Method for producing polyhydroxyalkanoate

Abstract

Polyhydroxyalkanoate-producing microorganisms are cultured to obtain bacterial cells that accumulate polyhydroxyalkanoate particles and have an average cell diameter of 2 μm or greater. The bacterial cells are then heat-treated to reduce the average particle size of the polyhydroxyalkanoate particles within the bacterial cells to 1.8 μm or greater and the average cell diameter of the particles is not greater. Furthermore, the heat-treated bacterial cells are disrupted, and PHA particles can be separated from the aqueous phase of the resulting cell disrupted liquid.

Description

Technical Field

[0001] The present invention relates to a method for producing polyhydroxyalkanoate by culturing a polyhydroxyalkanoate-producing microorganism. Background Art

[0002] Against the backdrop of growing awareness of environmental issues, food security, health and safety, and a growing desire for naturalness, the significance and importance of microbial-based material production (fermentation, biotransformation, etc.) are increasing. Microbial-based material production is also being applied to the production of protein pharmaceuticals and nucleic acids for gene therapy. For example, the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria is actively being used in industry.

[0003] As an example thereof, the production of polyhydroxyalkanoates (hereinafter also referred to as PHA) based on microorganisms can be cited, and the polyhydroxyalkanoates are expected to be industrially utilized as biodegradable plastics (refer to non-patent literature 1). PHA is a thermoplastic polyester produced and accumulated as an energy storage substance in the cells (hereinafter also referred to as bacteria) of multiple microbial species, and is biodegradable. At present, with the improvement of environmental awareness, plastics derived from non-petroleum have attracted much attention, especially PHA produced and accumulated by microorganisms in bacteria will be incorporated into the carbon cycle process in nature, so it is expected that the adverse effects on the ecosystem will be small, and its practical application is urgently desired. In the production of PHA using microorganisms, it is known that, for example, sugars, vegetable oils and fatty acids are given to bacteria of the genus Cupriaps as a carbon source, so that PHA is accumulated in the cells, thereby producing PHA (refer to non-patent literature 2 and 3).

[0004] However, in the production of substances using microorganisms, the separation and recovery of target products is complicated, which can increase production costs. Therefore, improving the separation and recovery efficiency of target products is a major issue in reducing production costs.

[0005] Non-Patent Document 4 reports that disruption of the phaP1 gene encoding the Phasin protein in Cupriavidus bacteria resulted in the accumulation of PHA particles of larger sizes than in the non-disrupted strain. However, this phaP1-disrupted strain showed a significant decrease in PHA accumulation, making it unsuitable for industrial production.

[0006] Non-Patent Document 5 also describes that applying temperature, pH, and other stimuli to PHA-accumulating bacteria causes PHA to aggregate within the cells. However, microscopic images only reveal a small number of PHA particles adhering to each other within the cells, and no changes in the average particle size of the PHA particles can be detected. Furthermore, the literature suggests that the effect on the average particle size of the PHA particles is limited, given the overall small size of the cells themselves.

[0007] Prior art literature

[0008] Non-patent literature

[0009] Non-patent document 1: Anderson AJ., et al., Int. J. Biol. Macromol., 12, 102-105 (1990)

[0010] Non-patent document 2: Sato S., et al., J. Biosci. Bioeng., 120(3), 246-251 (2015)

[0011] Non-patent document 3: Insomphun C., et al., Metab. Eng., 27, 38-45 (2015)

[0012] Non-patent document 4: Potter M., et al., Microbiology, 151 (Pt 3), 825-833 (2005)

[0013] Non-patent literature 5: Sedlacek P., et al., Appl. Microbiol. Biotechnol., 103(4), 1905-1917 (2019) Summary of the Invention

[0014] Problems to be solved by the invention

[0015] PHA accumulates in the form of particles inside microbial cells. In order to utilize PHA accumulated inside microbial cells as a biodegradable plastic, it is necessary to disrupt the cells, remove the PHA particles, separate them from other cell components and recover them. Separation and recovery methods can be roughly divided into methods based on organic solvent systems and methods based on water systems. The use of organic solvents will lead to high environmental burden and high costs, so methods based on water systems are preferred in industry. In the water-based method, for example, PHA particles can be separated from cell disrupted liquid containing PHA particles using a centrifuge, separation membrane, etc. In such a case, the efficiency of separation and recovery depends on the size of the PHA particles. That is, the larger the PHA particles before the separation process, the easier it is to implement separation and recovery using a centrifuge, separation membrane, etc., which can reduce production costs.

[0016] After disrupting microbial cells that have accumulated PHA particles, attempts have been made to agglomerate the PHA particles in the cell lysate before separation to increase their size. However, controlling the degree of agglomeration is difficult, and since PHA particles agglomerate while entraining impurities such as disrupted and fragmented cellular components, subsequent impurity removal is difficult. Therefore, agglomerating PHA particles in cell lysate is not suitable for industrial production.

[0017] In view of the above-mentioned situation, an object of the present invention is to provide a method for obtaining PHA particles having a large average particle size by aggregating PHA particles within microbial cells.

[0018] Solutions to Problems

[0019] The present inventors conducted intensive research and discovered that by culturing PHA-producing microorganisms to obtain bacterial cells that accumulate PHA particles and have an average cell diameter of 2 μm or greater, and then heat-treating these bacterial cells, PHA particles with an average particle diameter of 1.8 μm or greater can be formed within the bacterial cells. This led to the completion of the present invention.

[0020] Specifically, the present invention relates to a method for producing polyhydroxyalkanoate, comprising: culturing a polyhydroxyalkanoate-producing microorganism to obtain bacterial cells that accumulate polyhydroxyalkanoate particles and have an average cell diameter of 2 μm or greater; and heat-treating the bacterial cells at a temperature higher than the temperature during the culturing to reduce the average particle size of the polyhydroxyalkanoate particles in the bacterial cells to 1.8 μm or greater and below the average cell diameter.

[0021] The ratio of the average particle size of the polyhydroxyalkanoate particles after the heat treatment to the average particle size of the polyhydroxyalkanoate particles before the heat treatment is preferably 1.1 or more.

[0022] The weight ratio of the polyhydroxyalkanoate to the dry weight of the cultured bacterial cells is preferably 80% or more.

[0023] In the particle size distribution of the polyhydroxyalkanoate particles after the heat treatment, the proportion of polyhydroxyalkanoate particles having a particle diameter of 1 μm or less is preferably 2.0% by volume or less.

[0024] The average cell diameter of the bacterial cells accumulating the polyhydroxyalkanoate particles is preferably 2.2 μm or more.

[0025] The heat treatment is preferably performed at a temperature of 40 to 100° C. for 5 minutes or longer.

[0026] The heat treatment is preferably performed at a pH of 7.0 or higher.

[0027] The heat treatment is preferably performed on the culture solution containing the cultured bacterial cells.

[0028] Preferably, the production method further comprises: a step of disrupting the heat-treated bacterial cells to obtain a cell disrupted liquid; and a step of separating the polyhydroxyalkanoate particles from the aqueous phase of the cell disrupted liquid.

[0029] The polyhydroxyalkanoate is preferably a copolymer of two or more hydroxyalkanoic acids, more preferably a copolymer containing 3-hydroxyhexanoic acid as a monomer unit, and even more preferably a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0030] Preferably, the polyhydroxyalkanoate-producing microorganism belongs to the genus Cupriavidus, and more preferably, the polyhydroxyalkanoate-producing microorganism is a transformed microorganism of Cupriavidus necator.

[0031] Effects of the Invention

[0032] The present invention provides a method for agglomerating PHA particles within microbial cells to obtain PHA particles with a large average particle size. According to the present invention, PHA particles with a large average particle size can be formed within microbial cells before disrupting the cells. This prevents the PHA particles from agglomerating and entraining impurities such as fragmented cellular components. Furthermore, PHA particles with a large average particle size can be efficiently separated and recovered from cellular components, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are microscopic photographs of cells after culture and before (left) or after (right) heat treatment in Example 1.

[0034] Figure 2 These are microscopic photographs of cells after culture and before (left) or after (right) heat treatment in Example 2. DETAILED DESCRIPTION

[0035] Hereinafter, embodiments of the present invention will be described in detail.

[0036] This embodiment includes the steps of culturing PHA-producing microorganisms to obtain bacterial cells that accumulate PHA particles and have an average cell diameter of 2 μm or greater; and heat-treating the bacterial cells to form PHA particles within the bacterial cells that have an average particle diameter of 1.8 μm or greater. It should be noted that the average particle diameter in this application refers to the volume average particle diameter.

[0037] (PHA-producing microorganisms)

[0038] There are no particular limitations on PHA-producing microorganisms as long as they have the ability to accumulate PHA and can achieve an average cell diameter of 2 μm or more after PHA accumulation. The average cell diameter of the microorganism does not need to be always above 2 μm during cultivation, as long as it reaches 2 μm or more during the PHA accumulation stage before the microorganism is subjected to the heat treatment process. When the average cell diameter after PHA accumulation is less than 2 μm, it is difficult to form PHA particles with an average particle diameter of 1.8 μm or more even if the heat treatment described later is performed. The above-mentioned average cell diameter is more preferably above 2.2 μm, further preferably above 2.4 μm, further preferably above 2.6 μm, and particularly preferably above 2.8 μm. The upper limit of the above-mentioned average cell diameter is not particularly limited, and for example, it can be below 10 μm, and can also be below 5 μm.

[0039] The amount of PHA accumulated by the PHA-producing microorganism is not particularly limited. However, during the PHA accumulation stage prior to the heat treatment step, the proportion of PHA by weight relative to the dry weight of the microorganism is preferably 80% or greater, more preferably 85% or greater. The upper limit of this proportion is not particularly limited as long as it is less than 100%, and may be, for example, 98% or less, or 95% or less.

[0040] The PHA-producing microorganism is not particularly limited as long as it has a PHA synthase gene and accumulates PHA. Preferred examples include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Aeromonas, Escherichia, Alcaligenes, and Pseudomonas. From the viewpoint of safety and PHA productivity, bacteria belonging to the genera Ralstonia, Cupriavidus, Aeromonas, and Wautersia are more preferred, bacteria belonging to the genera Cupriavidus or Aeromonas are even more preferred, bacteria belonging to the genus Cupriavidus are even more preferred, and Cupriavidus necator is particularly preferred.

[0041] The PHA-producing microorganism may be a wild strain that originally accumulates PHA, a mutant obtained by artificially mutating such a wild strain, or a strain endowed with the ability to accumulate PHA by introducing a foreign PHA synthase gene through genetic engineering.

[0042] Examples of the PHA-producing microorganism include, but are not limited to, a minCD expression A2405 disrupted strain and an A1386 deletion disrupted strain, which are transformants of Cupriavidus necrotica described later.

[0043] The type of PHA produced by the PHA-producing microorganism 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 one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (for example, 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, 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. 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), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviated as 3HH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviated as 3HH), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviated as 4HB), and PHAs containing lactic acid (abbreviated as LA) as a component, such as a copolymer of 3HB and LA, P(LA-co-3HB). Of these, PHBH is preferred from the perspective of a wide range of applications as a polymer. The type of PHA to be produced can be appropriately selected depending on the intended purpose, such as the type of PHA synthase gene possessed by the microorganism to be used or introduced separately, the type of genes involved in the metabolic system involved in its synthesis, and the culture conditions.

[0044] (Cultivation of PHA-producing microorganisms)

[0045] By culturing the PHA-producing microorganisms described above, PHA particles can be accumulated within the microorganisms. Cultivation methods can be performed according to conventional microbial cultivation methods, as long as the culture is performed in a culture medium containing an appropriate carbon source. The culture medium composition, carbon source addition method, culture scale, aeration and stirring conditions, culture temperature, and culture time are not particularly limited. To ensure sufficient PHA accumulation, it is preferred that the carbon source be added to the culture medium continuously or intermittently.

[0046] Any carbon source can be used during cultivation as long as the PHA-producing microorganism can assimilate it. Examples include sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil, palm kernel oil (including low-melting-point fractions such as palm olein, palm diolein, and palm kernel olein obtained by fractionation), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil; their fractions; and purified byproducts; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid; and their derivatives; and glycerol, without particular limitation. Furthermore, if the PHA-producing microorganism can utilize gases such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, or alcohols, these gases can also be used as carbon sources.

[0047] When culturing the PHA-producing microorganisms, it is preferred to use a culture medium containing the carbon source, a nitrogen 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, but are not limited to, 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.

[0048] (Heat Treatment)

[0049] By heat-treating microbial cells that accumulate PHA particles and have an average cell diameter of 2 μm or greater at a temperature higher than the temperature at which the microbial cells were cultured, the average particle size of the PHA particles within the microbial cells can be increased. This heat treatment can be performed before disrupting the microbial cells and can be performed on a culture solution containing bacterial cells after culturing the PHA-producing microorganisms, or on a suspension obtained by recovering bacterial cells from the culture solution and resuspending them in water, a buffer solution, or the like. Heat treatment of the culture solution after culturing is preferred due to its ease of implementation.

[0050] The heat treatment conditions are not particularly limited as long as they can achieve an average particle size of PHA particles of 1.8 μm or greater within the microbial cells. However, conditions that disrupt the cytoskeleton and prevent PHA from leaking out of the cells are preferred. Specifically, the heat treatment temperature is higher than the temperature at which the microbial cells are cultured, preferably 40°C or higher, more preferably 50°C or higher, even more preferably 60°C or higher, and even more preferably 70°C or higher. The upper limit of the heat treatment temperature is not particularly limited, but can be, for example, 100°C or lower, preferably 90°C or lower.

[0051] The time for the heat treatment is preferably 5 minutes or more, more preferably 30 minutes or more, further preferably 180 minutes or more, and further preferably 360 minutes or more. The upper limit of the temperature for the heat treatment is not particularly limited, and may be, for example, 1 day or less, preferably 720 minutes or less.

[0052] During the heat treatment, the pH of the liquid containing the bacterial cells (e.g., a culture solution containing the bacterial cells or a suspension containing the bacterial cells) is not particularly limited and may be less than 7.0 or greater. However, from the perspective of increasing the average particle size of the PHA particles by heat treatment, it is preferably greater than 7.0, more preferably greater than 7.5, even more preferably greater than 8.0, and even more preferably greater than 8.5. The pH is not particularly limited to an upper limit as long as it does not disrupt the bacterial cells. For example, it may be less than 12, and preferably less than 11. It should be noted that the pH can be controlled by adding an appropriate amount of an acid, base, or the like to the liquid containing the bacterial cells.

[0053] The average particle size of the PHA particles in the heat-treated bacterial cells is not particularly limited as long as it is 1.8 μm or larger and smaller than the average cell diameter. The average particle size is preferably 1.9 μm or larger, more preferably 2.0 μm or larger, and even more preferably 2.1 μm or larger.

[0054] The heat treatment can increase the average particle size of the PHA particles within the bacterial cells. Specifically, the ratio of the average particle size of the PHA particles within the bacterial cells after the heat treatment to the average particle size of the PHA particles within the bacterial cells before the heat treatment (average particle size after heat treatment / average particle size before heat treatment) is greater than 1.0, preferably 1.1 or greater, more preferably 1.2 or greater, even more preferably 1.3 or greater, even more preferably 1.4 or greater, and most preferably 1.5 or greater.

[0055] Because the PHA particles aggregate within the bacterial cells during the heat treatment, the proportion of PHA particles with a small particle size relative to the total number of PHA particles is relatively small. Specifically, in the particle size distribution of the PHA particles within the bacterial cells after the heat treatment, the proportion of PHA particles with a particle size of 1 μm or less relative to the total number of PHA particles is preferably 2.5% by volume or less, more preferably 2.0% by volume or less, even more preferably 1.5% by volume or less, even more preferably 1.0% by volume or less, and particularly preferably 0.5% by volume or less.

[0056] In this embodiment, PHA particles accumulate and the average cell diameter of the bacterial cells is as large as 2 μm or larger. However, for bacterial cells with such large cell diameters, the proportion of small PHA particles before heat treatment tends to be high. Thus, even if the proportion of small PHA particles is high before heat treatment (e.g., 3.0% or more by volume or 4.0% or more by volume), heat treatment can reduce it to the aforementioned lower value. Thus, according to this embodiment, the reduction in the proportion of small PHA particles can provide the advantage of more efficient separation and recovery of PHA particles from cellular components.

[0057] (Cell disruption and PHA separation and recovery)

[0058] After the bacterial cells accumulating PHA particles are heat-treated as described above to increase the average particle size of the PHA particles within the bacterial cells, the bacterial cells can be disrupted using a known method, and the PHA particles can be separated and recovered from the aqueous phase of the resulting cell disrupted liquid.

[0059] The method for disrupting the bacterial cells is not particularly limited, and a known method can be applied. For example, cells can be disrupted by applying mechanical shear force or using a surfactant, alkali, enzyme, etc. to obtain a cell disrupted liquid in which cell components other than PHA are dissolved in water.

[0060] The method for separating and recovering PHA is not particularly limited, and known methods can be applied. For example, PHA particles can be separated from the aqueous phase by filtration and centrifugation of the cell lysate, followed by drying to recover PHA. The PHA particles having a large average particle size produced by this embodiment are preferred because they enable efficient separation and recovery in an aqueous system.

[0061] Example

[0062] The present invention will be described in more detail below using examples. However, the present invention is not limited to these examples. It should be noted that all genetic manipulations can be performed as described in, for example, Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). In addition, enzymes and cloning hosts used in genetic manipulations 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 manipulations.

[0063] (Production Example 1) Preparation of minCD-expressing A2405 disrupted strain

[0064] First, a plasmid for gene deletion was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 1) having the base sequences upstream and downstream of the A2405 structural gene was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which was also digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+A2405UD for gene deletion having the base sequences upstream and downstream of the A2405 structural gene.

[0065] Next, using the gene deletion plasmid vector pNS2X-sacB+A2405UD, an A2405 deletion disrupted strain was prepared as follows.

[0066] Escherichia coli S17-1 strain (ATCC47055) was transformed with the gene-deleted plasmid vector pNS2X-sacB+A2405UD, and the resulting transformed microorganism was mixed with the KNK-005 strain on Nutrient Agar medium (manufactured by Difco) for conjugation transfer. The KNK-005 strain is a transformant of the chromosome of the Cupria necrotica H16 strain into which a PHA synthase gene (a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2) derived from Aeromonas caviae was introduced, and can be prepared based on the method described in the specification of U.S. Patent No. 7,384,766.

[0067] The obtained cultured bacteria were inoculated into Simmons' agar medium (sodium citrate 2g / L, sodium chloride 5g / L, magnesium sulfate heptahydrate 0.2g / L, ammonium dihydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, agar 15g / L, pH 6.8) containing 250mg / L of kanamycin, and strains capable of growing on agar medium were selected to obtain a strain into which a plasmid was introduced on the chromosome of the KNK-005 strain. After the strain was cultured for two generations using Nutrient Broth medium (manufactured by Difco), it was diluted and spread on Nutrient Agar medium containing 15% sucrose, and the growing strain was obtained as a strain after plasmid shedding. Further, through analysis based on PCR and DNA sequencer, a strain was isolated in which the start codon to the stop codon of the A2405 structural gene on the chromosome was deleted. The A2405 gene deletion strain was named A2405 deletion destruction strain.

[0068] Next, the plasmid vector pNS2X-sacB-PA-minCD for expression of the minCD gene was prepared. Preparation was performed as follows. By PCR using synthetic oligoDNA, a DNA fragment (SEQ ID NO: 3) having a promoter sequence, the minCD gene sequence, and the base sequence of the introduction region on the genome was obtained. This DNA fragment was digested with the restriction endonuclease SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Laid-Open No. 2007-259708, which had also been digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-PA-minCD for expression of the minCD gene.

[0069] The minCD gene expression plasmid vector pNS2X-sacB-PA-minCD, prepared using the same conjugative transfer method as described above, was introduced into the A2405 deletion-disrupted strain. Furthermore, through the same culture and selection using Nutrient Agar medium containing 15% sucrose, a strain containing the promoter sequence and minCD gene sequence inserted into the chromosome was isolated. The resulting strain was designated the minCD-expressing A2405 disruption strain.

[0070] (Production Example 2) Preparation of A1386 Deletion-Disrupted Strain

[0071] First, a plasmid for gene deletion was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 4) having the upstream and downstream base sequences of the A1386 structural gene was obtained. The DNA fragment was digested with the restriction endonuclease SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which had also been digested with SwaI, and DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+A1386UD for gene deletion having the upstream and downstream base sequences of the A1386 structural gene.

[0072] Next, the A1386 gene-deleting plasmid vector pNS2X-sacB+A1386UD was introduced into the KNK-005 strain using the same method as in Production Example 1. Furthermore, a strain was isolated in which the start codon to the stop codon of the A1386 structural gene on the chromosome was deleted using the same method as in Production Example 1. This A1386 gene-deleted strain was designated the A1386 deletion-disrupted strain.

[0073] (Comparative Example 1) PHA production based on KNK-005 strain

[0074] The KNK-005 strain was cultured under the following conditions.

[0075] (Culture medium)

[0076] The composition of the seed 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, 0.15 w / v% KH2PO4, (pH 6.8).

[0077] The composition of the pre-culture medium was set to 1.1 w / v% Na2HPO4·12H2O, 0.19 w / v% KH2PO4, 1.29 w / v% (NH4)2SO4, 0.1 w / v% MgSO4·7H2O, 2.5 w / v% palm olein, and 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl3·6H2O, 1 w / v% CaCl2·2H2O, 0.02 w / v% CoCl2·6H2O, 0.016 w / v% CuSO4·5H2O, and 0.012 w / v% NiCl2·6H2O dissolved in 0.1N hydrochloric acid). Palm olein was added as a carbon source at a concentration of 10 g / L.

[0078] The composition of the PHA production medium is 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).

[0079] (Method for measuring the ratio of PHA accumulation to dry bacterial cells)

[0080] The ratio of PHA accumulation to dry bacterial cells was determined as follows. The cells were recovered from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain dry cells, whose weight was measured. 100 ml of chloroform was added to 1 gram of the obtained dry bacterial cells, and the mixture was stirred at room temperature for one day and one night to extract the PHA from the cells. After filtering out the cell residue, the mixture was concentrated to a total volume of 30 ml using an evaporator. Then, 90 ml of hexane was gradually added and the mixture was allowed to stand for 1 hour while slowly stirring. The precipitated PHA was filtered out and vacuum-dried at 50°C for 3 hours. The weight of the dry PHA was measured, and the ratio of PHA accumulation to the dry bacterial cell volume was calculated.

[0081] (Method for measuring average cell diameter)

[0082] The average cell diameter was measured as follows. After incubation, the culture solution was treated at 60°C for 10 minutes to inactivate the bacterial cells. The volume average particle size (MV) of the PHA-accumulating cells was then measured using a laser diffraction / scattering particle size distribution analyzer (Microtrac MT3300EXII). The measurement was performed using standard settings (particle transmittance: transparent, particle refractive index: 1.81, particle shape: non-spherical, solvent refractive index: 1.333).

[0083] (Measurement method of PHA average particle size)

[0084] The average particle size of PHA was measured as follows. After the culture was completed, 0.2 ml of the culture medium before or after heat treatment was collected and suspended in 20 ml of a 0.02 w / v% benzalkonium chloride aqueous solution. 10 ml of a 10 w / v% sodium lauryl sulfate aqueous solution was further added and mixed, and a PHA extract was obtained by ultrasonication. The obtained PHA extract was analyzed using a laser diffraction / scattering particle size distribution analyzer (Microtrac MT3300EXII) to determine the volume average particle size (MV) of the PHA particles and the proportion (volume %) of PHA particles with a particle size of less than 1 μm relative to the total PHA particles. The measurement was performed using standard settings (particle transmittance: transparent, particle refractive index: 1.81, particle shape: non-spherical, solvent refractive index: 1.333).

[0085] (PHA production and cultivation)

[0086] PHA production culture was carried out as described below. First, a glycerol stock solution (50 μl) of the KNK-005 strain was inoculated into a seed culture medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture solution was inoculated into a 3L fermentor (Jar fermentor) (MDL-300 type manufactured by Marubishi Bioengineering Co., Ltd.) to which 1.8L pre-culture medium was added at 1.0 v / v%. The operating conditions were set to a culture temperature of 33°C, a stirring speed of 500 rpm, and an aeration volume of 1.8 L / min. The pre-culture was carried out by culturing for 28 hours while controlling the pH to between 6.7 and 6.8. A 14% aqueous ammonium hydroxide solution was used for pH control.

[0087] Next, the pre-culture solution was inoculated at 5.0 v / v% into a 5 L fermenter (MDS-U50 manufactured by Marubishi Bioengineering Co., Ltd.) to which 2.5 L of PHA production medium was added. The operating conditions were set as a culture temperature of 33°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 solution of ammonium hydroxide was used for pH control. A carbon source was added intermittently. Palm olein was used as a carbon source. The culture was carried out until the PHA accumulation reached more than 80% relative to the dry bacteria. The obtained culture solution was heat-treated at the time and temperature described in Table 1-1.

[0088] As described above, the ratio of PHA accumulation to dry bacterial cells, average cell diameter, average PHA particle size before and after heat treatment, and the ratio of PHA particles 1 μm or smaller before and after heat treatment were measured. Furthermore, the increase rate of PHA average particle size due to heat treatment (average PHA particle size after heat treatment / average PHA particle size before heat treatment) was calculated. The results are shown in Table 1-1.

[0089] The average cell diameter of the KNK-005 strain after culture was 1.89 μm, and the proportion of PHA accumulation relative to the dry bacterial cells was 89%. The average PHA particle size before heat treatment was 1.72 μm, and the proportion of PHA particles smaller than 1 μm before heat treatment was 2.45% by volume. In this comparative example, the average cell diameter of the PHA-producing microorganism after culture was 1.89 μm. Therefore, even with heat treatment, as shown in Table 1-1, the average PHA particle size did not increase, and the proportion of PHA particles smaller than 1 μm did not decrease substantially.

[0090] (Example 1) PHA production based on minCD expression A2405 disrupted strain

[0091] Cultivation of the minCD-expressing A2405 disrupted strain was carried out under the same conditions as in Comparative Example 1. The obtained culture solution was heat-treated at the time and temperature described in Table 1-1. The ratio of the PHA accumulation to the dry bacterial body, the average cell diameter, the average PHA particle size before and after heat treatment, the ratio of PHA particles below 1 μm before and after heat treatment, and the increase rate of the PHA average particle size caused by heat treatment are shown in Table 1-1. In addition, after the cells before and after heat treatment were placed on a slide and dried, they were stained with fuchsin and observed under an optical microscope. The photos taken during microscopic observation are shown in Figure 1 The cells maintained their shape before and after heat treatment.

[0092] The average cell diameter of the minCD-expressing A2405 disrupted strain after culture was 2.95 μm, and the PHA accumulation ratio relative to the dry bacterial cells was 86%. The average PHA particle size before heat treatment was 1.62 μm, and the proportion of PHA particles 1 μm or smaller before heat treatment was 5.08% by volume. In this example, heat treatment increased the average PHA particle size, as shown in Table 1-1, with the maximum increase rate reaching 1.72%. Furthermore, the proportion of PHA particles 1 μm or smaller decreased significantly from 5.08% by volume before heat treatment, reaching a minimum of 0.00% by volume.

[0093] (Example 2) PHA production based on A1386 deletion disrupted strain

[0094] Cultivation using the A1386 deletion disrupted strain was carried out under the same conditions as in Comparative Example 1. The obtained culture solution was heat-treated at the time and temperature described in Table 1-2. The ratio of the PHA accumulation amount to the dry bacterial body, the average cell diameter, the average PHA particle size before and after heat treatment, the ratio of PHA particles below 1 μm before and after heat treatment, and the increase rate of the PHA average particle size caused by heat treatment are shown in Table 1-2. In addition, after the cells before and after heat treatment were placed on a slide and dried, they were stained with fuchsin and observed under an optical microscope. The photos taken during microscopic observation are shown in Figure 2 The cells maintained their shape before and after heat treatment.

[0095] The average cell diameter of the A1386 deletion-disrupted strain after culture was 2.48 μm, and the PHA accumulation ratio relative to the dry bacterial cells was 88%. The average PHA particle size before heat treatment was 1.48 μm, and the proportion of PHA particles 1 μm or smaller before heat treatment was 10.86% by volume. In this example, heat treatment increased the average PHA particle size, as shown in Table 1-2, with the maximum increase ratio reaching 1.59. Furthermore, the proportion of PHA particles 1 μm or smaller decreased significantly from 10.86% by volume before heat treatment, reaching a minimum of 0.00% by volume.

[0096] It should be noted that the PHA produced in Comparative Example 1 and Examples 1 and 2 was confirmed to be PHBH by HPLC analysis.

[0097] [Table 1-1]

[0098]

[0099] [Table 1-21

[0100]

[0101] (Example 3) pH conditions during heat treatment

[0102] The A1386 deletion-disrupted strain was cultured in the same manner as in Example 2, and the resulting culture medium was aliquoted. The pH of the aliquoted culture medium was controlled to the values ​​listed in Table 2 (within ±0.1), and heat-treated for the time and temperature listed therein. A 10% aqueous sodium hydroxide solution was used for pH control. Table 2 shows the average cell diameter, the average PHA particle size before and after heat treatment, the proportion of PHA particles 1 μm or smaller before and after heat treatment, and the rate of increase in the average PHA particle size due to heat treatment.

[0103] The average cell diameter of the A1386 deletion-disrupted strain after culture was 2.60 μm, and the PHA accumulation ratio relative to the dry bacterial cells was 89%. The average PHA particle size before heat treatment was 1.55 μm, and the proportion of PHA particles 1 μm or smaller before heat treatment was 7.08% by volume. In this example, controlling the pH to 7.0 or higher and performing heat treatment effectively increased the average PHA particle size, as shown in Table 2. It should be noted that after pH control, the change rate of the average PHA particle size before heat treatment was within 5%. During heat treatment, the increase rate reached 1.58% after treatment at 70°C for 30 minutes at pH 8.5. Furthermore, the proportion of PHA particles 1 μm or smaller was significantly reduced from 7.08% by volume before heat treatment to 0.02% by volume.

[0104] It should be noted that the PHA produced in Example 3 was confirmed to be PHBH by HPLC analysis.

[0105] [Table 2]

[0106] Sequence Listing <110> KANEKA CORPORATION <120> Method for producing polyhydroxyalkanoate <130> B190377 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 926 <212> DNA <213> Cupriavidus necator <400> 1 agtgaattcg gatttaaata cccctatacg cgcaagctga tggcggcggc gcaggtcggc 60 gctggctgac ccggaggtgt gcggcgcagc aacgccgcgg ccccgcctgg ctagcatccg 120 gttgttcgga tcaatccgat aaacaaggtg cgaattcccg cctatatcct tgattcgcca 180 gtcaaatccg gcgaatttgt aacgaacttt gacatgtgaa tgacaaccct ttacatccc 240 gcgagaacta gttttggggg tggtccgtcc gacgcattgg atcgtgcata gcacgtttgc 300 ggtgcaagac aggcccggaa agcctggtgc ggatgttgca tagggttcac cccgcaggtt 360 cacatgaatt tctcgcgaag ttcacgcgaa tttcacatat aaccagctgc cccggacttg 420 tgccggggct tgctttggaa cgatcaacgg gagaaccagt ttccggcgcg ctacaagcaa 480 aaaggactgc tgcgacagtc cttttttctt tggcggggcg tgctccccgg gctattgcac 540 tgcgaccgtt ccggccggtg ccaggcgggc ctgttccagc cgctgccgca gcgttgccat 600 caccgccgtg gtggcctgtt cgccggccag tatggctcgg ttgcgggcgt tgaagtcgct 660 gccgcccata tcgggcagct cggggcggat caccacgtcg gcgcgcgcca gtgccatctt 720 gttgatcgac tggcccatga tcgcggtggt ctgcagcagc acgccgctct ggcccgcgtt 780 cttctgcgcc gacgggtcgg ccgagatgtt gaccgcgatg acaaagtccg cgcccatgcc 840 gcgcgcggaa tccaccggca ccggctcgac caggccgccg tcgacatagt cgtgaccctg 900 gatcgacatt taaatggata gctcgg 926 <210> 2 <211> 594 <212> PRT <213> Aeromonas caviae <400> 2 Put Ser Gln Pro Ser Tyr Gly Pro Leu Phe Glu Ala Leu Ala His Tyr 1 5 10 15 Asn Asp Lys Leu Leu Ala Met Ala Lys Ala Gln Thr Glu Arg Thr Ala 20 25 30 Gln Ala Leu Leu Gln Thr Asn Leu Asp Asp Leu Gly Gln Val Leu Glu 35 40 45 Gln Gly Ser Gln Gln Pro Trp Gln Leu Ile Gln Ala Gln Met Asn Trp 50 55 60 Trp Gln Asp Gln Leu Lys Leu Met Gln His Thr Leu Leu Lys Ser Ala 65 70 75 80 Gly Gln Pro Ser Glu Pro Val Ile Thr Pro Glu Arg Ser Asp Arg Arg 85 90 95 Phe Lys Ala Glu Ala Trp Ser Glu Gln Pro Ile Tyr Asp Tyr Leu Lys 100 105 110 Gln Ser Tyr Leu Leu Thr Ala Arg His Leu Leu Ala Ser Val Asp Ala 115 120 125 Leu Glu Gly Val Pro Gln Lys Ser Arg Glu Arg Leu Arg Phe Phe Thr 130 135 140 Arg Gln Tyr Val Ser Ala Met Ala Pro Ser Asn Phe Leu Ala Thr Asn 145 150 155 160 Pro Glu Leu Leu Lys Leu Thr Leu Glu Ser Gly Gly Gln Asn Leu Val 165 170 175 Arg Gly Leu Ala Leu Leu Ala Glu Asp Leu Glu Arg Ser Ala Asp Gln 180 185 190 Leu Asn Ile Arg Leu Thr Asp Glu Ser Ala Phe Glu Leu Gly Arg Asp 195 200 205 Leu Ala Leu Thr Pro Gly Arg Val Val Gln Arg Thr Glu Leu Tyr Glu 210 215 220 Leu Ile Gln Tyr Ser Pro Thr Thr Glu Thr Val Gly Lys Thr Pro Val 225 230 235 240 Leu Ile Val Pro Pro Phe Ile Asn Lys Tyr Tyr Ile Met Asp Met Arg 245 250 255 Pro Gln Asn Ser Leu Val Ala Trp Leu Val Ala Gln Gly Gln Thr Val 260 265 270 Phe Met Ile Ser Trp Arg Asn Pro Gly Val Ala Gln Ala Gln Ile Asp 275 280 285 Leu Asp Asp Tyr Val Val Asp Gly Val Ile Ala Ala Leu Asp Gly Val 290 295 300 Glu Ala Ala Thr Gly Glu Arg Glu Val His Gly Ile Gly Tyr Cys Ile 305 310 315 320 Gly Gly Thr Ala Leu Ser Leu Ala Met Gly Trp Leu Ala Ala Arg Arg 325 330 335 Gln Lys Gln Arg Val Arg Thr Ala Thr Leu Phe Thr Thr Leu Leu Asp 340 345 350 Phe Ser Gln Pro Gly Glu Leu Gly Ile Phe Ile His Glu Pro Ile Ile 355 360 365 Ala Ala Leu Glu Ala Gln Asn Glu Ala Lys Gly Ile Met Asp Gly Arg 370 375 380 Gln Leu Ala Val Ser Phe Ser Leu Leu Arg Glu Asn Ser Leu Tyr Trp 385 390 395 400 Asn Tyr Tyr Ile Asp Ser Tyr Leu Lys Gly Gln Ser Pro Val Ala Phe 405 410 415 Asp Leu Leu His Trp Asn Ser Asp Ser Thr Asn Val Ala Gly Lys Thr 420 425 430 His Asn Ser Leu Leu Arg Arg Leu Tyr Leu Glu Asn Gln Leu Val Lys 435 440 445 Gly Glu Leu Lys Ile Arg Asn Thr Arg Ile Asp Leu Gly Lys Val Lys 450 455 460 Thr Pro Val Leu Leu Val Ser Ala Val Asp Asp His Ile Ala Leu Trp 465 470 475 480 Gln Gly Thr Trp Gln Gly Met Lys Leu Phe Gly Gly Glu Gln Arg Phe 485 490 495 Leu Leu Ala Glu Ser Gly His Ile Ala Gly Ile Ile Asn Pro Pro Ala 500 505 510 Ala Asn Lys Tyr Gly Phe Trp His Asn Gly Ala Glu Ala Glu Ser Pro 515 520 525 Glu Ser Trp Leu Ala Gly Ala Thr His Gln Gly Gly Ser Trp Trp Pro 530 535 540 Glu Met Met Gly Phe Ile Gln Asn Arg Asp Glu Gly Ser Glu Pro Val 545 550 555 560 Pro Ala Arg Val Pro Glu Glu Gly Leu Ala Pro Ala Pro Gly His Tyr 565 570 575 Val Lys Val Arg Leu Asn Pro Val Phe Ala Cys Pro Thr Glu Glu Asp 580 585 590 Ala Ala <210> 3 <211> 2478 <212> DNA <213> Cupriavidus necator <400> 3 agtgaattcg gatttaaatg ctaatggtga gtgtggtctt ggacatcgcg cctcctttac 60 tgcttgttgc cgctaatggc cgcgcaccta tgcagtgcat ccggcaggca ccagtctgaa 120 gccgctgcgc gcaacgcgcc gcgaagcggc gccatgccca tgcgccaggc gcatgcctcg 180 ctacttgcgc ggcattgtcc gcccgctcac agcacaatgc gcaaggcgcg tgccaggcat 240 aaactgatgg ccaattgtac gccgccccct gaccaggaac gccgggccag tcccggcgtt 300 ttttattct atagcgcaat taaccgccgt catattgcgt caccatgatt gccggatggc 360 cgcggcgatc ccttgctgga ggccggttcc aagaagattt aaagatgtca cggaattgtc 420 atacagggag catagagttc gtcttgtcaa aaatttgtca ttcccaacca atgttctctg 480 gaggacatat gtcccagaag aaatcgccac gcttcgagct gcgcagtggc aacgtagacg 540 cccctctct cgccctccag accgccgaca tggctgcgct gcgggatgac ctcctcgccc 600 gctttgaagc cacccccgac ttcttttcca atgacgtgat tgcgctggac ctgcgcgc 660 tggaagatga cagcgaagtc gcgcttggca ccgtgatcga gacgctggcc acgctcaggg 720 cccgcgccat cggcgtggtg gccgccccg gccagcgcga gtgggccgag cgcttcggcc 780 tgccgctgct ggacagccag gcccgccgcg gcagtggcgc cgatcgcgcc accgaccgtg 840 ccgccgaggc cagggccgca gccgcggcgg aacaggccgc agccgaccag gccgcgcgcg 900 aggaatccat ccgcgccgcc gcgcaggcca ccaccgacgc cgccgtggcc gctgccatcc 960 gccagaccca gaccatgctg atcgacaagc cgcttcgctc gggccagcag gtctacgcgc 1020 agggcgacgt ggtcatcctg gacgtggtca gctacggcgc cgaggtgatc gccgaaggca 1080 acatccatat ctatgccccg ctgcgcggcc gtgcgctggc gggcgtcaag ggcaacaccg 1140 gcgcgcgcat tttcagcacg tgcatggagc ctgaactgat ttccatcgcc ggcatctacc 1200 ggaccgcgga gcagacgctt ccggccgacg tgctcggcaa gaccgcccag gtgcgcctgg 1260 ccgatgaaaa actgatcctg gaagcgctgc ggctcaagta accgcggcag cccccgggac 1320 cgaattgcag agagcgcaag cttcaactta ttactggacc aaagagccat ggcaaaaatc 1380 atcgttgtga cctccggcaa gggaggcgtc ggcaagacca ccaccagcgc cagctttgcc 1440 gccggcctgg ccctgcgcgg ccacaagact gccgtgatcg acttcgacgt cggcctgcgc 1500 aaccttgacc tgatcatggg ttgcgagcgc cgcgtggtgt acgacctgat caacgtggtg 1560 cagggcgaag ccaacctgcg ccaggcgctg atcaaggaca agaagtgcga gaacctgttc 1620 atcctgccgg cctcgcagac gcgcgacaag gacgcgctca cgcgcgaagg cgtcgagaag 1680 gtcatcaacg gcctgatcga gatggatttc gaattcatca tctgcgactc gccggccggc 1740 atcgagtcgg gcgcgctgat ggcgatgtac ttcgccgacg aggcgctgat cgtgaccaac 1800 ccggaagtgt cgtcggtgcg cgattcggac cgcatcctgg gcatcctggc ctccaagacc 1860 aagcgcgcca gcgaaggcgg cgacccgatc aaagcaacacc tgctgatcac ccgctacaac 1920 cccaagcgtg tgcatggcgg cgaaatgctg tcgctgaccg acatccagga aatcctgcgc 1980 atcaagctga tcggcgtggt gccggagtct gaagccgtgc tgcacgctc gaaccagggc 2040 acgcccgcca tccacctgga aggcagcgac gtggccgacg cctatggcga cgtggtggac 2100 cgcttctcg gcaagcaa gccgatgcgt ttcaccgact accagaagcc gggtctgctc 2160 tcccgcatct tcggcaacaa gtaacctgcc ggcctggttc aaccagtcgg cagccgacta 2220 gtcccggcag ccgccagcgc gctggcctcg cttatcatgg cagctgcgcc gggcggcacg 2280 cgaacggcgc ggcaccaacg atcaacatgc cattgctacc gacaagac ttccagggcc 2340 agccgctggt ccggatcggc gatgccgaca cgttcctgct gctcgccccg caacacggcg 2400 ggcggctggt ccgctgggtg caccgcggac aggacatcct ctactggccg gacgctgcca 2460 tttaaatgga tagctcgg 2478 <210> 4 <211> 956 <212> DNA <213> Cupriavidus necator <400> 4 agtgaattcg gatttaaatg tatgcggcat gtcactgggt tttgccgacc cggaagcgat 60 cgagaaccag ctgaccacgg aacgtgagcc ggtcagcggg ttcgcgcgtt tcctctcata 120 gcaaagtttg agaaaagttt gtatcaatgt gtaacgatga gtgccgatat acaagacgac 180 gtccgtgtat tggctgggag tgttccaagg ggcagcaagg tgaacccggg tacgcctggg 240 gcaagccaga ggcggttcgc atgcaaacgt gaccttttgg ttgctttttc cgcaatgtgg 300 aaatgtttgc aaatcgaact ttaaggagcg tctgtaagtc tttaatcttg ctaacaattt 360 ctttctttcc tacactagcg ccattcctat gcgctgaacg aatcatgttc cggtctgatt 420 ccattttttc cagattcttc ggctccgcac ccctgtccgc tgttgccacc gcggtcctgg 480 tatcgtacgg aacgcctgac cgggaaaaaa cgcgccgtgc cacggcgcgt ttttcgttct 540 ggcggccgcg gccgtcagag cagcttgcct gggttcatga tcccggccgg gtcgaacacg 600 gccttgatct cgcgcatcag ccgcagttcc agcgggtcct tcatggtcag gaaggcatgg 660 cgcttgagct ggccgatgcc atgctcggcg ctgatgctgc cgccgtagcg catcacttcg 720 tccagcaccg cgtcggtcac cgcatcgcct tgcgtggccg cccagtcctt gggcgcgccg 780 gccgggcgcg acaggttgta gtgcaggttg ccgtcgccga agtgcccgaa gataaagggc 840 cggatggcgg gatcgagccc acgcagccgc gtttccatcg aggtcatgaa ggccggaatc 900 tgctcgatcg ggagcgagat gtcgtgcttc aggtgcgatt taaatggata gctcgg 956

Claims

1. A method for producing a polyhydroxyalkanoate, the method comprising: A step of culturing a polyhydroxyalkanoate-producing microorganism in which a gene encoding a PHA synthase represented by the amino acid sequence set forth in SEQ ID NO: 2 is introduced into the chromosome of Cupria necrotica H16 strain, and in which the A1386 gene or the A2405 gene is disrupted, to obtain bacterial cells that accumulate polyhydroxyalkanoate particles and have an average cell diameter of 2 μm or greater; a step of heat-treating the culture solution containing the cultured bacterial cells at a temperature higher than the temperature during the culture to adjust the average particle size of the polyhydroxyalkanoate particles in the bacterial cells to 1.8 μm or more and not more than the average cell diameter; In the case where the A2405 gene is disrupted, the minC gene from Cupriavidus necrotizingus and the minD gene from Cupriavidus necrotizingus are inserted.

2. The manufacturing method according to claim 1, wherein The ratio of the average particle size of the polyhydroxyalkanoate particles after the heat treatment to the average particle size of the polyhydroxyalkanoate particles before the heat treatment is 1.1 or more.

3. The manufacturing method according to claim 1 or 2, wherein: The weight of the polyhydroxyalkanoate accounts for 80% or more of the dry weight of the cultured bacterial cells.

4. The manufacturing method according to claim 1 or 2, wherein: In the particle size distribution of the polyhydroxyalkanoate particles after the heat treatment, the proportion of polyhydroxyalkanoate particles having a particle diameter of 1 μm or less is 2.0% by volume or less.

5. The manufacturing method according to claim 1 or 2, wherein: The average cell diameter of the bacterial cells accumulating the polyhydroxyalkanoate particles is 2.2 μm or more.

6. The manufacturing method according to claim 1 or 2, wherein: The heat treatment is performed at a temperature of 40 to 100° C. for 5 minutes or more.

7. The manufacturing method according to claim 1 or 2, wherein: The heat treatment is performed at a pH of 7.0 or higher.

8. The manufacturing method according to claim 1 or 2, further comprising: a step of disrupting the heat-treated bacterial cells to obtain a cell disrupted liquid, and a step of separating polyhydroxyalkanoate particles from the aqueous phase of the cell disrupted liquid.

9. The manufacturing method according to claim 1 or 2, wherein: The polyhydroxyalkanoate is a copolymer of two or more hydroxyalkanoic acids.

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

11. The manufacturing method according to claim 10, wherein: The polyhydroxyalkanoate is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

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