Method for producing copolymerized polyhydroxyalkanoate mixture, and transformed microorganism

Abstract

The present invention cultures a microorganism to produce a copolymerized polyhydroxyalkanoate mixture. The mixture contains a fraction (I) and a fraction (II), the fraction (I) contains a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is at least 9 mol% and less than 20 mol%; the fraction (II) contains a polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, the average 3-hydroxyhexanoic acid composition ratio is 0-8 mol%, and the weight ratio of the fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

Description

Technical Field

[0001] The present invention relates to a method for producing a copolymerized polyhydroxyalkanoate mixture and a transformation microorganism. Background Art

[0002] Polyhydroxyalkanoate (hereinafter sometimes referred to as "PHA") is a polyester-type organic polymer produced by a wide range of microorganisms. PHA is a biodegradable thermoplastic polymer that can be produced using renewable resources as raw materials. As a result, attempts have been made to use PHA as an environmentally friendly raw material or a biocompatible raw material for industrial production and use in various industries.

[0003] So far, it is known that a large number of microorganisms accumulate PHA in the bacteria as an energy storage material. As a representative example of P HA, poly-3-hydroxybutyric acid (hereinafter sometimes referred to as "P(3HB)"), which is a homopolymer of 3-hydroxybutyric acid (hereinafter sometimes referred to as "3HB"), can be cited. P(3HB) is a thermoplastic polymer, and because it is biodegradable in the natural environment, it has attracted much attention as an environmentally friendly plastic. However, P(3HB) has high crystallinity and therefore has hard and brittle properties, which limits its application range in actual use. In order to expand the scope of application, it is necessary to give P(3HB) flexibility.

[0004] In order to improve the flexibility of PHA, copolymerized polyhydroxyalkanoate (hereinafter also referred to as "P(3HB-co-3HH)") formed of 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as "3HH") and its production method have been studied (for example, refer to Patent Documents 1 and 2). In these reports, P(3HB-co-3HH) is produced by fermentation using a wild strain of Aeromonas caviae isolated from soil with fatty acids such as oleic acid and palmitic acid as a carbon source.

[0005] In addition, research has been conducted on the mass production of P(3HB-co-3HH) using Cupriavidus necator as a host and PHA synthase derived from Aeromonas caviae. By introducing an R-specific enoyl-CoA hydratase gene into Cupriavidus necator having PHA synthase derived from Aeromonas caviae, or by increasing the expression level of the R-specific enoyl-CoA hydratase gene on the host chromosome, P(3HB-co-3HH) is produced using plant oils and fats as raw materials, and the 3HH composition ratio of the P(3HB-co-3HH) can be increased to a maximum of about 14 mol% (see Patent Document 3, Patent Document 4 and Non-Patent Document 1).

[0006] In addition, there are also examples in which the 3HH composition ratio of P(3HB-co-3HH) was increased to more than 20 mol % by inhibiting the expression of a gene encoding β-ketothiolase for Cupric bacteria having a PHA synthase derived from Aeromonas caviae (see Patent Document 5), wherein the β-ketothiolase has thiolysis activity for β-ketoacyl-CoA having 6 carbon atoms (i.e., β-ketohexanoyl-CoA).

[0007] Research related to the physical properties of P(3HB-co-3HH) has also been conducted (see non-patent document 2). In this report, a fatty acid with a carbon number of 12 or more was used as the sole carbon source to culture Aeromonas caviae, and P(3HB-co-3HH) with various 3HH composition ratios was produced by fermentation. The crystallinity of P(3HB-co-3HH) decreases with the increase of the 3HH composition ratio, and therefore, the hard and brittle properties of P(3HB) gradually show soft properties. When the 3HH composition ratio is further increased, it shows higher softness than P(3HB-co-3HV). That is, P(3HB-co-3HH) can have a wide range of applicable properties from hard polymers to soft polymers by changing the 3HH composition ratio, and can be expected to be used in a wide range of fields.

[0008] On the other hand, although the crystallinity of P(3HB-co-3HH) decreases and the softness increases when the 3HH composition ratio is increased, there is a tendency for the processing characteristics to decrease. For example, although P(3HB-co-3HH) with a 3HH composition ratio increased to about 10 mol% is relatively soft, the crystallization rate is slow in the processes such as injection molding, film molding, blow molding, fiber spinning, extrusion foaming, and bead foaming, so there is a problem of low productivity.

[0009] In order to solve such a problem, research has been conducted to achieve a balance between processability and physical properties by mixing a plurality of PHAs having different monomer compositions (see Patent Document 6).

[0010] PHA produced by microorganisms accumulates in the microbial cells of the microorganisms. Therefore, in order to use PHA as a plastic, a process of isolating / purifying PHA from the microbial cells of the microorganisms is required. In the process of isolating / purifying PHA, after the microbial cells containing PHA are crushed or the biological components other than PHA are solubilized, PHA is taken out from the obtained aqueous suspension. At this time, separation operations such as centrifugation, filtration, and drying are performed. For the drying operation, a spray dryer, a fluidized bed dryer, a drum dryer, etc. can be used. From the perspective of ease of operation, a spray dryer is preferably used.

[0011] According to the experience of the inventors, by appropriately adjusting the hot air temperature during spray drying, the surface of the PHA particles is slightly melted and fused, and granulation can be performed into a particle size of the desired size. The granulated PHA is useful from the viewpoint of ease of handling and ease of transportation. The melting point of PHA depends on its copolymerization ratio. For example, PHA with a high 3HH composition ratio (about 9 mol% or more) has a low melting point, so the particle surface can be melted at a relatively low temperature for granulation. On the other hand, PHA with a low 3HH composition ratio (about 8 mol% or less) has a high melting point. In order to granulate it, the hot air temperature needs to be set to a high temperature. From the viewpoint of energy saving and safety, there is still room for improvement.

[0012] Patent Document 7 describes that a PHA powder having a large particle size can be produced even at a low hot air temperature by spray-drying an aqueous suspension containing PHA having a 3HH composition ratio of 0 to 8% and PHA having a 3HH composition ratio of 9 to 25% and having a pH of 7 or less.

[0013] In addition, there have been reports on the co-production of multiple PHAs in one microorganism (for example, see Patent Documents 8 to 10), but there is no disclosure on the production of PHAs that have a high melting point and are suitable for spray drying.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: Japanese Patent Application Laid-Open No. 5-93049

[0017] Patent Document 2: Japanese Patent Application Laid-Open No. 7-265065

[0018] Patent Document 3: International Publication No. 2011 / 105379

[0019] Patent Document 4: International Publication No. 2015 / 115619

[0020] Patent Document 5: International Publication No. 2019 / 142845

[0021] Patent Document 6: International Publication No. 2020 / 195550

[0022] Patent Document 7: International Publication No. 2021 / 251049

[0023] Patent Document 8: International Publication No. 2015 / 146195

[0024] Patent Document 9: International Publication No. 2017 / 056442

[0025] Patent Document 10: International Publication No. 2021 / 206155

[0026] Non-patent literature

[0027] Non-patent document 1: H. Arikawa, K. Matsumoto, Microb. Cell. Fact., 15, pp. 184 (2016)

[0028] Non-patent document 2: Y. Doi, S. Kitamura, H. Abe, Macromolecules, 28, pp. 4822-4823 (1995) Summary of the invention

[0029] Problems to be solved by the invention

[0030] As described above, there are problems in granulating PHA having a low 3HH composition ratio and a high melting point.

[0031] According to the method described in Patent Document 7, a PHA having a high melting point PHA as a main component and improved granulation properties can be obtained, but in this production, after two types of PHA are produced separately, each is purified, and after preparing respective aqueous suspensions, the combined aqueous suspensions are spray-dried (see each example). Since the two types of PHA need to be produced / purified separately, the efficiency is not high.

[0032] In view of the above-mentioned current situation, an object of the present invention is to provide an efficient method for producing a copolymerized polyhydroxyalkanoate mixture having a high melting point PHA as a main component and improved granulation properties.

[0033] Solution to the problem

[0034] The present inventors have conducted intensive studies to solve the above-mentioned problems and have found that a copolymerized polyhydroxyalkanoate mixture containing two polyhydroxyalkanoate fractions having a specific composition can be efficiently produced by culturing microorganisms, thereby completing the present invention.

[0035] That is, the present invention relates to a method for producing a copolymerized polyhydroxyalkanoate mixture, the method comprising: culturing a microorganism that produces the copolymerized polyhydroxyalkanoate mixture;

[0036] The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II).

[0037] The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 9 mol% or more and less than 20 mol%,

[0038] The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 0 mol% to 8 mol%,

[0039] The weight ratio of the polyhydroxyalkanoate fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

[0040] In addition, the present invention also relates to a transformed microorganism, which is a transformed microorganism that produces a copolymerized polyhydroxyalkanoate mixture.

[0041] The transformed microorganism has genes encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA.

[0042] The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II).

[0043] The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 9 mol% or more and less than 20 mol%,

[0044] The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 0 mol% to 8 mol%,

[0045] The weight ratio of the polyhydroxyalkanoate fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

[0046] Effects of the Invention

[0047] According to the present invention, a copolymerized polyhydroxyalkanoate mixture having a high melting point PHA as a main component and improved granulation properties can be efficiently produced. According to the present invention, it is not necessary to separately produce each polyhydroxyalkanoate contained in the copolymerized polyhydroxyalkanoate mixture and mix them, and the copolymerized polyhydroxyalkanoate mixture can be obtained by a single microbial culture / purification.

[0048] In addition, by simultaneously producing various polyhydroxyalkanoates in one microbial cell, a copolymerized polyhydroxyalkanoate mixture with an excellent mixing state can be obtained. Therefore, it is easy to ensure uniformity in the kneading / molding process for use as a plastic substitute product, which is also advantageous from the perspective of physical properties during actual use.

[0049] According to a preferred embodiment of the present invention, a powder of a copolymerized polyhydroxyalkanoate mixture can be obtained by spray drying. In addition, even at a relatively low drying temperature during spray drying, granulation into a desired particle size is possible, which is advantageous from the viewpoint of energy saving and safety. DETAILED DESCRIPTION

[0050] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments.

[0051] The present invention is a method for producing a copolymerized PHA mixture, comprising the step of culturing microorganisms that produce the copolymerized PHA mixture.

[0052] (Copolymer PHA mixture)

[0053] The copolymerized PHA mixture is composed of a PHA fraction (I) and a PHA fraction (II), wherein the PHA fraction (I) contains a copolymerized PHA having a 3HB structural unit and a 3HH structural unit, and the average 3HH composition ratio is 9 mol% or more and less than 20 mol%, and the PHA fraction (II) contains a PHA having a 3HB structural unit, and the average 3HH composition ratio is 0 mol% or more and 8 mol% or less. The PHA fraction (I) can be separated from the copolymerized PHA mixture by the MIBK fractionation method described later, and the average 3HH composition ratio of the PHA fraction (I) can be analyzed. In addition, by measuring the melting point using a differential scanning calorimeter (DSC), the average 3HH composition ratio of the PHA fraction (II) of the copolymerized PHA mixture can be analyzed.

[0054] The PHA fraction (I) is a fraction containing copolymerized PHA having at least 3HB structural units and 3HH structural units. The PHA fraction (I) may contain PHA containing hydroxyalkanoic acid structural units other than 3HB structural units and 3HH structural units, but is preferably a fraction containing copolymerized PHA containing only 3HB structural units and 3HH structural units without containing hydroxyalkanoic acid structural units other than 3HB structural units and 3HH structural units, that is, a fraction containing P(3HB-co-3HH).

[0055] Examples of hydroxyalkanoic acid structural units other than the 3HB structural unit and the 3HH structural unit include structural units of hydroxyalkanoic acids such as 3-hydroxypropionic acid, 3-hydroxyvaleric acid ("3HV"), 3-hydroxyalkanoic acids having 7 to 16 carbon atoms, 2-hydroxyalkanoic acids having 4 to 16 carbon atoms, 4-hydroxyalkanoic acid (e.g., 4-hydroxybutyric acid), 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid (e.g., 6-hydroxyhexanoic acid), and lactic acid, but are not limited thereto.

[0056] The average 3HH composition ratio in the PHA fraction (I) is 9 mol% or more, preferably 10 mol% or more. Thus, the PHA fraction (I) having a high average 3HH composition ratio has a low melting temperature. Therefore, even when the copolymerized PHA mixture is spray-dried at a relatively low drying temperature, it can be in a molten state. It can be inferred that the PHA fraction (I) in a molten state functions as a binder to aggregate the particles of the PHA fraction (II) in a non-molten state, resulting in a PHA powder with a large particle size even at a relatively low drying temperature.

[0057] The upper limit of the average 3HH composition ratio in the PHA fraction (I) is less than 20 mol %, preferably 19.9 mol % or less, more preferably 19 mol % or less, and further preferably 18 mol % or less.

[0058] The above-mentioned PHA fraction (II) is a fraction containing PHA having a 3HB structural unit. The PHA contained in the PHA fraction (II) may be a homopolymer having only a 3HB structural unit, or may be a copolymer PHA having a 3HB structural unit and a hydroxyalkanoic acid structural unit other than the 3HB structural unit. As the copolymer PHA, preferably a copolymer PHA having a 3HB structural unit, a 3HV structural unit and / or a 3HH structural unit, more preferably a copolymer PHA having a 3HB structural unit and a 3HH structural unit, and further preferably a copolymer PHA having only a 3HB structural unit and a 3HH structural unit, that is, P(3HB-co-3HH).

[0059] The average 3HH composition ratio in the PHA fraction (II) is 0 mol% or more and 8 mol% or less. Within this range, the balance between the softness and strength of the obtained copolymerized PHA mixture when it is made into a molded body can be made good. The upper limit of the average 3HH composition ratio is preferably 7 mol% or less, more preferably 6 mol% or less. As long as the lower limit of the average 3HH composition ratio is 0 mol% or more, it can be 0.1 mol% or more, or it can be 1 mol% or more. The average 3HH composition ratio can be 0 mol%.

[0060] The weight ratio of the PHA fraction (II) in the copolymerized PHA mixture is preferably 45 to 99%, more preferably 50 to 97%, further preferably 60 to 95%, and particularly preferably 70 to 90%. In addition, the weight ratio of the PHA fraction (I) in the copolymerized PHA mixture is preferably 1 to 55%, more preferably 3 to 50%, further preferably 5 to 40%, and particularly preferably 10 to 30%. When the ratio of each fraction is within the above range, the balance between the softness and strength of the obtained copolymerized PHA mixture when it is made into a molded body is good, and even at a relatively low drying temperature when the copolymerized PHA mixture is spray-dried, it can be granulated into a particle size of a desired size.

[0061] The average 3HH composition ratio of the copolymerized PHA mixture as a whole is preferably 0.5 to 14 mol%. When it is within this range, the balance between flexibility and strength when the copolymerized PHA mixture is formed into a molded body can be made good. The average 3HH composition ratio is more preferably 1 to 12 mol%, further preferably 1.5 to 10 mol%, further preferably 2 to 8 mol%, and particularly preferably 2.5 to 7 mol%.

[0062] (MIBK classification method)

[0063] A PHA fraction (I) having a high average 3HH composition ratio can be separated from the copolymerized PHA mixture by a solvent fractionation method utilizing the difference in solubility in methyl isobutyl ketone (MIBK). The higher the 3HH composition ratio of PHA, the higher the solubility in MIBK. Therefore, after the copolymerized PHA mixture is completely dissolved in high-temperature MIBK, the temperature is lowered to precipitate the PHA component having a low 3HH composition ratio, thereby obtaining the PHA fraction (I) as a soluble fraction.

[0064] The specific classification steps are described below. First, weigh about 100 mg of the copolymer PHA mixture into a screw-cap test tube, add 10 ml of MIBK and close the lid. Then, shake and mix at 140°C for about 0.5 to 3 hours and heat to completely dissolve the copolymer PHA mixture. After complete dissolution, place it at room temperature for 1 minute to lower the temperature below the boiling point, quickly transfer all the dissolved liquid to a centrifuge tube, and close the lid. The capped centrifuge tube is further placed in a 35°C water bath for 30 minutes to precipitate part of the dissolved matter. Separate the precipitate by centrifugation (9000 rpm, 5 minutes), and transfer all the centrifugal supernatant to a container such as an aluminum pan. Add 10 ml of MIBK to the centrifuge tube with the precipitate remaining, mix with a vortex mixer, centrifuge again (9000 rpm, 5 minutes), and add the centrifugal supernatant to the same aluminum pan or other container. At this time, there is precipitate remaining in the centrifuge tube. Then, the container was heated at 120°C for 30 minutes to volatilize MIBK and precipitate the dissolved matter in the centrifugal supernatant. Furthermore, the precipitate precipitated in the container and the precipitate remaining in the centrifuge tube were vacuum dried at 100°C for 6 hours. The precipitate precipitated in the container was recovered as PHA fraction (I). In addition, the precipitate remaining in the centrifuge tube was recovered as "PHA mainly composed of PHA fraction (II)".

[0065] (Method for measuring average 3HH composition ratio by gas chromatography)

[0066] The average 3HH composition ratio of each copolymer PHA mixture or PHA fraction (I) can be measured as follows. 1 ml of sulfuric acid-methanol mixed solution (15:85) and 1 ml of chloroform are added to about 1 to 20 mg of the dried copolymer PHA mixture or PHA fraction (I), and the mixture is sealed and heated at 100°C for 140 minutes to obtain the methyl ester of the PHA decomposition product. After cooling, 0.5 ml of deionized water is added thereto, mixed thoroughly, and allowed to stand until the aqueous layer and the organic layer are separated. Then, the monomer unit composition of the PHA decomposition product in the organic layer obtained is analyzed by capillary gas chromatography. The gas chromatograph used was GC-17A manufactured by Shimadzu Corporation, and the capillary column used was NEUTRA BOND-1 manufactured by GL Sciences (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). He was used as the carrier gas, the column inlet pressure was set to 100 kPa, and 1 μl of the sample was injected. The temperature conditions are as follows: the temperature is raised at an initial temperature of 50 to 200° C. at a rate of 8° C. / min, and further at a rate of 30° C. / min at 200 to 290° C. The average 3HH composition ratio of the copolymerized PHA mixture or the PHA fraction (I) is calculated from the peaks obtained by the analysis under the above conditions.

[0067] (Method for measuring average 3HH composition ratio by DSC)

[0068] The average 3HH composition ratio of the PHA fraction (II) can be measured as follows. Using a differential scanning calorimeter (DSC8500 manufactured by PerkinElmer), about 4 mg of "PHA mainly composed of PHA fraction (II)" was measured and measured. The heating / cooling conditions were set as follows: keep at 25°C for 1 minute, heat to 200°C, keep at 200°C for 1 minute, cool to 25°C, keep at 25°C for 1 minute, heat to 100°C, keep at 100°C for 60 minutes, cool to 25°C, keep at 25°C for 1 minute, heat to 200°C, and the heating / cooling rate was 10°C / min. The temperature of the maximum melting peak obtained at the last heating was determined. Using a calibration curve prepared in advance based on the melting peak analysis results of multiple PHAs with different 3HH composition ratios, the average 3HH composition ratio of the PHA fraction (II) was calculated based on the temperature of the above-mentioned maximum melting peak.

[0069] (Calculation method of weight ratio in copolymer PHA mixture)

[0070] The weight ratio of the PHA fraction (I) to the PHA fraction (II) in the copolymerized PHA mixture can be determined as follows.

[0071] Based on the calculated average 3HH composition ratios of the copolymer PHA mixture, PHA fraction (I) and PHA fraction (II), the weight ratio at which the PHA fraction (I) and the PHA fraction (II) are mixed to obtain the same average 3HH composition ratio of the copolymer PHA mixture is calculated, and the weight ratio of the PHA fraction (I) to the PHA fraction (II) in the copolymer PHA mixture is calculated.

[0072] (Copolymer PHA mixture production microorganisms)

[0073] The microorganism used in the production of the copolymerized PHA mixture (hereinafter also referred to as "copolymerized PHA mixture producing microorganism") is not particularly limited as long as it is a microorganism capable of fermenting and producing the copolymerized PHA mixture. It may be a wild strain that originally accumulates PHA, a mutant strain obtained by artificially mutating such a wild strain, or a strain endowed with the ability to accumulate PHA by introducing an external PHA synthase gene using genetic engineering methods.

[0074] The copolymerized PHA mixture producing microorganism, or when the microorganism is a transformant, the host of the transformant is not particularly limited, and for example, bacteria belonging to the genus Ralstonia, the genus Cupriavidus, the genus Wautersia, the genus Aeromonas, the genus Escherichia, the genus Alcaligenes, the genus Pseudomonas, etc. are listed as preferred examples. From the viewpoint of safety and PHA productivity, bacteria belonging to the genus Ralstonia, the genus Cupriavidus, the genus Aeromonas, and the genus Wautersia are more preferred, bacteria belonging to the genus Cupriavidus or the genus Aeromonas are further preferred, bacteria belonging to the genus Cupriavidus, and Cupriavidus necator is particularly preferred.

[0075] The copolymerized PHA mixture producing microorganism is preferably a microorganism that can efficiently produce two kinds of PHAs with different average composition ratios of 3HH, and has genes encoding two kinds of PHA synthases with different polymerization activities for (R)-3-hydroxyhexanoyl-CoA. (R)-3-Hydroxyhexanoyl-CoA is a precursor of the 3HH structural unit contained in PHA. By making the above-mentioned microorganism have genes encoding two kinds of PHA synthases with different polymerization activities for (R)-3-hydroxyhexanoyl-CoA, a mixture of PHAs with different average composition ratios of 3HH, that is, the copolymerized PHA mixture, can be fermented and produced in the cells of the microorganism. The above-mentioned microorganism only needs to have at least two genes encoding PHA synthases with different polymerization activities for (R)-3-hydroxyhexanoyl-CoA, and can also have more than three of the above genes as long as it can ferment and produce the above-mentioned copolymerized PHA mixture.

[0076] The two PHA synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA are not particularly limited, and the sequence identity of the amino acid sequences between the two PHA synthases is preferably 90% or less, more preferably 80% or less, and further preferably 70% or less. It is generally believed that PHA synthases function by forming multimers such as dimers. When the sequence identity of the amino acid sequences between the two PHA synthases is higher than 90%, it is believed that the two PHA synthases form a heterodimer and the copolymerized PHA mixture may not be produced.

[0077] Of the two types of PHA synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA, the gene encoding the PHA synthase having relatively high polymerization activity for (R)-3-hydroxyhexanoyl-CoA and synthesizing copolymerized PHA having 3HB structural units and 3HH structural units is set as gene (A), and the gene encoding the PHA synthase having lower polymerization activity for (R)-3-hydroxyhexanoyl-CoA than gene (A) is set as gene (B), and the expression level of gene (A) in the copolymerized PHA mixture producing microorganism is preferably lower than the expression level of gene (B). When the expression level of gene (A) is high, the ratio of high melting point PHA (PHA fraction (II)) to be the main component may be less than 45%.

[0078] The copolymerized PHA mixture producing microorganism is preferably a transformant adjusted so that the expression level of gene (A) is less than the expression level of gene (B). The method of adjusting the expression level of a gene is not particularly limited, and examples thereof include adjustment of the position of the target gene to be introduced, selection of the promoter type of the target gene, and the like.

[0079] The promoter used for regulating the expression amount includes, but is not particularly limited to, lac promoter, lacUV5 promoter, trp promoter, trc promoter, or variants thereof. In addition, instead of using an external promoter, a promoter sequence originally existing near the position where the target gene is inserted on genomic DNA may be used. In addition, the above-mentioned promoter sequence originally existing and an external promoter may be used in combination.

[0080] As the above-mentioned gene (A), for example, a polyhydroxyalkanoate synthase gene or a mutant thereof derived from a microorganism of the genus Aeromonas can be cited, and specifically, a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in any one of sequence numbers 1 to 8 (amino acid sequence of a PHA synthase mutant derived from bacteria of the genus Aeromonas) can be cited.

[0081] As another example of the above-mentioned gene (A), there can be cited a polyhydroxyalkanoate synthase gene or a mutant thereof identified by metagenome analysis, specifically a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in sequence number 9 or sequence number 44.

[0082] The sequence identity with respect to the gene (A) is preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and most preferably 99.5% or more.

[0083] As the above-mentioned gene (B), there can be mentioned a gene which is a combination of a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas and a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Cupria. Specifically, there can be mentioned a gene which encodes an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence recorded in sequence number 10 or sequence number 11.

[0084] In addition, as another example of the above-mentioned gene (B), a PHA synthase gene derived from a bacterium of the genus Chromobacterium or a mutant thereof can also be cited, specifically a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence recorded in SEQ ID NO: 12 or SEQ ID NO: 13.

[0085] As another example of the above-mentioned gene (B), a polyhydroxyalkanoate synthase gene or a mutant thereof identified by metagenomic analysis can be cited, specifically a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence recorded in sequence number 9 or sequence number 44.

[0086] The sequence identity with the gene (B) is preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0087] As the combination of gene (A) and gene (B), a combination may be selected in which the PHA synthase encoded by gene (B) has a lower polymerization activity for (R)-3-hydroxyhexanoyl-CoA than the PHA synthase encoded by gene (A).

[0088] In order to efficiently produce a copolymerized PHA mixture having a high average composition ratio of 3HH, the copolymerized PHA mixture producing microorganism is preferably a microorganism having a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity. R-specific enoyl-CoA hydratase has the function of converting hexenoyl-CoA to (R)-3-hydroxyhexanoyl-CoA in microbial cells. Therefore, it can be inferred that by making the above-mentioned microorganism have a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity, the amount of conversion to (R)-3-hydroxyhexanoyl-CoA will increase, resulting in an increase in the average 3HH composition ratio in the produced copolymerized PHA mixture.

[0089] The microorganism having a gene encoding a protein exhibiting the above-mentioned R-form-specific enoyl-CoA hydratase activity may be a microorganism originally having the gene or a microorganism into which an exogenous gene has been introduced by a genetic engineering method.

[0090] Examples of genes encoding a protein having R-specific enoyl-CoA hydratase activity that are inherent to the microorganism include, for example, phaJ4a gene and phaJ4b gene when the microorganism is Cupriavidus insecticidalis.

[0091] Examples of the exogenous gene encoding a protein having R-specific enoyl-CoA hydratase activity include a gene derived from Aeromonas caviae encoding an R-specific enoyl-CoA hydratase having an amino acid sequence as set forth in SEQ ID NO: 14, a gene derived from Cupriavidus insecticidalis encoding an R-specific enoyl-CoA hydratase having an amino acid sequence as set forth in SEQ ID NO: 15 or SEQ ID NO: 16, a Multifunctional enzyme type 2 (MFE2) gene derived from Yarrowia lipolytica encoding an enzyme having an amino acid sequence as set forth in SEQ ID NO: 17, a gene derived from Drosophila melanogaster encoding an enzyme having an amino acid sequence as set forth in SEQ ID NO: 18, and a gene derived from Drosophila melanogaster encoding an enzyme having an amino acid sequence as set forth in SEQ ID NO: 19. melanogaster), or a gene encoding a protein having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more to any of the amino acid sequences described in SEQ ID NOs: 14 to 18, and having R-specific enoyl-CoA hydratase activity, etc., but is not limited thereto.

[0092] In order to efficiently produce a copolymerized PHA mixture having a high average composition ratio of 3HH, the copolymerized PHA mixture producing microorganism is preferably a transformed microorganism that has undergone transformation such that the supply of (R)-3-hydroxyhexanoyl-CoA to PHA synthase in the cell is increased compared to a wild-type strain of the microorganism.

[0093] An example of a transformation microorganism transformed in a mode where the supply of (R)-3-hydroxyhexanoyl-CoA is increased can be a transformation microorganism transformed in a mode where the expression of a gene encoding a protein with R-specific enoyl-CoA hydratase activity is enhanced. In order to enhance the expression of this gene, for example, modification of an expression regulatory sequence (promoter sequence and / or SD sequence) for enhancing the expression of this gene can be performed as described in International Publication No. 2015 / 115619. In addition, for this gene, expression of a protein with R-specific enoyl-CoA hydratase activity can be improved by using a vector or by introducing chromosomal DNA as described in International Publication No. 2011 / 105379.

[0094] Another example of a transformed microorganism transformed in such a manner that the supply of (R)-3-hydroxyhexanoyl-CoA is increased is a transformed microorganism transformed in which the decomposition of the intermediate metabolite with a carbon number of 6 in the β-oxidation of oils or fatty acids is suppressed compared to a wild-type strain of the microorganism. As a result of suppressing the decomposition of the intermediate metabolite with a carbon number of 6 in the β-oxidation, the supply of (R)-3-hydroxyhexanoyl-CoA is increased, and the average 3HH composition ratio in the produced copolymer PHA mixture is increased.

[0095] As an example of a transformed microorganism transformed in such a manner that the decomposition of an intermediate metabolite having 6 carbon atoms in β-oxidation of fats and oils or fatty acids is suppressed, there can be mentioned, for example, a transformed microorganism transformed in such a manner that the expression of a gene encoding β-ketothiolase having thiolysis activity for β-ketohexanoyl-CoA, which is β-ketoacyl-CoA having 6 carbon atoms, is suppressed, as described in Patent Document 5. It should be noted that the transformed microorganism can be exemplified by two types of transformations: transformation for suppressing the expression of a gene encoding a β-ketothiolase having thiolysis activity for β-ketohexanoyl-CoA, and transformation for enhancing the expression of a gene encoding a protein having the above-mentioned R-body-specific enoyl-CoA hydratase activity.

[0096] Examples of the gene encoding β-ketothiolase include, for example, the bktB gene and the A1528 gene, and specifically, a gene encoding β-ketothiolase having an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence described in SEQ ID NO: 19 or SEQ ID NO: 20, but the present invention is not limited thereto. The sequence identity is preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0097] In order to inhibit the expression of the gene encoding β-ketothiolase, for example, there can be mentioned a method of completely deleting the enzyme gene in the transformed microorganism, a method of inserting a completely different gene such as a drug resistance gene into the sequence of the enzyme gene, or a method of deleting, replacing, adding or inserting a part of the sequence of the enzyme gene (preferably a region related to enzyme activity), etc. Gene disruption operations include, for example, homologous recombination techniques using vectors containing disruptive genes or disruptive DNA, techniques using transposons, etc. Alternatively, as other methods of destruction, there can be cited known techniques such as the CRISPR / Cas (e.g., Cas9) system for destroying target genes, and TALEN-based genome editing technology (Y. Wang et al., ACS Synth Biol. 2016, 5(7): 721-732; Bogdanove and Voytas, Science, 333: 1843-1846, 2011; Jinek, et al., Science, 337: 816-821, 2012; Shalem, et al., Science, 343: 84-87, 2014; Wang, et al., Science, 343: 80-84, 2014). For example, in the CRISPR / Cas9 system, the guide RNA (gRNA) has a sequence that can bind to a portion of the base sequence of the β-ketothiolase gene to be destroyed, and has the function of targeted transport of Cas9. In addition, the enzyme activity can be eliminated or reduced by mutations such as deletion, substitution, addition, or insertion of the base sequence around the gene, thereby reducing the gene transfer / translation efficiency and mRNA stability.

[0098] In the case where an exogenous gene is introduced into the copolymerized PHA mixture producing microorganism, the introduced gene may be present on the chromosome possessed by the microorganism serving as the host, or on the DNA of a plasmid, a megaplasmid, etc. From the viewpoint of maintaining the introduced gene, it is preferably present on the chromosome or megaplasmid possessed by the microorganism, and more preferably on the chromosome possessed by the microorganism. In addition, in the case of increasing the expression level of a gene originally maintained by the microorganism serving as the host, the expression level of the gene may also be increased by replacing, deleting or adding the base sequence upstream of the gene.

[0099] Methods for site-specifically replacing or inserting arbitrary DNA into DNA possessed by a microorganism, or methods for deleting arbitrary sites in DNA possessed by a microorganism are well known to those skilled in the art and can be used when producing the transformed microorganism of the present invention. Although not particularly limited, representative methods include: a method utilizing a transposon and homologous recombination mechanism (Ohman et al., J. Bacteriol., vol. 162: p. 1068 (1985)), a method based on the principle of site-specific introduction caused by a homologous recombination mechanism and shedding based on a second-stage homologous recombination (Noti et al., Methods Enzymol., vol. 154, p. 197 (1987)), and a method in which a microbial strain in which the sacB gene derived from Bacillus subtilis coexists and the gene is shedding by a second-stage homologous recombination is easily separated into a sucrose-supplemented medium-tolerant strain (Schweizer, Mol. Microbiol., vol. 6, p. 1195 (1992); Lenz et al., J. Bacteriol., vol. 176, p. 4385 (1994)). The method for introducing the vector into cells is not particularly limited, and examples thereof include the calcium chloride method, the electroporation method, the polyethylene glycol method, and the spheroplast method.

[0100] For gene cloning and gene recombination techniques, techniques described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989 or 2001) and the like can be used.

[0101] The promoter used to express the introduced gene is not particularly limited. The promoter of the phaC1 gene of Cupricinus entomopathogenis, the promoter of the phaP1 gene, the trp promoter, the lac promoter, the lacUV5 promoter, the trc promoter, the tic promoter, the tac promoter derived from Escherichia coli, or the artificially prepared lacN17 promoter having the modified base sequence derived from Escherichia coli shown in SEQ ID NO: 21, the artificially prepared lacN19 promoter having the modified base sequence derived from Escherichia coli shown in SEQ ID NO: 22, etc. can be used.

[0102] (Cultivation of Microorganisms)

[0103] By culturing the copolymerized PHA mixture producing microorganism, the copolymerized PHA mixture can be accumulated in the microbial cells. As a method for culturing the copolymerized PHA mixture producing microorganism, it can be based on a common microbial culture method, as long as it is cultured in a culture medium with an appropriate carbon source. There are no particular limitations on the composition of the culture medium, the method of adding the carbon source, the culture scale, the aeration and stirring conditions, the culture temperature, the culture time, etc. The carbon source is preferably added to the culture medium continuously or intermittently.

[0104] As a carbon source during cultivation, any carbon source can be used as long as the copolymer PHA mixture producing microorganism can assimilate it. Although not particularly limited, examples thereof include: sugars such as glucose, fructose, sucrose, and xylose; palm oil, palm kernel oil (including palm olein, palm double olein, and palm kernel olein, etc., which are low-melting-point fractions obtained by separating them), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, their fractionated oils, or their purified byproducts; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, their derivatives, or glycerol, etc. In addition, in the case where the copolymer PHA mixture producing microorganism can utilize gases such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, and alcohols, these can also be used as carbon sources. Among them, the carbon source preferably contains oils (especially vegetable oils) or fatty acids.

[0105] In the manufacture of the copolymerized PHA mixture, it is preferred to culture the microorganisms using a culture medium containing the carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Although not limited to the following, examples of nitrogen sources include: ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like. Examples of inorganic salts include: potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, and the like. Examples of other organic nutrient sources include: amino acids such as glycine, alanine, serine, threonine, and proline, vitamins such as vitamin B1, vitamin B12, and vitamin C, and the like.

[0106] After the copolymer PHA mixture producing microorganism is cultured for an appropriate period of time so that the copolymer PHA mixture is accumulated in the microbial cells, the copolymer PHA mixture is recovered using a known method. There is no particular limitation on the recovery method, but industrially, recovery based on separation / purification in a water system with low environmental burden is preferred. For example, after the culture is completed, the cells are broken by applying a mechanical shear force or using a surfactant, alkali, enzyme, etc., thereby obtaining a cell lysate in which the cell components other than PHA are dissolved in water. The copolymer PHA mixture is separated from the aqueous phase by filtering and centrifuging the cell lysate and then dried, and the copolymer PHA mixture can be recovered.

[0107] (Spray Drying)

[0108] According to a preferred embodiment of the present invention, the copolymerized PHA mixture can be purified by disrupting the cells after the culture is completed, and an aqueous suspension of the copolymerized PHA mixture is obtained, and the aqueous suspension is spray-dried to obtain a powder of the copolymerized PHA mixture. In addition, since PHA can be obtained in the form of powder, PHA with excellent handling properties can be obtained with high efficiency.

[0109] The aqueous suspension of the copolymerized PHA mixture is a liquid obtained by dispersing the high-purity copolymerized PHA mixture after removing cell-derived components other than PHA in an aqueous medium. The aqueous suspension contains water as an aqueous medium, and may contain an organic solvent (e.g., ethanol, ethanol, acetone, etc.) that is compatible with water in addition to water.

[0110] The concentration of the copolymerized PHA mixture in the aqueous suspension can be set from the viewpoint of improving productivity during spray drying and the fluidity of the aqueous suspension, and is specifically preferably 30 to 65% by weight, more preferably 40 to 60% by weight.

[0111] In addition, the aqueous suspension may further contain a dispersant. Specific examples of the dispersant include oxyalkylene dispersants described in International Publication No. 2021 / 251049, polyvinyl alcohol, and the like.

[0112] The aqueous suspension can be obtained, for example, as follows. The copolymer PHA mixture-producing microorganism is cultured to allow the copolymer PHA mixture to accumulate in microbial cells, and then the cells are disrupted to obtain a cell disrupted liquid. Next, after dehydration by filtration, centrifugation, etc. as needed, purification treatment is performed to decompose or remove cell-derived components other than PHA. After washing the obtained copolymer PHA mixture with water or the like as needed, an aqueous medium containing water is added or removed as needed to adjust the concentration, thereby obtaining an aqueous suspension of the copolymer PHA mixture. For detailed steps, reference can be made to, for example, International Publication No. 2010 / 067543.

[0113] The aqueous suspension of the copolymerized PHA mixture is spray-dried to obtain a powder of the copolymerized PHA mixture. The spray drying can be performed, for example, by supplying the aqueous suspension of the copolymerized PHA mixture in the form of fine droplets to a dryer, and drying it by contacting it with hot air in the dryer.

[0114] The temperature of the hot air used in the spray drying is not particularly limited, and can be appropriately selected within the range of 100 to 300° C. From the viewpoint of reducing the energy used in the spray drying, the upper limit is preferably 200° C. or less, and more preferably 180° C. or less. In addition, the exhaust temperature (exhaust air temperature) when discharged from the spray dryer is not particularly limited, but from the viewpoint of controlling the particle size by fusing the surface of the PHA particles, it is preferably 80° C. or more, and more preferably 90° C. or more.

[0115] According to a preferred embodiment, a PHA powder having a large particle size can be obtained at a lower drying temperature during spray drying, and as a result, the cost (equipment cost, practicality) of the drying process can be reduced.

[0116] Preferred aspects of the present disclosure are listed in the following items, but the present invention is not limited to the following items.

[0117] [Project 1]

[0118] A method for producing a copolymerized polyhydroxyalkanoate mixture, the method comprising:

[0119] a step of culturing a microorganism that produces the copolymerized polyhydroxyalkanoate mixture,

[0120] The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II),

[0121] The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 9 mol% or more and less than 20 mol%,

[0122] The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 0 mol% to 8 mol%,

[0123] The weight ratio of the polyhydroxyalkanoate fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

[0124] [Project 2]

[0125] The manufacturing method according to item 1, wherein:

[0126] In the copolymerized polyhydroxyalkanoate mixture, an average 3-hydroxyhexanoic acid composition ratio in the entire mixture is 0.5 to 14 mol %.

[0127] [Item 3]

[0128] The manufacturing method according to item 1 or 2, wherein:

[0129] The microorganism has genes encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA.

[0130] [Item 4]

[0131] The manufacturing method according to item 3, wherein:

[0132] The amino acid sequence identity between the two polyhydroxyalkanoate synthetases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA is 90% or less.

[0133] [Item 5]

[0134] The manufacturing method according to item 3 or 4, wherein:

[0135] The genes encoding the two polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA are gene (A) and gene (B),

[0136] The gene (A) encodes a polyhydroxyalkanoate synthase that synthesizes a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit,

[0137] The gene (B) encodes a polyhydroxyalkanoate synthase having a lower polymerization activity for (R)-3-hydroxyhexanoyl-CoA than the gene (A).

[0138] [Item 6]

[0139] The manufacturing method according to item 5, wherein:

[0140] The microorganism is regulated so that the expression level of the gene (A) is lower than the expression level of the gene (B).

[0141] [Item 7]

[0142] The manufacturing method according to item 5 or 6, wherein:

[0143] The gene (A) is a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas or a mutant thereof.

[0144] [Item 8]

[0145] The manufacturing method according to item 7, wherein:

[0146] The gene (A) is a gene encoding an amino acid sequence having a sequence identity of 99.5% to 100% with respect to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8.

[0147] [Item 9]

[0148] The production method according to any one of items 5 to 8, wherein

[0149] The gene (B) is composed of a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas and a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Cupriavidus.

[0150] [Item 10]

[0151] The manufacturing method according to item 9, wherein:

[0152] The gene (B) is a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0153] [Item 11]

[0154] The production method according to any one of items 5 to 8, wherein

[0155] The gene (B) is a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Chromobacterium or a mutant thereof.

[0156] [Item 12]

[0157] The manufacturing method according to item 11, wherein:

[0158] The gene (B) is a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13.

[0159] [Item 13]

[0160] The production method according to any one of items 5 to 12, wherein

[0161] Any one of the gene (A) and the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 44.

[0162] [Item 14]

[0163] The production method according to any one of items 1 to 13, wherein

[0164] The microorganism is a microorganism having a gene encoding a protein exhibiting R-body-specific enoyl-CoA hydratase activity.

[0165] [Item 15]

[0166] The production method according to any one of items 1 to 14, wherein

[0167] The microorganism is a transformed microorganism that has been transformed so that the supply of (R)-3-hydroxyhexanoyl-CoA to polyhydroxyalkanoate synthase in the cell is increased compared to a wild-type strain of the microorganism.

[0168] [Item 16]

[0169] The manufacturing method according to item 15, wherein:

[0170] The transformed microorganism is transformed so that the expression of a gene encoding a protein exhibiting R-body-specific enoyl-CoA hydratase activity is enhanced.

[0171] [Item 17]

[0172] The manufacturing method according to item 15 or 16, wherein:

[0173] The transformed microorganism is transformed in which the expression of a gene encoding β-ketothiolase having a thiolytic activity on β-ketohexanoyl-CoA, which is β-ketoacyl-CoA having 6 carbon atoms, is suppressed.

[0174] [Item 18]

[0175] The production method according to any one of items 1 to 17, wherein

[0176] In the culturing step, a carbon source including oil or fatty acid is added.

[0177] [Item 19]

[0178] The production method according to any one of items 1 to 18, wherein

[0179] The microorganism belongs to the genus Cupriavidus, or is a transformant of a microorganism of the genus Cupriavidus.

[0180] [Item 20]

[0181] The manufacturing method according to item 19, wherein:

[0182] The microorganism is Copperobacterium spp. or a transformant of Copperobacterium spp.

[0183] [Item 21]

[0184] The manufacturing method according to any one of items 1 to 20, comprising:

[0185] After culturing the microorganism, the cells of the microorganism are disrupted and purified to obtain an aqueous suspension of the copolymerized polyhydroxyalkanoate mixture; and

[0186] A step of obtaining a powder of the copolymerized polyhydroxyalkanoate mixture by spray-drying the aqueous suspension.

[0187] [Item 22]

[0188] A transformed microorganism which produces a copolymerized polyhydroxyalkanoate mixture,

[0189] The transformed microorganism has genes encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA.

[0190] The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II),

[0191] The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 9 mol% or more and less than 20 mol%,

[0192] The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 0 mol% to 8 mol%,

[0193] The weight ratio of the polyhydroxyalkanoate fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

[0194] Example

[0195] Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited by these examples. It should be noted that all gene manipulations can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). In addition, the enzymes and cloning hosts used in gene manipulations can be purchased from suppliers in the market and used according to their instructions. It should be noted that, as enzymes, as long as they can be used in gene manipulations, there is no particular limitation.

[0196] (Microbial strain preparation example 1) Preparation of microbial strain (1) for producing copolymerized PHA mixture

[0197] First, a plasmid for PHA synthase gene disruption was prepared as follows.

[0198] By using PCR with synthetic oligo DNA, a DNA fragment (SEQ ID NO: 23) having the upstream and downstream base sequences of the phaC1 structural gene (PHA synthase gene) of the H16 strain of Copperworm was obtained. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+phaC1UD for PHA synthase gene disruption.

[0199] Next, a PHA synthase gene-disrupted strain was prepared as follows using the PHA synthase gene-disrupted plasmid vector pNS2X-sacB+phaC1UD.

[0200] Escherichia coli S17-1 strain (ATCC47055) was transformed with the PHA synthase gene disruption plasmid vector pNS2X-sacB+phaC1UD, and the transformed microorganisms obtained and KNK005trc-phaJ4b / ΔphaZ1, 2, 6 strains (hereinafter also referred to as "KNK005dZ / trc-J4b strains") were mixed cultured on Nutrient Agar medium (manufactured by Difco) to perform conjugation transfer.

[0201] It should be noted that the KNK005dZ / trc-J4b strain is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-corrupted bacteria H16 strain are deleted, the PHA synthase gene on the chromosome is replaced with a modified version of the PHA synthase gene derived from the genus Aeromonas (a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 2, i.e., the N149S / D171G mutant (NSDG) gene), and the expression of the R-body-specific enoyl-CoA hydratase gene (phaJ4b gene) on the chromosome is enhanced. It can be prepared according to the method described in PCT International Publication No. 2015 / 115619.

[0202] The obtained culture solution was inoculated on Simmons' agar medium (sodium citrate 2g / L, sodium chloride 5g / L, magnesium sulfate heptahydrate 0.2g / L, ammonium dihydrogen phosphate 1g / L, dipotassium hydrogen phosphate 1g / L, agar 15g / L, pH 6.8) containing 250mg / L of kanamycin, and strains that can grow on agar medium were selected to obtain strains into which plasmids were introduced on the chromosome of KNK005dZ / trc-J4b strain. After the strains were cultured for two generations with Nutrient Broth medium (manufactured by Difco), they were diluted and coated on Nutrient Agar medium containing 15% sucrose, and strains that can grow were obtained as strains after plasmid shedding. Further, through analysis based on PCR and DNA sequencer, a strain in which the PHA synthase gene on the chromosome was deleted was isolated. The gene-destroyed strain was named KNK005dZ / dNSDG / trc-J4b strain.

[0203] Furthermore, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0204] By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 24) was obtained, which has the base sequence of the upstream and downstream of the B1168 structural gene (unknown function) of the H16 strain of Copperworm, the trp promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in sequence number 12. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which was also digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1168U-trp-phaCcsA479W-B1168D for introducing the PHA synthase gene.

[0205] Next, the plasmid vector pNS2X-sacB+B1168U-trp-phaCcsA479W-B1168D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the trp promoter and the gene encoding the PHA synthase having the amino acid sequence recorded in SEQ ID NO: 12 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W strain. It should be noted that it was confirmed that the deletion of the B1168 gene did not affect the growth and PHA biosynthesis.

[0206] Furthermore, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0207] By using PCR with synthetic oligo DNA, a DNA fragment (SEQ ID NO. 25) was obtained, which has a base sequence upstream of the A2712 structural gene (function unknown) of the H16 strain of Cupricobacterium spp. and a part of the A2712 structural gene, and a base sequence of a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+A2712U-phaCa mSGLVNE-A2712 for introducing a PHA synthase gene.

[0208] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNE-A2712, a PHA synthase gene-transferred strain was prepared as follows.

[0209] The plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNE-A2712 for PHA synthase gene introduction was introduced into the KNK005dZ / dNSD G / trc-J4b / B1168::trp-phaCcsA479W strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain in which a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO: 2 was introduced upstream of the A2712 gene on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / B1168::trp-pha CcsA479W / PA2712-phaCamSGLVNE strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (1)").

[0210] The copolymer PHA mixture producing microbial strain (1) is the following strain: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene (phaJ4b gene) on the chromosome is enhanced, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 are introduced into the strain.

[0211] (Microbial strain preparation example 2) Preparation of microbial strain (2) for producing copolymerized PHA mixture

[0212] First, a plasmid for bktB gene disruption was prepared as follows.

[0213] By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 26) was obtained, which has the upstream and downstream base sequences of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Copperworm. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which was also digested with SwaI, by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+bktBUD for bktB gene disruption.

[0214] Next, using the plasmid vector pNS2X-sacB+bktBUD for bktB gene disruption, a bktB gene disrupted strain was prepared as follows.

[0215] The plasmid vector pNS2X-sacB+bktBUD for bktB gene disruption was introduced into the KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W / PA2712-phaCamSGLVNE strain (copolymer PHA mixture producing microbial strain (1)) by the same method of conjugation transfer as described above. Furthermore, a strain in which the bktB gene on the chromosome was deleted was isolated by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W / PA2712-phaCamSGLVNE / dbkt B strain (hereinafter also recorded as "copolymer PHA mixture producing microbial strain (2)").

[0216] The copolymer PHA mixture producing microbial strain (2) is the following strain: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 are introduced, and the bktB gene on the chromosome is deleted.

[0217] (Microbial strain preparation example 3) Preparation of microbial strain (3) for producing copolymerized PHA mixture

[0218] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0219] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 27) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricobacterium spp. and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sac B described in Japanese Patent Application Laid-Open No. 2007-259708 which had also been digested with SwaI by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNE-bktBD for introducing the PHA synthase gene.

[0220] Next, using the plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNE-bktBD for PHA synthase gene introduction, a PHA synthase gene-introduced strain was prepared as follows.

[0221] The plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNE-bktBD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the bktB gene on the chromosome was deleted and a gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 was introduced at the position where the bktB gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W / bktB::phaCamSGLVNE strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (3)").

[0222] The copolymer PHA mixture producing microbial strain (3) is the following strain: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 are introduced, and the bktB gene on the chromosome is deleted.

[0223] (Microbial strain preparation example 4) Preparation of microbial strain (4) for producing copolymerized PHA mixture

[0224] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0225] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 28) was obtained, which had a base sequence upstream of the A2712 structural gene (function unknown) of the H16 strain of Cupricinus entomopathogenis and a part of the A2712 structural gene, a lac promoter, and a base sequence of a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with restriction enzyme SwaI, and the obtained 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 using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+A2712U-lac-phaCamSGLVNE-A2712 for introducing a PHA synthase gene.

[0226] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+A2712U-la c-phaCamSGLVNE-A2712, a PHA synthase gene-transferred strain was prepared as follows.

[0227] The plasmid vector pNS2X-sacB+A2712U-lac-phaCamSGLVNE-A2712 for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain was isolated in which a lac promoter and a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO: 2 were introduced upstream of the A2712 gene on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W / PA2712-lac-phaCamSGLVNE strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (4)").

[0228] The copolymer PHA mixture producing microbial strain (4) is the following strain: the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 are introduced into the strain.

[0229] (Microbial strain preparation example 5) Preparation of microbial strain (5) for producing copolymerized PHA mixture

[0230] First, using the PHA synthase gene disruption plasmid vector pNS2X-sacB+phaC1UD, a PHA synthase gene disrupted strain was prepared as follows.

[0231] The plasmid vector pNS2X-sacB+phaC1UD for PHA synthase gene disruption was introduced into the KNK005dZ / trc-J4b / dbktB strain by the same method using conjugation as described above.

[0232] It should be noted that the KNK005dZ / trc-J4b / dbktB strain is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-corrupted bacteria H16 strain are deleted, the PHA synthase gene on the chromosome is replaced with a modified version of the PHA synthase gene derived from the genus Aeromonas (a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 2, i.e., the N149S / D171G mutant (NSDG) gene), the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, and the bktB structural gene is deleted. It can be prepared according to the method described in PCT International Publication No. 2019 / 142845.

[0233] Furthermore, a strain in which the PHA synthase gene on the chromosome was deleted was isolated by the same culture and selection using a Nutrient Agar medium containing 15% sucrose as described above. This gene-disrupted strain was named KNK005dZ / dNSDG / trc-J4b / dbktB strain.

[0234] Furthermore, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0235] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 29) was obtained, which has the base sequence of the upstream of the B1185 structural gene (function unknown) of the H16 strain of Copperworm, the trp promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 12. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1185U-trp-phaCcsA479W-B1185 for introducing the PHA synthase gene.

[0236] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+B1185U-tr p-phaCcsA479W-B1185, a PHA synthase gene-transferred strain was prepared as follows.

[0237] The plasmid vector pNS2X-sacB+B1185U-trp-phaCcsA479W-B1185 for PHA synthase gene introduction was introduced into the KNK005dZ / dNS DG / trc-J4b / dbktB strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the trp promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 12 were introduced upstream of the B1185 gene on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-trp-phaCcsA479W strain.

[0238] Furthermore, using the plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNE-A2712 for introducing the PHA synthase gene, a PHA synthase gene-introduced strain was prepared as follows.

[0239] The PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-trp-phaCcsA479W strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain in which a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2 was introduced upstream of the A2712 gene on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-trp-phaCcsA479W / PA2712-phaCamSGLVNE strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (5)").

[0240] The copolymer PHA mixture producing microbial strain (5) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 are introduced.

[0241] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (6) for Producing Copolymerized PHA Mixture

[0242] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0243] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 30) was obtained, which has the base sequence of the upstream of the B1185 structural gene (function unknown) of the H16 strain of Copperworm, and a part of the B1185 structural gene, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1185U-phaCamSGLV NE-B1185 for introducing the PHA synthase gene.

[0244] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+B1185U-phaCamSGLVNE-B1185, a PHA synthase gene-transferred strain was prepared as follows.

[0245] The plasmid vector pNS2X-sacB+B1185U-phaCamSGLVNE-B1185 for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain in which a gene encoding PHA synthase having the amino acid sequence described in SEQ ID NO: 2 was introduced upstream of the B1185 gene on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-phaCamSGLVNE strain.

[0246] Further, a plasmid for expression of the PHA synthase gene was prepared. The preparation was carried out as described below. By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 31) having a lacUV5 promoter as a modified version of the lac promoter of Escherichia coli was obtained. The DNA fragment was digested with restriction enzymes EcoRI and MunI, and the obtained DNA fragment was connected to the product obtained by cutting the plasmid vector pCUP2 recorded in International Publication No. 2007 / 049716 with MunI, and the product connected in the direction in which the restriction enzyme SpeI recognition sequence of pCUP2 was located downstream of the lacUV5 promoter was screened out to obtain pCUP2-lacUV5. Next, by using PCR with synthetic oligo DNA, a DNA fragment (sequence number 32) was obtained, which had a base sequence of a gene encoding a PHA synthase having an amino acid sequence recorded in sequence number 12. The DNA fragment was digested with restriction enzymes EcoRI and SpeI, and the resulting DNA fragment was ligated with a product obtained by cleaving pCUP2-lacUV5 with MunI and SpeI to obtain a plasmid pCUP2-lacUV5-phaCcsA479W for expressing the PHA synthase gene.

[0247] Next, the PHA synthase gene expression plasmid pCUP2-lacUV5-phaCcsA479W was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-phaCamSGLVNE strain and named KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-phaCamSGLVNE / pCUP2-lacUV5-phaCcsA479W strain (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (6)").

[0248] As described below, the plasmid vector was introduced into the cells by electroporation. The gene introduction device used was a Gene Pulser manufactured by Biorad, and the sample pool used a 0.2 cm gap manufactured by Biorad. 400 μl of competent cells and 20 μl of expression vector were injected into the sample pool and placed in a pulse device, and electric pulses were applied under the conditions of electrostatic capacitance 25 μF, voltage 1.5 kV, and resistance value 800 Ω. After the pulse, the bacterial liquid in the sample pool was shaken and cultured for 3 hours at 30°C using Nutrient Broth medium (manufactured by DIFCO), and cultured for 2 days at 30°C using a selection plate (Nutrient Agar medium (manufactured by DIFCO), kanamycin 100 mg / L), to obtain a growing copolymer PHA mixture production microbial strain (6).

[0249] The copolymer PHA mixture producing microbial strain (6) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 are introduced.

[0250] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (7) for Producing Copolymerized PHA Mixture

[0251] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0252] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 33) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricinus entomopathogenis, the lacN19 promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 2. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+bktBU-lacN19-phaCamSGLVNE-bktBD for introducing the PHA synthase gene.

[0253] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktBU-lac N19-phaCamSGLVNE-bktBD, a PHA synthase gene-transferred strain was prepared as follows.

[0254] The plasmid vector pNS2X-sacB+bktBU-lacN19-phaCamSGLVNE-bktBD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the bktB gene on the chromosome was deleted and the lacN19 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced into the position where the bktB gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE strain.

[0255] Furthermore, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0256] By using PCR with synthetic oligo DNA, a DNA fragment (SEQ ID NO. 34) was obtained, which has the base sequences upstream and downstream of the B1168 structural gene (function unknown) of the H16 strain of Copperworm, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 11. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1168U-AmNSRe12A506M-B1168D for introducing the PHA synthase gene.

[0257] Next, the plasmid vector pNS2X-sacB+B1168U-AmNSRe12A506M-B1168D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and a gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 11 was introduced at the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE / B1168::AmNSRe12A506M strain (hereinafter also referred to as "copolymer PHA mixture-producing microbial strain (7)").

[0258] The copolymer PHA mixture producing microbial strain (7) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant are introduced, and the PHA synthase mutant is composed of a part of the PHA synthase gene derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 11 and a part of the PHA synthase gene derived from the genus Cupric bacteria.

[0259] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (8) for Producing Copolymerized PHA Mixture

[0260] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0261] By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 35) was obtained, which has the base sequence of the upstream and downstream of the B1168 structural gene (unknown function) of the H16 strain of Copperworm, the trp promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in sequence number 11. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which was also digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1168U-trp-AmNSRe12A506M-B1168D for introducing the PHA synthase gene.

[0262] Next, the plasmid vector pNS2X-sacB+B1168U-trp-AmNSRe12A506M-B1168D for PHA synthase gene introduction was introduced into KN K005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and selection based on Nutrient Agar medium containing 15% sucrose, a strain in which the B1168 gene on the chromosome was deleted, the trp promoter was introduced at the position where the B1168 gene originally existed, and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 11 was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE / B1168::trp-AmNSRe12A506M strain (hereinafter also referred to as "copolymer PHA mixture-producing microbial strain (8)").

[0263] The copolymer PHA mixture producing microbial strain (8) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant are introduced, wherein the PHA synthase mutant is composed of a part of the PHA synthase gene derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 11 and a part of the PHA synthase gene derived from the genus Cupric bacteria.

[0264] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (9) for Producing Copolymerized PHA Mixture

[0265] First, a plasmid for expressing the PHA synthase gene was prepared as follows.

[0266] By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 36) was obtained, which has the base sequence of the gene encoding the PHA synthase having the amino acid sequence recorded in sequence number 10. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated with the product obtained by cutting the plasmid vector pCUP2 recorded in International Publication No. 2007 / 049716 with MunI and SpeI, thereby obtaining pCUP2-AmNSRe12. Next, by using PCR with synthetic oligo DNA, a DNA fragment (sequence number 37) having the trp promoter was obtained. The DNA fragment was digested with restriction enzyme Mun I, and the product obtained by cutting pCUP2-AmNSRe12 with MunI was ligated, and the product ligated in the direction in which the gene encoding the PHA synthase having the amino acid sequence recorded in sequence number 10 is located downstream of the trp promoter was screened, thereby obtaining pCUP2-trp-AmNSRe12.

[0267] Next, the PHA synthase gene expression plasmid pC UP2-trp-AmNSRe12 was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGL VNE strain by the same electroporation method as above, and the strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE / p CUP2-trp-AmNSRe12 (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (9)").

[0268] The copolymer PHA mixture producing microbial strain (9) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant are introduced, and the PHA synthase mutant is composed of a part of the PHA synthase gene derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 10 and a part of the PHA synthase gene derived from the genus Cupric bacteria.

[0269] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (10) for Producing Copolymerized PHA Mixture

[0270] First, using the plasmid vector pNS2X-sacB+bktBU-phaCa mSGLVNE-bktBD for PHA synthase gene introduction, a PHA synthase gene-introduced strain was prepared as follows.

[0271] The plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNE-bktBD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the bktB gene on the chromosome was deleted and a gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 was introduced at the position where the bktB gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE strain.

[0272] Next, a plasmid for expressing the PHA synthase gene was prepared as follows.

[0273] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 38) having a base sequence encoding a gene for a PHA synthase having an amino acid sequence as described in SEQ ID NO: 11 was obtained. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated with a product obtained by cleaving pCUP2-lacUV5 with MunI and SpeI to obtain a plasmid pCUP2-lacUV5-AmNSRe12A506M for expressing a PHA synthase gene.

[0274] Next, the PHA synthase gene expression plasmid pC UP2-lacUV5-AmNSRe12A506M was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::phaCa mSGLVNE strain by the same electroporation method as above, and the strain was named KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE / pC UP2-lacUV5-AmNSRe12A506M (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (10)").

[0275] The copolymer PHA mixture producing microbial strain (10) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant are introduced, and the PHA synthase mutant is composed of a part of the PHA synthase gene derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 11 and a part of the PHA synthase gene derived from the genus Cupric bacteria.

[0276] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (11) for Producing Copolymerized PHA Mixture

[0277] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0278] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 39) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricinus entomopathogenis, the lacN19 promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 3. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+bktBU-lacN19-phaCamSGLVNES389G-bktBD for introducing the PHA synthase gene.

[0279] Next, using the plasmid vector pNS2X-sacB+bktBU-lac N19-phaCamSGLVNES389G-bktBD for PHA synthase gene introduction, a PHA synthase gene-introduced strain was prepared as follows.

[0280] The plasmid vector pNS2X-sacB+bktBU-lacN19-phaCamSGLVNES389G-bktBD for PHA synthase gene introduction was introduced into KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the bktB gene on the chromosome was deleted, the lacN19 promoter was introduced into the position where the bktB gene originally existed, and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 3 was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNES389G strain.

[0281] Furthermore, the PHA synthase gene expression plasmid pC UP2-lacUV5-AmNSRe12A506M was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNES389G strain by the same electroporation method as above, and the strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNES389G / pCUP2-lacUV5-AmNSRe12A506M (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (11)").

[0282] The copolymer PHA mixture producing microbial strain (11) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 3 and a gene encoding a PHA synthase mutant are introduced, and the PHA synthase mutant is composed of a part of the PHA synthase gene derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 11 and a part of the PHA synthase gene derived from the genus Cupric bacteria.

[0283] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (12) for Producing Copolymerized PHA Mixture

[0284] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0285] By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 40) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupricobacterium spp. and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in sequence number 4. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389P-bktBD for introducing the PHA synthase gene.

[0286] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389P-bktBD, a PHA synthase gene-transferred strain was prepared as follows.

[0287] The plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389P-bktBD for introducing the PHA synthase gene was introduced into the KNK005dZ / trc-J4b / dbktB / dA1528 strain by the same method using conjugation as described above.

[0288] It should be noted that the KNK005dZ / trc-J4b / dbktB / dA1528 strain is a strain in which the phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the PHA synthase gene on the chromosome is replaced with a modified version of the PHA synthase gene derived from the genus Aeromonas (a gene encoding a PHA synthase having the amino acid sequence recorded in sequence number 2, i.e., the N149S / D171G mutant (NSDG) gene), the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, and the bktB structural gene and the A1528 structural gene are deleted. It can be prepared according to the method described in PCT International Publication No. 2019 / 142845.

[0289] Furthermore, by the same culture as above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the bktB gene on the chromosome was deleted and a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 4 was introduced into the position where the bktB gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dA1528 / bktB::phaCamSGLVNES389P strain.

[0290] Next, a plasmid for expressing the PHA synthase gene was prepared as follows.

[0291] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 41) having a base sequence encoding a gene of a PHA synthase having an amino acid sequence as described in SEQ ID NO: 13 was obtained. The DNA fragment was digested with restriction enzymes EcoRI and SpeI, and the obtained DNA fragment was ligated with a product obtained by cleaving pCUP2-lacUV5 with MunI and SpeI to obtain a plasmid pCUP2-lacUV5-phaCcsA479M for expressing a PHA synthase gene.

[0292] Next, the PHA synthase gene expression plasmid pCUP2-lacUV5-phaCcsA479M was introduced into the KNK005dZ / dNSDG / trc-J4b / dA1528 / bktB::phaCamSGLVNES389P strain by the same electroporation method as above, and the strain was named KNK005dZ / dNSDG / trc-J4b / dA1528 / bktB::phaCamSGLVNES389P / pCUP2-lacUV5-phaCcsA479M (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (12)").

[0293] The copolymer PHA mixture producing microbial strain (12) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the A1528 gene and bktB gene on the chromosome are deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 4 and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 13 are introduced.

[0294] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (13) for Producing Copolymerized PHA Mixture

[0295] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0296] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 42) was obtained, which had the base sequences upstream and downstream of the B1168 structural gene (function unknown) of the H16 strain of Copperworm, the trp promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 9. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708 which had also been digested with SwaI by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1168U-trp-phaCbp-B1168D for introducing the PHA synthase gene.

[0297] Next, the PHA synthase gene introduction plasmid vector pNS2X-sacB+B1168U-trp-phaCbp-B1168D was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the trp promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 9 were introduced at the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE / B1168::trp-phaCbp strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (13)").

[0298] The copolymer PHA mixture producing microbial strain (13) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0299] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (14) for Producing Copolymerized PHA Mixture

[0300] The plasmid vector pNS2X-sacB+B1168U-trp-phaCbp-B1168D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the trp promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 9 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE / B1168::trp-phaCbp strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (14)").

[0301] The copolymer PHA mixture producing microbial strain (14) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0302] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (15) for Producing Copolymerized PHA Mixture

[0303] First, a plasmid for expressing the PHA synthase gene was prepared as follows.

[0304] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 43) having the base sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 9 was obtained. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated with the product obtained by cleaving pCUP2-lacUV5 with MunI and SpeI to obtain a plasmid pCUP2-lacUV5-phaCbp for expressing the PHA synthase gene.

[0305] Next, the PHA synthase gene expression plasmid pCUP2-lacUV5-phaCbp was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE strain by the same electroporation method as above and named the strain KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE / pCUP2-lacUV5-phaCbp (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (15)").

[0306] The copolymer PHA mixture producing microbial strain (15) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0307] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (16) for Producing Copolymerized PHA Mixture

[0308] The PHA synthase gene expression plasmid pCUP2-lacUV5-phaCbp was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE strain by the same electroporation method as above and named KNK005dZ / dNSDG / trc-J4b / bktB::lacN19-phaCamSGLVNE / pCUP2-lacUV5-phaCbp strain (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (16)").

[0309] The copolymer PHA mixture producing microbial strain (16) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0310] (Example of Preparation of Microbial Strain) Preparation of Microbial Strain (17) for Producing Copolymerized PHA Mixture

[0311] The PHA synthase gene expression plasmid pCUP2-lacUV5-phaCbp was introduced into the KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W strain by the same electroporation method as above and named KNK005dZ / dNSDG / trc-J4b / B1168::trp-phaCcsA479W / pCUP2-lacUV5-phaCbp strain (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (17)").

[0312] The copolymer PHA mixture producing microbial strain (17) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, and a gene encoding a PHA synthase mutant derived from the genus Chromobacterium having the amino acid sequence recorded in sequence number 12 and a gene encoding a PHA synthase derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0313] (Microbial strain preparation example 18) Preparation of microbial strain (18) for producing copolymerized PHA mixture

[0314] First, the plasmid vector pNS2X-sacB+B1168U-trp-AmNSRe12A506M-B1168D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the trp promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 11 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::trp-AmNSRe12A506M strain.

[0315] Next, the PHA synthase gene expression plasmid pCUP2-lacUV5-phaCbp was introduced into KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::trp-AmNSRe12A506M by the same electroporation method as above, and the strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::trp-AmNSRe12A506M / pCUP2-lacUV5-phaCbp (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (18)").

[0316] The copolymer PHA mixture producing microbial strain (18) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal Cupric bacteria H16 strain are deleted, the expression of the R-body specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant and a gene encoding a PHA synthase derived from a soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced, and the PHA synthase mutant is composed of a part of the PHA synthase gene derived from a microorganism of the genus Aeromonas having the amino acid sequence recorded in sequence number 11 and a part of the PHA synthase gene derived from a microorganism of the genus Cupric bacteria.

[0317] (Microbial strain preparation example 19) Preparation of microbial strain (19) for producing copolymerized PHA mixture

[0318] First, a plasmid for expressing the PHA synthase gene was prepared as follows.

[0319] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 45) having a base sequence encoding a gene of a PHA synthase having an amino acid sequence as described in SEQ ID NO: 44 was obtained. The DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated with a product obtained by cleaving pCUP2-lacUV5 with MunI and SpeI to obtain a plasmid pCUP2-lacUV5-phaCbpT357P for expressing a PHA synthase gene.

[0320] Next, the PHA synthase gene expression plasmid pCUP2-lacUV5-phaCbpT357P was introduced into the KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE strain by the same electroporation method as above and named KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE / pCUP2-lacUV5-phaCbpT357P strain (hereinafter also recorded as "copolymer PHA mixture producing microbial strain (19)").

[0321] The copolymer PHA mixture producing microbial strain (19) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0322] (Microbial strain preparation example 20) Preparation of microbial strain (20) for producing copolymerized PHA mixture

[0323] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0324] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 46) was obtained, which had the base sequences upstream and downstream of the B1168 structural gene (unknown function) of the H16 strain of Copperworm, the lacUV5 promoter, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 44. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1168U-lacUV5-phaCbpT357P-B1168D for introducing the PHA synthase gene.

[0325] Next, the plasmid vector pNS2X-sacB+B1168U-lacUV5-phaCbpT357P-B1168D for PHA synthase gene introduction was introduced into KNK 005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the lacUV5 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 44 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / bktB::phaCamSGLVNE / B1168::lacUV5-phaCbpT357P strain (hereinafter also referred to as "copolymer PHA mixture-producing microbial strain (20)").

[0326] The copolymer PHA mixture producing microbial strain (20) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0327] (Microbial strain preparation example 21) Preparation of microbial strain (21) for producing copolymerized PHA mixture

[0328] First, the plasmid vector pNS2X-sacB+B1168U-lacUV5-phaCbpT357P-B1168D for PHA synthase gene introduction was introduced into KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-phaCamSGLVNE by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and selection based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the lacUV5 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 44 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / PB1185-phaCamSGLVNE / B1168::lacUV5-phaCbpT357P strain (hereinafter also referred to as "copolymer PHA mixture-producing microbial strain (21)").

[0329] The copolymer PHA mixture producing microbial strain (21) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0330] (Microbial strain preparation example 22) Preparation of microbial strain (22) for producing copolymerized PHA mixture

[0331] First, the plasmid vector pNS2X-sacB+B1168U-lacUV5-phaCbpT357P-B1168D for PHA synthase gene introduction was introduced into KNK005dZ / dNSDG / trc-J4b / dbktB by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and selection based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the lacUV5 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 44 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain.

[0332] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNE-A2712, a PHA synthase gene-transferred strain was prepared as follows.

[0333] The PHA synthase gene introduction plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNE-A2712 was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain by the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain in which a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2 was introduced upstream of the A2712 gene on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P / PA2712-phaCamSGLVNE strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (22)").

[0334] The copolymer PHA mixture producing microbial strain (22) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 2 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0335] (Microbial strain preparation example 23) Preparation of microbial strain (23) for producing copolymerized PHA mixture

[0336] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0337] By PCR using synthetic oligo DNA, a DNA fragment (sequence number 47) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupric spp. and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in sequence number 5. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389C-bktBD for introducing the PHA synthase gene.

[0338] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389C-bktBD, a PHA synthase gene-transferred strain was prepared as follows.

[0339] The plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389C-bktBD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO: 5 was introduced into the position on the chromosome where the bktB gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::phaCamSGLVNES389C / B1168::lacUV5-phaCbpT357P strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (23)").

[0340] The copolymer PHA mixture producing microbial strain (23) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 5 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0341] (Microbial strain preparation example 24) Preparation of microbial strain (24) for producing copolymerized PHA mixture

[0342] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0343] By using PCR with synthetic oligo DNA, a DNA fragment (SEQ ID NO. 52) was obtained, which has the base sequence of the upstream of the B1185 structural gene (function unknown) of the H16 strain of Copperworm, and a part of the B1185 structural gene, and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO. 7. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+B1185U-phaCamSGLVNES389V-B1185D for introducing the PHA synthase gene.

[0344] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+B1185U-phaCamSGLVNES389V-B1185D, a PHA synthase gene-transferred strain was prepared as follows.

[0345] The plasmid vector pNS2X-sacB+B1185U-phaCamSGLVNES389V-B1185D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and selection based on Nutrient Agar medium containing 15% sucrose, a strain in which a gene encoding PHA synthase having the amino acid sequence described in SEQ ID NO: 7 was introduced at the position on the chromosome where the bktB gene originally existed was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1185::phaCamSGLVNES389V / B1168::lacUV5-phaCbpT357P strain (hereinafter also referred to as "copolymer PHA mixture-producing microbial strain (24)").

[0346] The copolymer PHA mixture producing microbial strain (24) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 7 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0347] (Microbial strain preparation example 25) Preparation of microbial strain (25) for producing copolymerized PHA mixture

[0348] First, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0349] By using PCR with synthetic oligo DNA, a DNA fragment (sequence number 49) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupric spp. and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in sequence number 7. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708 which had also been digested with SwaI by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389V-bktBD for introducing the PHA synthase gene.

[0350] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389V-bktBD, a PHA synthase gene-transferred strain was prepared as follows.

[0351] The plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389V-bktBD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO: 7 was introduced at the position on the chromosome where the bktB gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::phaCamSGLVNES389V / B1168::lacUV5-phaCbpT357P strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (25)").

[0352] The copolymer PHA mixture producing microbial strain (25) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 7 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0353] (Microbial strain preparation example 26) Preparation of microbial strain (26) for producing copolymerized PHA mixture

[0354] First, using the plasmid vector pNS2X-sacB+B1168U-trp-phaCbp-B1168D for PHA synthase gene introduction, a PHA synthase gene-introduced strain was prepared as follows.

[0355] The plasmid vector pNS2X-sacB+B1168U-trp-phaCbp-B1168D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method of conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the trp promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 9 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::trp-phaCbp strain.

[0356] Next, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0357] By PCR using synthetic oligo DNA, a DNA fragment (sequence number 50) was obtained, which has the base sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the H16 strain of Cupric spp. and the base sequence of the gene encoding the PHA synthase having the amino acid sequence described in sequence number 8. The DNA fragment was digested with the restriction enzyme SwaI, and the obtained 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 by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare the plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389T-bktBD for introducing the PHA synthase gene.

[0358] Next, using the PHA synthase gene-transferred plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389T-bktBD, a PHA synthase gene-transferred strain was prepared as follows.

[0359] The PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::trpUV5-phaCbp strain using the same conjugation transfer method as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain in which a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 8 was introduced into the position on the chromosome where the bktB gene originally existed was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::phaCamSGLVNES389T / B1168::trp-phaCbp strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (26)").

[0360] The copolymer PHA mixture producing microbial strain (26) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 8 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0361] (Microbial strain preparation example 27) Preparation of microbial strain (27) for producing copolymerized PHA mixture

[0362] Using the plasmid vector pNS2X-sacB+bktBU-phaCamSGL VNES389V-bktBD for PHA synthase gene introduction, a PHA synthase gene-introduced strain was prepared as follows.

[0363] The plasmid vector pNS2X-sacB+bktBU-phaCamSGLVNES389V-bktBD for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::trp-phaCbp strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on NutrientAgar medium containing 15% sucrose, a strain in which a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 7 was introduced into the position on the chromosome where the bktB gene originally existed was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::phaCamSGLVNES389V / B1168::trp-phaCbp strain (hereinafter also described as "copolymer PHA mixture producing microbial strain (27)").

[0364] The copolymer PHA mixture producing microbial strain (27) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 7 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 9 are introduced.

[0365] (Microbial strain preparation example 28) Preparation of microbial strain (28) for producing copolymerized PHA mixture

[0366] First, using a plasmid vector for introducing a PHA synthase gene, pNS2X-sacB+B1168U-lacUV5-phaCbpT357P-B1168D, a PHA synthase gene-introduced strain was prepared as follows.

[0367] The plasmid vector pNS2X-sacB+B1168U-lacUV5-phaCbpT357P-B1168D for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and selection based on Nutrient Agar medium containing 15% sucrose, a strain was isolated in which the B1168 gene on the chromosome was deleted and the lacUV5 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 44 were introduced into the position where the B1168 gene originally existed. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain.

[0368] Next, a plasmid for introducing the PHA synthase gene was prepared as follows.

[0369] By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO. 51) was obtained, which has a base sequence upstream of the A2712 structural gene (function unknown) of the H16 strain of Cupricinus entomopathogenis and a part of the A2712 structural gene, and a base sequence of a gene encoding a PHA synthase having an amino acid sequence described in SEQ ID NO. 7. The DNA fragment was digested with restriction enzyme SwaI, and the obtained DNA fragment was ligated with the vector pNS2X-sacB described in Japanese Patent Application Publication No. 2007-259708 which had also been digested with SwaI by DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.) to prepare a plasmid vector pNS2X-sacB+A2712U-phaCamS GLVNES389V-A2712 for introducing a PHA synthase gene.

[0370] Next, using the plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNES389V-A2712 for introducing the PHA synthase gene, the PHA synthase gene-introduced strain was prepared as follows.

[0371] The plasmid vector pNS2X-sacB+A2712U-phaCamSGLVNES389V-A2712 for PHA synthase gene introduction was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P strain by the same method using conjugation transfer as described above. Furthermore, by the same culture as described above and screening based on Nutrient Agar medium containing 15% sucrose, a strain in which a gene encoding PHA synthase having the amino acid sequence described in SEQ ID NO: 7 was introduced upstream of the A2712 gene on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB / B1168::lacUV5-phaCbpT357P / PA2712-phaCamSGLVNES389V strain (hereinafter also referred to as "copolymer PHA mixture producing microbial strain (28)").

[0372] The copolymer PHA mixture producing microbial strain (28) is a strain in which the phaC1 gene (PHA synthase gene), phaZ1 gene, phaZ2 gene and phaZ6 gene on the chromosome of the insecticidal copper-greedy bacteria H16 strain are deleted, the expression of the R-body-specific enoyl-CoA hydratase gene on the chromosome is enhanced, the bktB gene on the chromosome is deleted, and a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence recorded in sequence number 7 and a gene encoding a PHA synthase mutant derived from the soil metagenome having the amino acid sequence recorded in sequence number 44 are introduced.

[0373]

[0374]

[0375] (Example 1) PHA production based on copolymerized PHA mixture producing microbial strain (1)

[0376] Cultivation studies of the production microbial strain (1) using the copolymerized PHA mixture were conducted under the following conditions.

[0377] (Culture medium)

[0378] The composition of the mother culture medium was set to 1 w / v% Meat-extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast-extract, 0.9 w / v% Na2HPO4·12H2O, 0.15 w / v% KH2PO4, (pH 6.8).

[0379] The composition of the pre-culture medium is set to 1.1w / v% Na2HPO4·12H2O, 0.19w / v% KH2PO4, 1.29w / v% (NH4)2SO4, 0.1w / v% MgSO4·7H2O, 2.5w / v% palm olein, and 0.5v / v% trace metal salt solution (a solution prepared by dissolving 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 in 0.1N hydrochloric acid).

[0380] 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 (a solution prepared by dissolving 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 in 0.1N hydrochloric acid).

[0381] (Measurement method of PHA accumulation ratio)

[0382] The ratio of PHA accumulation to dry bacterial cells was determined as follows. The bacterial cells were recovered from the culture solution by centrifugation, washed with ethanol, freeze-dried, and dried bacterial cells were obtained and the weight was measured. 100 ml of chloroform was added to 1 g of the obtained dry bacterial cells, and the mixture was stirred at room temperature for a day and a night to extract the PHA (copolymer PHA mixture) in the bacterial cells. After filtering out the bacterial residue, the mixture was concentrated with an evaporator to a total volume of 30 ml, and then 90 ml of hexanol was gradually added, slowly stirred and allowed to stand for 1 hour. After filtering out the precipitated PHA, it was vacuum dried at 50°C for 3 hours. The weight of the dry PHA was measured to calculate the ratio of the PHA accumulation to the amount of dry bacterial cells.

[0383] (Measurement method of PHA accumulation ratio)

[0384] The ratio of PHA accumulation to dry bacterial cells was determined as follows. The bacterial cells were recovered from the culture solution by centrifugation, washed with ethanol, freeze-dried, and dried bacterial cells were obtained and the weight was measured. 100 ml of chloroform was added to 1 g of the obtained dry bacterial cells, and the mixture was stirred at room temperature for a day and a night to extract the PHA (copolymer PHA mixture) in the bacterial cells. After filtering out the bacterial residue, the mixture was concentrated with an evaporator to a total volume of 30 ml, and then 90 ml of hexanol was gradually added, slowly stirred and allowed to stand for 1 hour. After filtering out the precipitated PHA, it was vacuum dried at 50°C for 3 hours. The weight of the dry PHA was measured to calculate the ratio of the PHA accumulation to the amount of dry bacterial cells.

[0385] (Analysis of copolymerized PHA)

[0386] The “PHA fraction (I)” and the “PHA mainly composed of the PHA fraction (II)” in the copolymerized PHA mixture were separated by the above-mentioned MIBK fractionation method.

[0387] The average 3HH composition ratio of each of the copolymerized PHA mixture and the PHA fraction (I) was analyzed by the above-mentioned method for measuring the average 3HH composition ratio by gas chromatography.

[0388] The average 3HH composition ratio of the PHA fraction (II) was analyzed by the above-mentioned method for measuring the average 3HH composition ratio by DSC.

[0389] The weight ratio of the PHA fraction (I) to the PHA fraction (II) in the copolymerized PHA mixture is calculated by the above-mentioned method for calculating the weight ratio in the copolymerized PHA mixture.

[0390] (PHA production and cultivation)

[0391] PHA production culture was carried out as described below. First, a glycerol stock solution (50 μl) of the copolymerized PHA mixture producing microbial strain (1) was inoculated into a mother culture medium (10 ml) and cultured for 24 hours to carry out mother culture. Next, the mother culture solution was inoculated at 1.0 v / v% into a 3L fermenter (MDL-300 manufactured by BEMARUBISHI) to which 1.8L of pre-culture medium was added. The operating conditions were set to a culture temperature of 30°C, a stirring speed of 500 rpm, and an aeration volume of 1.8 L / min. The pH was controlled between 6.7 and 6.8, and cultured for 28 hours to carry out pre-culture. A 14% ammonium hydroxide aqueous solution was used for pH control.

[0392] Next, the pre-culture solution was inoculated at 5.0 v / v% into a 5 L fermenter (MDS-U50 manufactured by BEMARUBISHI) to which 2.5 L of PHA production medium was added. The operating conditions were set at a culture temperature of 33°C, a stirring speed of 420 rpm, and 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 ratio of PHA accumulation to the amount of dry bacteria reached more than 80%. The ratio of PHA accumulation to dry bacteria, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), and the weight ratio of PHA fractions (I) and (II) were measured as described above. The results are shown in Table 3.

[0393] (Examples 2 to 28) PHA production based on copolymerized PHA mixtures produced by microbial strains (2) to (28)

[0394] Cultivation studies of the microbial strains (2) to (28) using the copolymerized PHA mixture were conducted under the same conditions as in Example 1. Table 3 shows the ratio of the PHA accumulation to the dry bacterial cells, the average composition ratio of 3HH in the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), and the weight ratio of PHA fractions (I) and (II).

[0395] [Table 3]

[0396]

[0397] As can be seen from Table 3, in Examples 1 to 28, a copolymerized PHA mixture containing a PHA fraction (I) having an average 3HH composition ratio of 9 mol% or more and less than 20 mol% and a PHA fraction (II) having an average 3HH composition ratio of 0 to 8 mol% was produced by culturing microorganisms, and the weight proportion of the PHA fraction (II) was 45% or more.

[0398] (Reference Example 1) Evaluation of the granulation properties of PHA produced by the microbial strain (4) producing the copolymerized PHA mixture

[0399] The spray drying and granulation properties of PHA obtained by culturing the PHA-producing microbial strain (4) producing a copolymerized PHA mixture were evaluated as follows.

[0400] (Spray Drying)

[0401] The culture solution of the copolymer PHA mixture producing microbial strain (4) was treated by the method described in paragraphs

[0085] to

[0087] of International Publication No. 2021 / 251049 to obtain a PHA aqueous suspension.

[0402] The obtained PHA aqueous suspension was adjusted to 50% by weight, and 1.0 phr (1 part by weight relative to 100 parts by weight of PHA present in the aqueous suspension) of an ethylene oxide / propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name PLONON 208) was added and mixed to obtain a mixed solution. Then, after the mixed solution was stirred for 120 minutes, 0.4 ml of 10% by weight sulfuric acid was added per 100 g of the mixed solution to obtain a PHA aqueous suspension with a pH of 3.0. The obtained PHA aqueous suspension was spray-dried using a spray dryer (OC-16) manufactured by Ohkawara Chemical Industry Co., Ltd. (hot air temperature: 160°C, exhaust air temperature: 105°C, rotary atomizer speed: 13000 rpm) to obtain a PHA powder.

[0403] (Evaluation of granulation properties)

[0404] The evaluation of granulation properties was performed as follows using the fine powder amount as an index.

[0405] 0.05 g of a surfactant sodium dodecyl sulfate was added as a dispersant to 20 ml of ion exchange water to obtain a surfactant aqueous solution. Then, 0.2 g of PHA powder was added to the surfactant aqueous solution and dispersed to obtain a dispersion for measurement. The prepared dispersion was introduced into a laser diffraction / scattering particle size distribution measuring device (LA-950 (HORIBA)) to measure the particle size. Particles with a diameter of 20 μm or less were regarded as fine powders, and the amount of fine powder in the PHA powder at this time is shown in Table 4.

[0406] (Reference Examples 2-3) Evaluation of the granulation properties of PHA produced by the microbial strains (5) and (8) producing copolymerized PHA mixtures

[0407] The spray drying and granulation properties of PHA obtained by culturing PHA production by copolymerizing PHA mixture producing microorganism strains (5) and (8) were evaluated under the same conditions as in Reference Example 1. Particles with a diameter of 20 μm or less were considered fine powders, and the amount of fine powder in the PHA powders at this time is shown in Table 4.

[0408] (Reference Examples 4-5) Evaluation of the granulation properties of PHA produced by the microbial strains (13) and (15) producing copolymerized PHA mixtures

[0409] The spray drying and granulation properties of PHA obtained by culturing the PHA production of the copolymerized PHA mixture producing microorganism strains (13) and (15) were evaluated under the same conditions as in Reference Example 1 except that the hot air temperature was changed to 140°C and the exhaust air temperature was changed to 90°C. PHA particles with a particle size of 20 μm or less were regarded as fine powders, and the amount of fine powder in the PHA powder at this time is shown in Table 4.

[0410] (Comparative Example 1) Evaluation of granulation properties of P(3HB)

[0411] The spray drying and granulation evaluation of PHA obtained by culturing the P(3HB)-producing microbial strain (Cupriavidusnecator H16 strain) were attempted under the same conditions as in Reference Example 1. In this example, granulation was substantially not observed, and evaluation could not be performed due to the risk of dust explosion. The results are shown in Table 4.

[0412] (Comparative Examples 2-3) Evaluation of granulation properties of P(3HB-co-3HH)

[0413] The spray drying and granulation properties of PHA obtained by culturing PHA production of two microbial strains (transformed strains of Cupriavidus necator) producing P(3HB-co-3HH) were evaluated under the same conditions as in Reference Example 1. Particles with a particle size of 20 μm or less were regarded as fine powders, and the amount of fine powder in the PHA powder at this time is shown in Table 4. It should be noted that the microbial strain has one active polyhydroxyalkanoate synthase and does not produce the copolymerized polyhydroxyalkanoate mixture of the present invention.

[0414]

[0415] According to Table 4, it was confirmed that the amount of fine powder was significantly reduced in Reference Examples 1 to 5 compared with Comparative Examples 1 to 3 under the same drying conditions.

Claims

1. A method for producing a copolymerized polyhydroxyalkanoate mixture, the method comprising: a step of culturing a microorganism that produces the copolymerized polyhydroxyalkanoate mixture, The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II), The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 9 mol% or more and less than 20 mol%, The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 0 mol% to 8 mol%, The weight ratio of the polyhydroxyalkanoate fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

2. The manufacturing method according to claim 1, wherein: In the copolymerized polyhydroxyalkanoate mixture, an average 3-hydroxyhexanoic acid composition ratio in the entire mixture is 0.5 to 14 mol %.

3. The manufacturing method according to claim 1 or 2, wherein: The microorganism has genes encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA.

4. The manufacturing method according to claim 3, wherein: The amino acid sequence identity between the two polyhydroxyalkanoate synthetases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA is 90% or less.

5. The manufacturing method according to claim 3, wherein: The genes encoding the two polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA are gene (A) and gene (B), The gene (A) encodes a polyhydroxyalkanoate synthase that synthesizes a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, The gene (B) encodes a polyhydroxyalkanoate synthase having a lower polymerization activity for (R)-3-hydroxyhexanoyl-CoA than the gene (A).

6. The manufacturing method according to claim 5, wherein: The microorganism is regulated so that the expression level of the gene (A) is lower than the expression level of the gene (B).

7. The manufacturing method according to claim 5, wherein: The gene (A) is a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas or a mutant thereof.

8. The manufacturing method according to claim 7, wherein: The gene (A) is a gene encoding an amino acid sequence having a sequence identity of 99.5% to 100% with respect to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 8.

9. The manufacturing method according to claim 5, wherein: The gene (B) is composed of a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Aeromonas and a portion of a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Cupriavidus.

10. The manufacturing method according to claim 9, wherein: The gene (B) is a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO:

11.

11. The manufacturing method according to claim 5, wherein: The gene (B) is a polyhydroxyalkanoate synthase gene derived from a microorganism of the genus Chromobacterium or a mutant thereof.

12. The manufacturing method according to claim 11, wherein: The gene (B) is a gene encoding an amino acid sequence having a sequence identity of 90 to 100% with respect to the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO:

13.

13. The manufacturing method according to claim 5, wherein: Any one of the gene (A) and the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO:

44.

14. The manufacturing method according to claim 1 or 2, wherein: The microorganism is a microorganism having a gene encoding a protein exhibiting R-body-specific enoyl-CoA hydratase activity.

15. The manufacturing method according to claim 1 or 2, wherein: The microorganism is a transformed microorganism that has been transformed so that the supply of (R)-3-hydroxyhexanoyl-CoA to polyhydroxyalkanoate synthase in the cell is increased compared to a wild-type strain of the microorganism.

16. The manufacturing method according to claim 15, wherein: The transformed microorganism is transformed so that the expression of a gene encoding a protein exhibiting R-body-specific enoyl-CoA hydratase activity is enhanced.

17. The manufacturing method according to claim 15, wherein: The transformed microorganism is transformed in which the expression of a gene encoding β-ketothiolase having a thiolytic activity on β-ketohexanoyl-CoA, which is β-ketoacyl-CoA having 6 carbon atoms, is suppressed.

18. The manufacturing method according to claim 1 or 2, wherein: In the culturing step, a carbon source including oil or fatty acid is added.

19. The manufacturing method according to claim 1 or 2, wherein: The microorganism belongs to the genus Cupriavidus, or is a transformant of a microorganism of the genus Cupriavidus.

20. The manufacturing method according to claim 19, wherein: The microorganism is Copperobacterium spp. or a transformant of Copperobacterium spp.

21. The manufacturing method according to claim 1 or 2, comprising: After culturing the microorganism, the cells of the microorganism are disrupted and purified to obtain an aqueous suspension of the copolymerized polyhydroxyalkanoate mixture; as well as A step of obtaining a powder of the copolymerized polyhydroxyalkanoate mixture by spray-drying the aqueous suspension.

22. A transformed microorganism which produces a copolymerized polyhydroxyalkanoate mixture. The transformed microorganism has genes encoding two types of polyhydroxyalkanoate synthases having different polymerization activities for (R)-3-hydroxyhexanoyl-CoA. The copolymerized polyhydroxyalkanoate mixture contains a polyhydroxyalkanoate fraction (I) and a polyhydroxyalkanoate fraction (II), The polyhydroxyalkanoate fraction (I) comprises a copolymerized polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 9 mol% or more and less than 20 mol%, The polyhydroxyalkanoate fraction (II) contains polyhydroxyalkanoate having a 3-hydroxybutyric acid structural unit, and the average 3-hydroxyhexanoic acid composition ratio is 0 mol% to 8 mol%, The weight ratio of the polyhydroxyalkanoate fraction (II) in the copolymerized polyhydroxyalkanoate mixture is 45% or more.

Images