Method for producing polyhydroxyalkanoate

Abstract

This method for producing a polyhydroxyalkanoate comprises: a step for culturing polyhydroxyalkanoate-producing microorganisms to obtain microbial cells in which polyhydroxyalkanoates are accumulated; and a step for treating the microbial cells with an enzyme. The microbial cells have a number-average aspect ratio of a cell size of at least 2.0, and the enzyme includes a cell wall-degrading enzyme. The enzyme may further include a proteolytic enzyme.

Description

Method for producing polyhydroxyalkanoic acid

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid.

[0002] Against the backdrop of growing awareness of environmental issues, food issues, health and safety, and a growing preference for natural or natural products, the significance and importance of substance production using microorganisms (fermentation production, bioconversion, etc.) is increasing, and substance production using microorganisms is also being applied to the production of protein pharmaceuticals, nucleic acids for gene therapy, etc. For example, the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria is being actively applied industrially.

[0003] One example is the production of polyhydroxyalkanoic acid (hereinafter also referred to as PHA) by microorganisms, which is expected to be used industrially as a biodegradable plastic (see Non-Patent Document 1). PHA is a thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species, and is biodegradable. Currently, non-petroleum-derived plastics are attracting attention due to increased environmental awareness. In particular, PHA produced and accumulated within the cells of microorganisms is expected to have little adverse impact on the ecosystem because it is incorporated into the natural carbon cycle process, and its practical application is eagerly awaited. In PHA production using microorganisms, for example, it is known that PHA is produced by feeding sugars, vegetable oils, or fatty acids as carbon sources to Capriavidus bacteria, allowing PHA to accumulate within the cells (see Non-Patent Documents 2 and 3).

[0004] Since PHA produced by microorganisms is usually accumulated in the form of granules within the cells of the microorganisms, in order to use PHA as a plastic, a process for separating and recovering PHA from the cells of the microorganisms is required. Furthermore, in order to use PHA as a plastic, it is desirable to increase the purity of PHA and reduce the content of impurities such as cell components as much as possible.

[0005] As a method for decomposing and / or removing biological components other than PHA, methods have been proposed in which biological components other than PHA are solubilized and removed by physical, chemical, or biological treatment. For example, Patent Documents 1 and 2 disclose a method in which a treatment for disrupting PHA-containing microbial cells is combined with a surfactant treatment, and a method in which an alkali is added, heat treatment is performed, and then a disruption treatment is performed. Furthermore, Patent Document 3 discloses a method in which an aqueous suspension of microbial cells is treated with sodium hypochlorite, an enzyme, or the like to solubilize biological components other than PHA, thereby obtaining PHA.

[0006] After disrupting the cells of the PHA-containing microorganism or solubilizing biological components other than PHA, methods for recovering the PHA from the resulting aqueous suspension include, for example, separation procedures such as centrifugation or filtration, and drying procedures using a spray dryer or drum dryer.

[0007] JP-T-08-502415A International Publication No. 2004 / 065608 JP-A-2005-348640 JP-A-2019-97518 JP-A-2020 / 174988 JP-A-2021 / 049207

[0008] Anderson AJ. , et al. , Int. J. Biol. Macromol. , 12, 201-105 (1990) Sato S. , et al. , J. Biosci. Bioeng. , 120(3), 246-251 (2015) Insomphun C. , et al. , Metab. Eng. , 27, 38-45 (2015)

[0009] In the drying operation of the aqueous suspension, it is desirable that the PHA concentration of the aqueous suspension is high in order to perform drying efficiently. However, as the PHA concentration increases, the fluidity of the aqueous suspension decreases, making the drying operation more difficult. In other words, with respect to the PHA concentration of the aqueous suspension, there is a trade-off between the efficiency and ease of the drying process, and it has been difficult to improve the fluidity of the aqueous suspension while maintaining a high PHA concentration.

[0010] Patent Document 4 discloses a method of achieving good dispersibility by adding polyvinyl alcohol as a dispersant to a PHA aqueous dispersion. However, the addition of a dispersant in large amounts is undesirable because it can affect the processability of the recovered PHA.

[0011] One possible way to improve the fluidity of an aqueous suspension while maintaining its concentration at a high level is to optimize the shape of the particles contained in the aqueous suspension. Generally, the particle shape that maximizes the fluidity of an aqueous suspension is spherical. The fluidity of an aqueous suspension tends to decrease as the particle shape increases in surface area per unit volume, such as oval spheres, plates, rods, and rods.

[0012] The shape of PHA particles depends on the shape of the microbial cells that accumulate the particles. In many cases, microbial cells that have accumulated sufficient PHA are spherical. For example, when about 90% of PHA is accumulated in the KNK-005 strain, the cells become spherical, and the PHA particles obtained by disrupting the cells are also spherical (see Reference Example 1 described below).

[0013] However, depending on the type of microorganism producing PHA and the type of genetic modification introduced, the shape of PHA-accumulating cells may not be spherical. For example, as in Patent Documents 5 and 6, when the cell division inhibitor minCD is overexpressed or when some cell wall decomposition enzyme genes are disrupted, PHA-accumulating cells become rod-shaped. PHA particles accumulated in rod-shaped cells tend to be rod-shaped. Therefore, the fluidity of aqueous PHA suspensions obtained from rod-shaped PHA-accumulating cells is likely to decrease.

[0014] In view of the above-mentioned current situation, an object of the present invention is to provide a method for producing PHA that enables recovery of PHA particles that are closer to spherical in shape from rod-shaped PHA-accumulating cells.

[0015] The present inventors have found that when rod-shaped microbial cells in which PHA has accumulated are treated with a specific enzyme, the PHA particles become spherical, leading to the present invention.

[0016] That is, the present invention relates to a method for producing polyhydroxyalkanoic acid, which comprises the steps of culturing a polyhydroxyalkanoic acid-producing microorganism to obtain microbial cells that have accumulated polyhydroxyalkanoic acid, and treating the microbial cells with an enzyme, wherein the microbial cells have a number-average aspect ratio of cell diameter of 2.0 or more, and the enzyme includes a cell wall-degrading enzyme.

[0017] According to the present invention, it is possible to recover more spherical PHA particles from rod-shaped PHA-accumulating cells. Because the PHA particles are nearly spherical, an aqueous suspension containing the PHA particles can exhibit relatively high fluidity even at high PHA concentrations. This can improve the efficiency of subsequent drying processes.

[0018] Micrograph of PHA-accumulating cells obtained in Reference Example 1 Micrograph of PHA-accumulating cells obtained in Comparative Example 1 Micrograph of PHA-accumulating cells obtained in Comparative Example 2

[0019] An embodiment of the present invention is described in detail below. The embodiment of the present invention relates to a method for producing PHA, which includes a step of culturing a PHA-producing microorganism to obtain microbial cells of a specific shape in which PHA has accumulated, and a step of treating the microbial cells with an enzyme.

[0020] (PHA) The type of PHA is not particularly limited as long as it is a PHA that can be produced by a microorganism, and it may be a homopolymer composed of one type of hydroxyalkanoic acid, or a copolymer composed of two or more types of hydroxyalkanoic acids. Specific examples include homopolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, copolymers 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 copolymers of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms.

[0021] Among these, homopolymers or copolymers containing 3-hydroxybutyric acid as a monomer unit are preferred. Examples of such polymers include P(3HB), a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), P(3HB-co-3HH) (abbreviation: PHBH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), P(3HB-co-4HB), and PHA containing lactic acid (abbreviation: LA) as a constituent component, such as P(LA-co-3HB), a copolymer of 3HB and LA. However, this is not limited to these. Among these, PHBH is preferred from the viewpoint of its wide range of applications as a polymer.

[0022] The type of PHA to be produced can be appropriately selected depending on the purpose, such as the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic gene involved in the synthesis, and the culture conditions.

[0023] (PHA-producing microorganism) The PHA-producing microorganism may be a microorganism capable of producing PHA. The microorganism may be a microorganism having a PHA synthase gene. The microorganism may be a wild-type strain that inherently has the PHA synthase gene, a mutant strain obtained by artificially mutating such a wild-type strain, or a strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques.

[0024] In this embodiment, the PHA-producing microorganism has a cell shape that becomes non-spherical, preferably rod-shaped, when it accumulates PHA. The PHA-producing microorganism may be a wild-type strain whose cell shape becomes non-spherical when it accumulates PHA, or may be a strain that has been mutated by artificial mutation or genetic engineering techniques so that its cell shape becomes non-spherical when it accumulates PHA.

[0025] The shape change of PHA-accumulating cells due to artificial mutation or genetic engineering techniques may be intentional or unintentional. For example, a strain may be mutated so that the PHA-accumulating cells become non-spherical as a result of transformation to increase their size, with the aim of improving the efficiency of the process of recovering PHA-accumulating cells or PHA particles by filtration or centrifugation. Techniques for increasing the size of PHA-accumulating cells include, for example, genetic modification to enhance the expression of genes encoding MinC and / or MinD, proteins that control cell division, as disclosed in Patent Document 5 or Patent Document 6, and genetic modification to reduce the expression of the A1386 gene and / or the 2405 gene, genes thought to function as peptidoglycan hydrolases. In strains that have undergone these genetic modifications, the cell shape may change to a non-spherical shape. However, genetic modification that changes the cell shape to a non-spherical shape is not limited to these examples.

[0026] Furthermore, even in the case of a strain whose cells become spherical when PHA is sufficiently accumulated, it is conceivable that the cells may become non-spherical during accumulation. For such strains, the effects of the present invention can be achieved by interrupting the culture at the stage when the cells become non-spherical and performing the enzymatic treatment according to the present disclosure.

[0027] The PHA-producing microorganism or the host of the microorganism is not particularly limited, but is preferably a bacillus, more preferably a gram-negative bacillus. Preferred examples of the bacillus include bacteria belonging to the family Burkholderiaceae, such as the genera Ralstonia, Cupriavidus, Wautersia, and Burkholderia, as well as bacteria belonging to the genus Pseudomonas, Halomonas, and Escherichia.

[0028] From the viewpoints of safety and PHA productivity, bacteria belonging to the genus Ralstonia, Cupriavidus, or Escherichia are more preferred, bacteria belonging to the genus Cupriavidus or Escherichia are even more preferred, and Cupriavidus necator or Escherichia coli are particularly preferred.

[0029] (PHA synthase gene) The PHA synthase gene possessed by the PHA-producing microorganism is not particularly limited, and examples thereof include PHA synthase genes derived from organisms similar to the genera Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, as well as modified versions thereof. Examples of the modified versions include nucleotide sequences encoding PHA synthases in which one or more amino acid residues have been deleted, added, inserted, or substituted. Examples include genes having a nucleotide sequence encoding a polypeptide represented by the amino acid sequence set forth in any of SEQ ID NOS: 1 to 5, and genes having a nucleotide sequence encoding a polypeptide represented by an amino acid sequence having 85% or more sequence identity to the amino acid sequence and having PHA synthase activity. The sequence identity is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0030] (Culturing) The step of culturing the PHA-producing microorganism can be carried out by a person skilled in the art based on common technical knowledge, and there are no particular limitations on the medium composition, method of adding a carbon source, culture scale, aeration and stirring conditions, culture temperature, culture time, etc. By carrying out the culture for an appropriate period of time, PHA can be accumulated within the cells of the PHA-producing microorganism. Hereinafter, PHA-producing microorganisms that have accumulated PHA within their cells are also referred to as PHA-accumulating cells.

[0031] (Shape of PHA-accumulating cells) The shape of PHA-accumulating cells, which is non-spherical, preferably rod-shaped, can be confirmed by microscopic observation. Quantitatively, the shape of PHA-accumulating cells is defined by the number-average aspect ratio of the cell diameter of PHA-accumulating cells observed under a microscope. Here, the aspect ratio is the ratio (Lb / La) of the shortest minor axis length (La) to the longest major axis length (Lb) of a line passing through the center of a PHA-accumulating cell, and the number-average aspect ratio is the number-average value of the aspect ratio. The closer the number-average aspect ratio is to 1, the closer the shape of the PHA-accumulating cell is to a spherical shape, and the larger the number-average aspect ratio, the more elongated and rod-shaped the PHA-accumulating cell is. In this embodiment, the number-average aspect ratio of the cell diameter of PHA-accumulating cells is preferably 2.0 or more, more preferably 2.5 or more. The upper limit of the number-average aspect ratio of the cell diameter is not particularly limited, but may be, for example, 10 or less, or 5 or less.

[0032] The number-average aspect ratio of the cell diameter of PHA-accumulating cells is calculated as follows. In an image observed with an optical microscope, for each of 50 or more PHA-accumulating cells, the shortest minor axis length (La) and the longest major axis length (Lb) of the lines passing through the center of the cell are measured, and the aspect ratio (Lb / La) is calculated. The number-average aspect ratio of the cell diameter of PHA-accumulating cells can then be determined by calculating the number-average value of the obtained aspect ratios.

[0033] (Optional Aggregation Step) PHA-accumulating cells typically contain one or more PHA particles. The number of PHA particles in the PHA-accumulating cells according to this embodiment is not particularly limited. However, since the fluidity of the aqueous suspension improves as the PHA particle diameter increases for a given total amount of PHA, it is preferable to carry out a process to aggregate multiple PHA particles in the PHA-accumulating cells prior to the enzyme treatment. Examples of a means for aggregating PHA particles in PHA-accumulating cells include heat or alkali treatment methods, such as those disclosed in Appl Microbial Biotechnol (2019 February; 103(4): 1905-1917). Heat treatment methods are particularly preferred. The conditions for the heat treatment are not particularly limited, but a treatment in which the PHA-accumulating cells are maintained at 50 to 90°C for approximately 10 minutes to 7 hours is preferred.

[0034] In this embodiment, the PHA-accumulating cells satisfying the above-described number-average aspect ratio of cell diameter are treated with an enzyme. As the enzyme, it is preferable to use at least a cell wall-degrading enzyme, and it is more preferable to use a protease in addition to the cell wall-degrading enzyme.

[0035] The cell wall-degrading enzyme is not particularly limited as long as it is an enzyme that degrades bacterial cell walls, and examples thereof include lysozyme, amylase, cellulase, maltase, saccharase, α- and β-glycosinase, etc. Lysozyme is particularly preferred.

[0036] Commercially available cell wall-degrading enzymes include, for example, "egg white lysozyme" (manufactured by Nagase ChemteX Corporation), "Lysozyme" (manufactured by Huayuan Jingmao Co., Ltd., Shandong Province), "Biozyme A," "Cellulase A "Amano" 3," "Cellulase T "Amano" 4," "α-Glucosidase "Amano"" (all manufactured by Amano Enzyme Co., Ltd.), "Termamyl," and "Cellsoft" (all manufactured by Novozymes). Furthermore, an enzyme composition containing, in addition to the cell wall-degrading enzyme, a stabilizer for the enzyme, a surfactant, an anti-redeposition agent, and the like, may also be used.

[0037] The proteolytic enzyme is not particularly limited, but examples thereof include alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, and carboxypeptidase.

[0038] Examples of commercially available protease products include "Protease A," "Protease P," and "Protease N" (all manufactured by Amano Enzyme Co., Ltd.), "Alcalase," "Esperase," "Zavinase," and "Evalase" (all manufactured by Novozymes). These commercially available products are also suitable for use in terms of decomposition activity. Furthermore, enzyme compositions containing, in addition to the protease, a stabilizer for the enzyme, a surfactant, an anti-redeposition agent, and the like, may also be used.

[0039] The temperature and pH conditions for the enzyme treatment are preferably within the optimum range for the enzyme used. For example, when Alcalase (manufactured by Novozymes) is used, the enzyme treatment is preferably carried out at a temperature of 50 to 60°C and a pH of 8 to 9. When Esperase (manufactured by Novozymes) is used, the enzyme treatment is preferably carried out at a temperature of 55 to 65°C and a pH of 8 to 10. When egg white lysozyme (manufactured by Nagase ChemteX Corporation) is used, the enzyme treatment is preferably carried out at a temperature of 40 to 50°C and a pH of 6 to 7.

[0040] The time for the enzyme treatment can be appropriately set taking into consideration the effects of the invention, and is not particularly limited, but may be within the range of, for example, 0.5 to 8 hours.

[0041] The amount of the enzyme used is not particularly limited and may be appropriately determined depending on the type and activity of the enzyme used, taking into consideration the effects of the invention. For example, the amount is preferably within the range of 0.001 to 10 parts by weight, and more preferably 0.001 to 5 parts by weight, per 100 parts by weight of the bacterial cell components.

[0042] In the enzymatic treatment according to this embodiment, it is possible to spheroidize PHA particles even when only a cell wall-degrading enzyme is used as the enzyme, but by using a cell wall-degrading enzyme and a protease in combination, it is possible to make the PHA particles more spherical. When using a cell wall-degrading enzyme and a protease in combination, the enzymatic treatment may be carried out using one enzyme and then the other enzyme, or both enzymes may be used simultaneously. However, since the cell wall-degrading enzyme itself is a protein and can be decomposed by the protease, it is preferable to carry out the enzymatic treatment using the cell wall-degrading enzyme, and then add the protease and carry out the enzymatic treatment with the protease.

[0043] (Cell disruption process and PHA recovery process) After the enzyme treatment, a disruption process can be carried out by a well-known method to disrupt microbial cells to obtain a cell disruption solution, and a recovery process can be carried out to recover PHA particles from the cell disruption solution.

[0044] Since the enzyme treatment can also disrupt microbial cells to some extent, a PHA recovery step can be carried out directly after the enzyme treatment. However, in order to further remove cellular components, it is preferable to carry out a disruption step separately from the enzyme treatment and then carry out the PHA recovery step after the disruption step. Such a disruption step can be carried out by any known method, and is not particularly limited. Examples of such a disruption step include a method using an enzyme other than a cell wall-degrading enzyme and a protease, a method using mechanical shearing force, and a method using a surfactant or alkali. By carrying out such a disruption step, PHA-accumulating cells are further disrupted, and a cell lysate in which cellular components other than PHA are dissolved in water can be obtained.

[0045] A known method can also be applied to the PHA recovery process, and is not particularly limited. For example, by repeatedly concentrating the PHA particles by filtration or centrifugation of the cell lysate and resuspending them in an aqueous system, the PHA particles can be recovered as a PHA aqueous suspension in which the cell components are diluted. The PHA aqueous suspension can also be used directly for processing. Alternatively, the PHA particles can be separated from the PHA aqueous suspension by filtration or centrifugation and dried to recover the PHA particles as a powder.

[0046] According to the present invention, since the PHA particles are spherical, the fluidity of the aqueous suspension is relatively good even when the PHA particles are contained at a high concentration, and the concentration step or drying step described above can be carried out efficiently.

[0047] (Spheronization of PHA particles) The spheronization of PHA particles by the above-mentioned enzyme treatment can be evaluated by the particle size span value obtained from the measurement result of a laser diffraction particle size distribution analyzer.Specifically, the spheronization of PHA particles by enzyme treatment can be evaluated based on the degree of reduction in the particle size span value of the PHA obtained by crushing PHA-accumulating cells after enzyme treatment, compared with the particle size span value of the PHA obtained by crushing PHA-accumulating cells before enzyme treatment.

[0048] In this embodiment, the particle size span value of the PHA after enzyme treatment is preferably 10% or more smaller than the particle size span value of the PHA before enzyme treatment, and more preferably 15% or more smaller. Specifically, the value calculated by 100 × (particle size span value of the PHA before enzyme treatment - particle size span value of the PHA after enzyme treatment) / (particle size span value of the PHA before enzyme treatment) is preferably 10% or more, and more preferably 15% or more. The upper limit is not particularly limited, but may be 70% or less, or may be 50% or less.

[0049] Here, the particle size span value indicates the particle size distribution width and is a value defined by the formula (D90-D10) / D50. Here, D10, D50, and D90 represent particle sizes corresponding to 10%, 50%, and 90% of the integrated value of the particle size distribution, respectively. In calculating the particle size span value, it is desirable to treat the culture solution after culture but before enzyme treatment at 70°C for 1 hour to inactivate the bacterial cells and aggregate the PHA particles within the cells.

[0050] The principle of evaluating changes in PHA particle shape based on changes in particle size span value utilizes the properties of a laser diffraction particle size distribution analyzer. Generally, laser diffraction particle size distribution analyzers are designed to measure spherical particles, so when measuring non-spherical particles, the particle size distribution width tends to be larger than the actual particle size distribution width. Naturally, the particle size distribution width also varies depending on the variation in particle volume. Therefore, the particle size distribution width measured by a laser diffraction particle size distribution analyzer depends on the variation in volume and shape of the particles being measured. In other words, the smaller the variation in particle volume and the closer the particle shape is to a sphere, the smaller the particle size span value.

[0051] The absolute value of the particle size span of the PHA after the enzyme treatment is difficult to define uniquely because the volume of PHA particles accumulated in microbial cells varies depending on the type of microorganism used and the method of culturing the microorganism, but is usually preferably 0.85 or less, more preferably 0.70 or less. The lower limit of the absolute value of the particle span is not particularly limited, but may be, for example, 0.3 or more, 0.4 or more, or 0.5 or more.

[0052] According to the present invention, PHA particles having a more spherical shape can be recovered from rod-shaped microbial cells that have accumulated PHA, and as a result, the concentration or drying process of the aqueous PHA suspension can be easily and efficiently carried out.

[0053] The following items list preferred aspects of the present disclosure, but the present invention is not limited to them. [Item 1] A method for producing polyhydroxyalkanoic acid, comprising: culturing a polyhydroxyalkanoic acid-producing microorganism to obtain microbial cells that have accumulated polyhydroxyalkanoic acid; and treating the microbial cells with an enzyme, wherein the microbial cells have a number-average aspect ratio of cell diameter of 2.0 or more, and the enzyme includes a cell wall-degrading enzyme. [Item 2] The production method according to Item 1, wherein the enzyme further includes a protease. [Item 3] The production method according to Item 1 or 2, wherein the particle size span value of the polyhydroxyalkanoic acid after the enzyme treatment step is 10% or more smaller than the particle size span value of the polyhydroxyalkanoic acid before the enzyme treatment step. [Item 4] The production method according to any one of Items 1 to 3, wherein the particle size span value of the polyhydroxyalkanoic acid after the enzyme treatment step is 0.85 or less. [Item 5] The production method according to any one of Items 1 to 4, wherein the microorganism is a transformed microorganism of a Bacillus. [Item 6] The production method according to any one of Items 1 to 5, wherein the microorganism is a transformed microorganism of a Gram-negative bacterium. [Item 7] The production method according to Item 6, wherein the microorganism belongs to the genus Capriavidus. [Item 8] The production method according to Item 7, wherein the microorganism is a transformed microorganism of Capriavidus necator. [Item 9] The production method according to Item 6, wherein the microorganism belongs to the genus Escherichia. [Item 10] The production method according to Item 9, wherein the microorganism is a transformed microorganism of Escherichia coli. [Item 11] The production method according to any one of Items 1 to 10, further comprising a step of heat-treating the microbial cells after the step of obtaining the microbial cells and before the step of treating with the enzyme. [Item 12] The production method according to any one of Items 1 to 11, further comprising a step of disrupting the microbial cells to obtain a cell lysate solution and a step of recovering polyhydroxyalkanoic acid particles from the cell lysate solution after the step of treating with the enzyme. [Item 13] The method according to any one of Items 1 to 12, wherein the polyhydroxyalkanoic acid is a polymer containing 3-hydroxybutyric acid as a monomer unit.[Item 14] The method according to any one of Items 1 to 13, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid. [Item 15] The method according to Item 14, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit. [Item 16] The method according to Item 15, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0054] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. The overall genetic manipulation can be carried out as described in, for example, Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, and the like used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. The enzymes used are not particularly limited as long as they can be used in genetic manipulation.

[0055] (Production Example 1) Preparation of BW25113 phaCAB-Expressing Strain First, a gene expression plasmid was prepared. This was done as follows. PCR using synthetic oligo DNA was used to obtain a DNA fragment (SEQ ID NO: 6) containing a promoter sequence, the sequence of the phaCAB genes (three genes involved in PHA synthesis), and the nucleotide sequence of a terminator. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated with the vector pCUP2 described in Japanese Patent No. 5,650,368, which had also been digested with MunI and SpeI, using DNA ligase (Ligation High (Toyobo Co., Ltd.)), to prepare the phaCAB gene expression plasmid vector pCUP2-REP-phaCAB. Next, the gene expression plasmid vector pCUP2-REP-phaCAB was introduced into Escherichia coli BW25113 strain by the heat shock method.

[0056] Reference Example 1 Evaluation of the Shape of Cells and PHA Particles of KNK-005 Strain The KNK-005 strain was cultured under the following conditions to allow PHA to accumulate in the cells, and then the shapes of the cells and PHA particles were evaluated. The KNK-005 strain is a transformant in which a PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 3) has been introduced onto the chromosome of the Capriavidus necator H16 strain, and can be prepared in accordance with the method described in U.S. Patent No. 7,384,766.

[0057] (Culture medium) The composition of the seed culture medium was 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). The preculture medium consisted of 1.1 w / v% NaHPO.12H0, 0.19 w / v% KHPO, 1.29 w / v% (NH)SO, 0.1 w / v% MgSO.7H0, 2.5 w / v% palm olein oil, and 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl.6H0, 1 w / v% CaCl.2H0, 0.02 w / v% CoCl.6H0, 0.016 w / v% CuSO.5H0, and 0.012 w / v% NiCl.6H0 in 0.1 N hydrochloric acid). Palm olein oil was added as a carbon source at a concentration of 10 g / L. The composition of the PHA production medium was 0.385 w / v% NaHPO.12H0, 0.067 w / v% KHPO, 0.291 w / v% (NH)SO, 0.1 w / v% MgSO.7H0, and 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl.6H0, 1 w / v% CaCl.2H0, 0.02 w / v% CoCl.6H0, 0.016 w / v% CuSO.5H0, and 0.012 w / v% NiCl.6H0 dissolved in 0.1 N hydrochloric acid).

[0058] (Method for measuring the ratio of PHA accumulation to dry bacterial cells) The ratio of PHA accumulation to dry bacterial cells (hereinafter also referred to as PHA content) was measured as follows. Bacterial cells were collected from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain dry bacterial cells. 100 ml of chloroform was added to 1 g of the obtained dry bacterial cells, and the mixture was stirred at room temperature for one day and one night to extract the PHA within the bacterial cells. The bacterial cell residue was filtered and then concentrated in an evaporator to a total volume of 30 ml. 90 ml of hexane was then gradually added, and the mixture was left to stand for 1 hour with slow stirring. The precipitated PHA was filtered and then vacuum-dried at 50°C for 3 hours. The weight of the dried PHA was measured, and the ratio of the dry PHA weight to the dry bacterial cell weight was calculated to obtain the PHA content.

[0059] (Microscopic observation of cells and calculation method of number-average aspect ratio of cell diameter) Microscopic observation of cells was performed as follows. The culture medium was appropriately diluted, placed on a slide glass, dried, and then stained with fuchsin. The stained cells were observed under an optical microscope. Fifty cells were randomly selected from the observed cells, and the La and Lb of the cells were measured, and La / Lb was calculated. The average value of La / Lb was used as the number-average aspect ratio of the cell diameter. The average values ​​of La and Lb were also measured.

[0060] (Method for measuring PHA particle size span) The PHA particle size span was measured as follows. After the bacterial cell inactivation treatment described below and before or after enzyme treatment, the culture solution was suspended in a sodium dodecyl sulfate aqueous solution with a final concentration of 3.3%, and the bacterial cells were disrupted by ultrasonic treatment. The result was analyzed using a laser diffraction / scattering particle size distribution analyzer (Microtrac MT3300EXII manufactured by Microtrac-Bell) to measure the particle sizes (D10, D50, D90) of the PHA particles. The measurement was performed under standard settings (particle transmittance: transmission, particle refractive index: 1.81, particle shape: aspherical, solvent refractive index: 1.333). From the measured D10, D50, and D90 values, (D90-D10) / D50 was calculated and used as the PHA particle size span.

[0061] (PHA production culture) PHA production culture was carried out as follows. First, a glycerol stock (50 μl) of the KNK-005 strain was inoculated into a seed medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (MDL-300 model, manufactured by Marubishi Bioengine) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 33°C, an agitation speed of 500 rpm, and an aeration rate of 1.8 L / min, and the culture was continued for 28 hours while controlling the pH between 6.7 and 6.8 to carry out preculture. A 14% aqueous ammonium hydroxide solution was used for pH control.

[0062] Next, the preculture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (Marubishi Bioengine MDS-U50) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 33°C, an agitation speed of 420 rpm, and an aeration rate of 2.1 L / min. The pH was controlled between 6.7 and 6.8. A 25% aqueous solution of ammonium hydroxide was used for pH control. The carbon source was added intermittently. Palm olein oil was used as the carbon source. The culture was continued until the PHA content reached approximately 90%. After completion of the culture, the culture solution was heat-treated at 70°C for 1 hour to inactivate the somatic cells and thermally aggregate the intracellular PHA particles. The number-average aspect ratio of the cell size and the PHA particle size span were measured as described above. The results are shown in Table 1. Figure 1 also shows a photograph of the cells taken during optical microscopy observation as described above. As a result of the investigation, it was found that the number average aspect ratio of the cell diameter of the KNK-005 strain after PHA accumulation was 1.55, and the cell shape was spherical.

[0063] (Comparative Example 1) Evaluation of Cell and PHA Particle Shape of minCD-Expressing A2405-Disrupted Strain The minCD-expressing A2405-disrupted strain was cultured under the same conditions as in Reference Example 1 to allow PHA to accumulate in the cells, and the culture solution was heat-treated at 70 ° C. for 1 hour to inactivate the bacterial cells and thermally aggregate the PHA particles in the cells. The minCD-expressing A2405-disrupted strain is a transformant in which the minCD gene (a gene encoding the cell division inhibitor enzyme described in SEQ ID NO: 7) derived from Capriavidus necator is introduced onto the chromosome of the KNK-005 strain, and the A2405 gene (a gene encoding the cell wall-degrading enzyme described in SEQ ID NO: 8) is disrupted, and can be produced in accordance with the method described in WO 2021 / 049207. The number-average aspect ratio of the cell diameter and the PHA particle diameter span were measured as described above. The results are shown in Table 1. Furthermore, a microscopic photograph of the cells observed as described above is shown in FIG. 2.

[0064] As a result of the investigation, the number-average aspect ratio of the cell diameter after PHA accumulation was 3.51, the cell shape was rod-like, and the PHA particle diameter span was 46% or more larger than that of Reference Example 1.

[0065] Example 1 Evaluation of PHA particle shape of minCD-expressing A2405 disruptant strain after cell wall-degrading enzyme treatment After the culture solution from Comparative Example 1 in which the bacterial cells had been inactivated was cooled to 50°C, egg white lysozyme (manufactured by Nagase ChemteX Corporation), a cell wall-degrading enzyme, was added in an amount equivalent to 0.0036% by weight of the bacterial cell components contained in the culture solution, and the mixture was stirred for 2 hours while controlling the pH at 6 to 7. The PHA particle size span was measured as described above. The results are shown in Table 1.

[0066] As a result of the investigation, it was found that the PHA particle size span in Example 1 was reduced by 13% or more compared to Comparative Example 1, and that the PHA particles were sphericalized by treatment with the cell wall-degrading enzyme.

[0067] Example 2 Evaluation of PHA particle shape of minCD-expressing A2405 disruptant strain after cell wall-degrading enzyme treatment and protease treatment While maintaining the culture solution after cell wall-degrading enzyme treatment in Example 1 at 50°C, the protease Alcalase (Novozymes) was added in an amount equivalent to 1.1% by weight of the amount of bacterial components contained in the culture solution, and the mixture was stirred for 2 hours while controlling the pH at 8.2 to 8.8. The PHA particle size span was measured as described above. The results are shown in Table 1.

[0068] As a result of the study, it was found that in Example 2, the PHA particle size span was reduced by more than 32% compared to Comparative Example 1, indicating that the PHA particles were further sphericalized by treatment with cell wall-degrading enzymes and protease.

[0069] It was confirmed by HPLC analysis that the PHA produced in Reference Example 1, Comparative Example 1, and Examples 1 and 2 was PHBH.

[0070] Comparative Example 2 Evaluation of Cell Shape and PHA Particle Shape of BW25113 phaCAB-Expressing Strain The BW25113 phaCAB-expressing strain was cultured under the following conditions to allow PHA to accumulate in the cells, and then the shapes of the cells and PHA particles were evaluated.

[0071] (Culture medium) The composition of the pre-culture medium was 1 w / v% Bacto-Tryptone, 0.5 w / v% Yeast extract, 1 w / v% NaCl12H2O (pH 6.8). The composition of the PHA production medium was 1 w / v% Bacto-Tryptone, 0.5 w / v% Yeast extract, 1 w / v% NaCl12H2O (pH 6.8), 2% Glucose.

[0072] (PHA Production Culture) PHA production culture was performed as follows. First, a glycerol stock (50 μl) of the BW25113 phaCAB-expressing strain was inoculated into a preculture medium (5 ml) and cultured at 37°C for 18 hours to perform seed culture. Next, the preculture solution was inoculated at 1.0 v / v% into a 500 mL Sakaguchi flask containing 100 mL of PHA production medium. Culture was performed at a culture temperature of 37°C and a shaking speed of 120 rpm. Culture was continued until the PHA content reached approximately 60%. After completion of culture, the culture solution was heat-treated at 60°C for 1 hour to inactivate the somatic cells and thermally aggregate the intracellular PHA particles. The number-average aspect ratio of the cell diameter and the PHA particle diameter span were measured as described above. The results are shown in Table 1. Furthermore, a photograph taken during microscopic observation of the cells as described above is shown in Figure 3.

[0073] As a result of the investigation, the number-average aspect ratio of the cell diameter after PHA accumulation was 3.02, and the cell shape was rod-like.

[0074] Example 3 Evaluation of PHA particle shape of BW25113 phaCAB-expressing strain after cell wall-degrading enzyme treatment After the culture solution from Comparative Example 2, which had been subjected to bacterial cell inactivation treatment, was cooled to 50°C, and egg white lysozyme (manufactured by Nagase ChemteX Corporation), a cell wall-degrading enzyme, was added in an amount equivalent to 0.0036% by weight of the bacterial cell components contained in the culture solution. The mixture was stirred for 2 hours while controlling the pH at 6 to 7. The PHA particle size span was measured as described above. The results are shown in Table 1.

[0075] As a result of the investigation, it was found that in Example 3, the PHA particle size span was reduced by 11% or more compared to Comparative Example 2, and it was found that the PHA particles were sphericalized by treatment with the cell wall-degrading enzyme.

[0076] Example 4 Evaluation of Cell and PHA Particle Shape of BW25113 phaCAB-Expressing Strain After Cell Wall-Degrading Enzyme Treatment and Protease Treatment While maintaining the culture solution after cell wall-degrading enzyme treatment in Example 3 at 50°C, the protease Alcalase (Novozymes) was added in an amount equivalent to 1.1% by weight of the amount of bacterial components contained in the culture solution, and the mixture was stirred for 2 hours while controlling the pH at 8.2 to 8.8. The PHA particle size span was measured as described above. The results are shown in Table 1.

[0077] As a result of the study, it was found that in Example 4, the PHA particle size span was reduced by more than 17% compared to Comparative Example 2, indicating that the PHA particles were further sphericalized by treatment with cell wall-degrading enzymes and protease.

[0078] It was confirmed by HPLC analysis that the PHA produced in Comparative Example 2 and Examples 3 and 4 was P(3HB).

[0079]

Claims

1. a step of culturing a polyhydroxyalkanoic acid-producing microorganism to obtain microbial cells that have accumulated polyhydroxyalkanoic acid; a step of heat-treating the microbial cells after the step of obtaining the microbial cells; and a step of treating the microbial cells with an enzyme after the heat treatment step; The microorganism belongs to the genus Capriavidus or Escherichia, The microbial cells have a number average aspect ratio of cell diameter of 2.0 or more, A method for producing a polyhydroxyalkanoic acid, wherein the enzyme comprises a cell wall-degrading enzyme.

2. The method according to claim 1 , wherein the enzyme further comprises a protease.

3. 3. The method according to claim 1, wherein the particle size span value of the polyhydroxyalkanoic acid after the enzyme treatment step is 10% or more smaller than the particle size span value of the polyhydroxyalkanoic acid before the enzyme treatment step.

4. The method according to claim 1 or 2, wherein the particle size span value of the polyhydroxyalkanoic acid after the enzyme treatment step is 0.85 or less.

5. The method according to claim 1 or 2, wherein the microorganism belongs to the genus Capriavidus.

6. The method according to claim 5, wherein the microorganism is a transformed microorganism of Capriavidus necator.

7. The method according to claim 1 or 2, wherein the microorganism belongs to the genus Escherichia.

8. The method according to claim 7, wherein the microorganism is a transformed Escherichia coli microorganism.

9. The method according to claim 1 or 2, further comprising, after the enzyme treatment step, a step of disrupting the microbial cells to obtain a cell lysate, and a step of recovering polyhydroxyalkanoic acid particles from the cell lysate.

10. 3. The method according to claim 1, wherein the polyhydroxyalkanoic acid is a polymer containing 3-hydroxybutyric acid as a monomer unit.

11. The method according to claim 1 or 2, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids.

12. The method according to claim 11, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

13. The method according to claim 12, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.