the enhancement of its expression has a greater effect on improving PHA productivity than the introduction of a gene encoding other molecular chaperones, such as GroESL or DnaKJ, or the enhancement of their expression.

[0160] (Example 3) PHA production based on PHA-producing microbial strain (5)

[0161] Cultivation studies using the PHA-producing microbial strain (5) were conducted under the following conditions.

[0162] (PHA production culture under high-density culture)

[0163] The composition of the mother culture medium was set to 1 w / v% Meat-extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast-extract, 0.9 w / v% Na2HPO4･12H2O, and 0.15 w / v% KH2PO4 (pH 6.8).

[0164] The composition of the pre-culture medium was set to 1.1w / v% Na2HPO4･12H2O, 0.19w / v% KH2PO4, 1.29w / v% (NH4)2SO4, 0.1w / v% MgSO4･7H2O, 2.5w / v% palm olein, and 0.5v / v% trace metal salt solution (1.6w / v% FeCl3･6H2O, 1w / v% CaCl2･2H2O, 0.02w / v% CoCl2･6H2O, 0.016w / v% CuSO4･5H2O, and 0.012w / v% NiCl2･6H2O dissolved in 0.1N hydrochloric acid).

[0165] The composition of the PHA production medium was set to 0.385w / v% Na2HPO4･12H2O, 0.067w / v% KH2PO4, 0.291w / v% (NH4)2SO4, 0.1w / v% MgSO4･7H2O, and 0.5v / v% trace metal salt solution (1.6w / v% FeCl3･6H2O, 1w / v% CaCl2･2H2O, 0.02w / v% CoCl2･6H2O, 0.016w / v% CuSO4･5H2O, and 0.012w / v% NiCl2･6H2O dissolved in 0.1N hydrochloric acid).

[0166] PHA production culture was carried out as described below. First, the glycerol raw material (50 μl) of the PHA production microbial strain (5) was inoculated into the mother culture medium (10 ml) and cultured for 24 hours, thereby carrying out the mother culture. Then, the mother culture solution was inoculated at 1.0 v / v% into a 3 L Jar fermenter (MDL-300 type manufactured by Marubi Bioengineering) to which 1.8 L of pre-culture medium was added. The operating conditions were set to a culture temperature of 30°C, a stirring speed of 500 rpm, and a ventilation volume of 1.8 L / min, and the pH was controlled between 6.7 and 6.8 and cultured for 28 hours, thereby carrying out the pre-culture. A 14% ammonium hydroxide aqueous solution was used for pH control.

[0167] Next, the pre-culture solution was inoculated at 5.0 v / v% into a 5 L Jarfermenter (MDS-U50 manufactured by Marubi Bioengineering) to which 2.5 L of PHA production medium was added. The operating conditions were set to a culture temperature of 36°C, a stirring speed of 420 rpm, an aeration volume of 2.1 L / min, and the pH was controlled between 6.7 and 6.8. A 25% aqueous ammonium hydroxide solution was used for pH control. A carbon source was added intermittently. Palm olein was used as a carbon source. The culture was carried out for 48 hours. The PHA production was determined as described above. The PHA production of Comparative Example 4, in which the KNK005 / dZ / trc-J4b strain was cultured under the same conditions, was used as a baseline value, and the PHA production was shown in Table 2 as a relative value relative to the baseline value.

[0168] (Example 4) PHA production based on PHA-producing microbial strain (6)

[0169] Cultivation studies using the PHA-producing microbial strain (6) were conducted under the same conditions as in Example 3, and PHA production was measured as described above. The PHA production was performed as in Example 3 and is shown in Table 2 as a relative value to the reference value.

[0170] (Comparative Examples 4 to 7)

[0171] Cultivation studies were conducted 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 PHA production was measured as described above. PHA production was measured as in Example 3, as relative to the baseline value, and is shown in Table 2.

[0172]

[0173] The following can be seen from Table 2. When comparing Examples 3 and 4 with Comparative Example 5, it can be seen that the PHA production amounts in Examples 3 and 4 were higher than in Comparative Example 5. This suggests that PHA productivity is improved by introducing a gene encoding a molecular chaperone belonging to the ClpB family or enhancing its expression.

[0174] Furthermore, when comparing Examples 3 and 4 with Comparative Examples 6 and 7, it was found that Examples 3 and 4 had higher PHA productivity than Comparative Examples 6 and 7. This indicates that the introduction of a gene encoding a molecular chaperone belonging to the ClpB family or the enhancement of its expression is more effective in improving PHA productivity than the introduction of a gene encoding other molecular chaperones, such as GroESL or DnaKJ, or the enhancement of their expression.

Claims

1. A transformed microorganism having the ability to produce polyhydroxyalkanoate, The transformed microorganism has a polyhydroxyalkanoate synthase gene, The transformed microorganism has a gene encoding a molecular chaperone belonging to the ClpB family introduced therein, or its expression is enhanced.

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

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

5. The transformed microorganism according to claim 1 or 2, wherein The transformed microorganism belongs to the genus Cupriavidus. The transformed microorganism according to claim 1 or 2, wherein a gene encoding PhaA derived from a thermophilic bacterium and / or a gene encoding PhaB derived from a thermophilic bacterium has been introduced.

7. The transformed microorganism according to claim 6, wherein The thermophilic bacteria is Cupriavidus sp. strain S-6.

8. A method for producing polyhydroxyalkanoate, comprising the step of culturing the transformed microorganism according to claim 1 or 2.

9. The method for producing a polyhydroxyalkanoate according to claim 8, wherein Polyhydroxyalkanoate is a copolymer of two or more hydroxyalkanoic acids.

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

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