Method for producing copolymerized polyhydroxyalkanoic acid mixture and transformed microorganism

Abstract

A method for manufacturing a copolymer polyhydroxyalkanoic acid mixture. The method includes a step for culturing a microorganism that produces the copolymer polyhydroxyalkanoic acid mixture. The mixture contains a fraction (I) which includes a copolymer polyhydroxyalkanoic acid having 3-hydroxybutyric acid structural units and 3-hydroxyhexanoic acid structural units and in which the average composition ratio of 3-hydroxyhexanoic acid is 20 mol% or greater, and a fraction (II) which includes a polyhydroxyalkanoic acid having 3-hydroxybutyric acid structural units and in which the average composition ratio of 3-hydroxyhexanoic acid is 0-15 mol% nclusive. In the copolymer polyhydroxyalkanoic acid mixture, the average composition ratio of 3-hydroxyhexanoic acid is 22 mol% or less.

Description

[Technical field]

[0001] The present invention relates to a method for producing a copolymerized polyhydroxyalkanoic acid mixture and a transformed microorganism. [Background technology]

[0002] Polyhydroxyalkanoates (hereinafter sometimes referred to as "PHAs") are polyester-type organic polymers produced by a wide range of microorganisms. PHAs are biodegradable thermoplastic polymers that can be produced from renewable resources. For these reasons, attempts are being made to industrially produce PHAs as environmentally friendly or biocompatible materials and to use them in a variety of industries.

[0003] It is known that many microorganisms accumulate PHAs as an energy storage substance within their cells. A representative example of PHA is poly-3-hydroxybutyrate (hereinafter sometimes referred to as "P(3HB")), a homopolymer of 3-hydroxybutyric acid (hereinafter sometimes referred to as "3HB"). P(3HB) is a thermoplastic polymer that is biodegradable in the natural environment and has attracted attention as an environmentally friendly plastic. However, P(3HB) is highly crystalline and therefore hard and brittle, limiting its range of practical applications. To expand its range of applications, it was necessary to impart flexibility to P(3HB).

[0004] Therefore, a copolymer PHA (hereinafter referred to as "P(3HB-co-3HV)") consisting of 3HB and 3-hydroxyvaleric acid (hereinafter referred to as "3HV") and a method for producing the same have been developed (see, for example, Patent Documents 1 and 2). P(3HB-co-3HV) is more flexible than P(3HB), and it was thought that it could be used in a wide range of applications. However, in reality, even if the 3HV molar fraction in P(3HB-co-3HV) is increased, there is little change in the physical properties associated with it, and the flexibility is not improved to the extent required for processing into films, sheets, soft packaging containers, etc., and therefore it is only used in limited fields of hard molded bodies such as shampoo bottles and disposable razor handles.

[0005] In order to increase the flexibility of PHA, research has been conducted on copolymerized polyhydroxyalkanoates (hereinafter sometimes referred to as "P(3HB-co-3HH)") consisting of 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as "3HH") and methods for producing the same (see, for example, Patent Documents 3 and 4). In these reports, P(3HB-co-3HH) is produced by fermentation using a wild-type strain of Aeromonas caviae isolated from soil, with fatty acids such as oleic acid and palmitic acid as the carbon source.

[0006] Research has also been conducted on the high 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, which has the 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 vegetable oil as a raw material, and the 3HH composition ratio of the P(3HB-co-3HH) has been increased to a maximum of about 14 mol% (see Patent Document 5, Patent Document 6, and Non-Patent Document 1).

[0007] Furthermore, in one example, the 3HH composition ratio of P(3HB-co-3HH) was increased to 20 mol% or more by suppressing the expression of a gene encoding a β-ketothiolase enzyme having thiolysis activity against β-ketoacyl-CoA (i.e., β-ketohexanoyl-CoA) with six carbon atoms in Capriavidus necator, which has a PHA synthesis enzyme derived from Aeromonas caviae (see Patent Document 7).

[0008] Research on the physical properties of P(3HB-co-3HH) has also been conducted (see Non-Patent Document 2). In this report, Aeromonas caviae was cultured using fatty acids with 12 or more carbons as the sole carbon source, and P(3HB-co-3HH) with various 3HH composition ratios was produced by fermentation. It was revealed that P(3HB-co-3HH) exhibits gradually flexible properties from the hard and brittle properties of P(3HB), as the crystallinity of P(3HB-co-3HH) decreases with increasing 3HH composition ratio, and that it exhibits flexibility exceeding that of P(3HB-co-3HV) as the 3HH composition ratio increases. In other words, P(3HB-co-3HH) can have a wide range of physical properties applicable to both hard and soft polymers by changing the 3HH composition ratio, and is therefore expected to be applicable in a wide range of fields.

[0009] On the other hand, when the 3HH composition ratio of P(3HB-co-3HH) is increased, the crystallinity decreases, improving flexibility, but the processing characteristics tend to decrease. For example, P(3HB-co-3HH) with an increased 3HH composition ratio of about 10 mol% is relatively soft, but the crystallization rate is slow in injection molding, film molding, blow molding, fiber spinning, extrusion foaming, bead foaming, and other processes, resulting in low productivity. In order to solve this problem, research has been conducted to improve melt processability and processing speed by co-producing the relatively soft P(3HB-co-3HH) and a copolymer PHA with a low 3HH composition ratio and a high melting point (i.e., high crystallinity) in the same cell (see Patent Document 8).

[0010] However, in the PHA mixture described in Patent Document 8, the melting point of the low-melting point component (the PHA component thought to have the highest 3HH composition ratio) exceeds 100°C, and therefore it is presumed that the PHA mixture does not contain a PHA component with a high 3HH composition ratio of 20 mol % or more, and the mechanical properties such as tear strength are insufficient, leaving room for improvement. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Publication No. 57-150393 [Patent Document 2] Japanese Patent Application Publication No. 59-220192 [Patent Document 3] Japanese Patent Application Publication No. 5-93049 [Patent Document 4] Japanese Patent Application Publication No. 7-265065 [Patent Document 5] International Publication No. 2011 / 105379 [Patent Document 6] International Publication No. 2015 / 115619 [Patent Document 7] International Publication No. 2019 / 142845 [Patent Document 8] International Publication No. 2017 / 056442 [Non-patent literature]

[0012] [Non-Patent Document 1] H.Arikawa,K.Matsumoto,Microb.Cell.Fact.,15,pp.184 (2016) [Non-Patent Document 2] Y. Doi, S. Kitamura, H. Abe, Macromolecules, 28, pp. 4822-4823 (1995) Summary of the Invention [Problem to be solved by the invention]

[0013] As described above, it was difficult to obtain a molded product using P(3HB-co-3HH) that satisfied both good processability and mechanical properties.

[0014] In view of the above-mentioned current situation, an object of the present invention is to provide a method for producing a copolymerized polyhydroxyalkanoic acid that has both excellent processability and mechanical properties. [Means for solving the problem]

[0015] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that by culturing microorganisms to produce a copolymerized polyhydroxyalkanoic acid mixture containing two types of polyhydroxyalkanoic acid fractions having specific compositions, it is possible to obtain a copolymerized polyhydroxyalkanoic acid mixture that combines excellent processability and mechanical properties, and thus completed the present invention.

[0016] That is, the present invention relates to a method for producing a copolymerized polyhydroxyalkanoic acid mixture, comprising a step of culturing a microorganism that produces the copolymerized polyhydroxyalkanoic acid mixture, wherein the copolymerized polyhydroxyalkanoic acid mixture contains a polyhydroxyalkanoic acid fraction (I) containing a copolymerized polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit and having an average 3-hydroxyhexanoic acid composition ratio of 20 mol % or more, and a polyhydroxyalkanoic acid fraction (II) containing a polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and having an average 3-hydroxyhexanoic acid composition ratio of 0 mol % or more and 15 mol % or less, and the copolymerized polyhydroxyalkanoic acid mixture has an average 3-hydroxyhexanoic acid composition ratio of 22 mol % or less. Preferably, the weight ratio of the polyhydroxyalkanoic acid fraction (I) in the copolymerized polyhydroxyalkanoic acid mixture is 10 to 90%. Preferably, the copolymerized polyhydroxyalkanoic acid mixture has an average composition ratio of 3-hydroxyhexanoic acid of 10 to 22 mol %. Preferably, the microorganism has genes encoding two types of polyhydroxyalkanoic acid synthases that differ from each other in polymerization activity toward 3-hydroxyhexanoyl-CoA. Preferably, the amino acid sequences of the two types of polyhydroxyalkanoic acid synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA have a sequence identity of 90% or less. Preferably, the genes encoding the two types of polyhydroxyalkanoic acid synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA are a gene (A) encoding a polyhydroxyalkanoic acid synthase having a higher polymerization activity for 3-hydroxyhexanoyl-CoA than the wild-type polyhydroxyalkanoic acid synthase derived from Aeromonas caviae having the amino acid sequence set forth in SEQ ID NO:1, and a gene (B) encoding a polyhydroxyalkanoic acid synthase having a lower polymerization activity for 3-hydroxyhexanoyl-CoA than the wild-type polyhydroxyalkanoic acid synthase derived from Aeromonas caviae. Preferably, the gene (A) is a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas or a mutant thereof, and more preferably, the gene (A) is a gene encoding an amino acid sequence having 99.5 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, the gene (B) is constructed by combining a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas with a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Capriavidus. More preferably, the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO:6. Preferably, the gene (B) is a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Chromobacterium or a mutant thereof, and more preferably, the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5. Preferably, the gene (B) is a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Bacillus or a mutant thereof, and more preferably, the gene (B) is a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8. Preferably, the microorganism is a transformed microorganism transformed to increase the supply of 3-hydroxyhexanoyl-CoA to intracellular polyhydroxyalkanoic acid synthase compared to a wild-type strain of the microorganism. More preferably, the transformed microorganism is transformed to suppress the decomposition of a C6 intermediate metabolite in the C6 oxidation of fats and oils or fatty acids. Even more preferably, the transformed microorganism is transformed to suppress the expression of a gene encoding a C6 β-ketothiolase enzyme having thiolysis activity against C6 β-ketohexanoyl-CoA, a C6 β-ketoacyl-CoA. Preferably, the β-ketothiolase enzyme has an amino acid sequence that shows 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10. Preferably, the microorganism is a microorganism having a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity. Preferably, a carbon source containing fats and oils or fatty acids is added in the culture step. More preferably, the carbon source containing fats and oils or fatty acids is a carbon source containing a medium-chain fatty acid having 6 to 12 carbon atoms or a glyceride of the medium-chain fatty acid. Even more preferably, the medium-chain fatty acid is hexanoic acid. Preferably, the microorganism belongs to the genus Capriavidus or is a transformant of a Capriavidus microorganism. More preferably, the microorganism is Capriavidus necator or is a transformant of Capriavidus necator. The present invention also relates to a transformed microorganism that produces a copolymerized polyhydroxyalkanoic acid mixture, the transformed microorganism having genes encoding two types of polyhydroxyalkanoic acid synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA, and that is transformed so as to increase the supply of 3-hydroxyhexanoyl-CoA to the intracellular polyhydroxyalkanoic acid synthase compared to a wild-type strain of the transformed microorganism, the copolymerized polyhydroxyalkanoic acid mixture comprising a polyhydroxyalkanoic acid fraction (I) containing a copolymerized polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit and having an average 3-hydroxyhexanoic acid composition ratio of 20 mol % or more, and a polyhydroxyalkanoic acid fraction (II) containing a polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and having an average 3-hydroxyhexanoic acid composition ratio of 0 mol % or more and 15 mol % or less, and the copolymerized polyhydroxyalkanoic acid mixture has an average 3-hydroxyhexanoic acid composition ratio of 22 mol % or less. Effect of the Invention

[0017] According to the present invention, it is possible to produce a copolymerized polyhydroxyalkanoic acid mixture that has both excellent processability and mechanical properties. According to a preferred embodiment of the present invention, the copolymerized polyhydroxyalkanoic acid mixture produced is easy to handle, and can be easily isolated and purified industrially from microorganisms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, an embodiment of the present invention will be described, however, the present invention is not limited to the following embodiment. The present invention is a method for producing a copolymeric PHA mixture, comprising the step of culturing a microorganism that produces the copolymeric PHA mixture.

[0019] (Copolymerized PHA mixture) The copolymer PHA mixture is composed of a PHA fraction (I) containing a copolymer PHA having a 3HB structural unit and a 3HH structural unit and having an average 3HH composition ratio of 20 mol% or more, and a PHA fraction (II) containing a PHA having a 3HB structural unit and having an average 3HH composition ratio of 0 mol% or more and 15 mol% or less. The copolymer PHA mixture can be fractionated into the PHA fraction (I) and the PHA fraction (II) by the MIBK fractionation method described later.

[0020] The PHA fraction (I) is a fraction containing a copolymerized PHA having at least 3HB structural units and 3HH structural units, and may contain a PHA containing hydroxyalkanoic acid structural units other than 3HB structural units and 3HH structural units, but is preferably a fraction containing a copolymerized PHA having only 3HB structural units and 3HH structural units, i.e., P(3HB-co-3HH), without containing hydroxyalkanoic acid structural units other than 3HB structural units and 3HH structural units.

[0021] 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, 3HV, 3-hydroxyalkanoic acids having 7 to 16 carbon atoms, 2-hydroxyalkanoic acids having 4 to 16 carbon atoms, 4-hydroxyalkanoic acids (e.g., 4-hydroxybutyric acid), 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids (e.g., 6-hydroxyhexanoic acid), and lactic acid, but are not limited to these.

[0022] The average 3HH composition ratio in the PHA fraction (I) is 20 mol% or more, preferably 22 mol% or more, more preferably 24 mol% or more. The upper limit of the average 3HH composition ratio is not particularly limited, but is preferably 35 mol% or less, more preferably 32 mol% or less, and particularly preferably 30 mol% or less.

[0023] The PHA fraction (II) is a fraction containing PHA having 3HB structural units. The PHA contained in the PHA fraction (II) may be a homopolymer having only 3HB structural units, or may be a copolymer PHA having 3HB structural units and other hydroxyalkanoic acid structural units. Considering mechanical properties, a copolymer PHA having 3HB structural units and other hydroxyalkanoic acid structural units is preferred. As the copolymer PHA, a copolymer PHA having 3HB structural units and 3HV structural units and / or 3HH structural units is preferred, a copolymer PHA having 3HB structural units and 3HH structural units is more preferred, and a copolymer PHA having only 3HB structural units and 3HH structural units, i.e., P(3HB-co-3HH), is even more preferred.

[0024] The average 3HH composition ratio in the PHA fraction (II) is 0 mol% or more and 15 mol% or less. The lower limit of the average 3HH composition ratio is preferably 0.1 mol% or more, more preferably 1 mol% or more, even more preferably 2 mol% or more, and even more preferably 3 mol% or more. The upper limit of the average 3HH composition ratio is preferably 12 mol% or less, more preferably 10 mol% or less.

[0025] The weight ratio of the PHA fraction (I) in the copolymer PHA mixture is preferably 10% to 90%, more preferably 20% to 80%, and particularly preferably 30% to 70%. The weight ratio of the PHA fraction (II) in the copolymer PHA mixture is preferably 10% to 90%, more preferably 20% to 80%, and particularly preferably 30% to 70%.

[0026] The average 3HH composition ratio of the copolymer PHA mixture as a whole is 22 mol% or less. If the average 3HH composition ratio exceeds 22 mol%, the copolymer PHA mixture becomes highly viscous, and problems such as the formation of unintended aggregates and adhesion or clogging of piping or pump equipment are likely to occur in the isolation and purification process of the copolymer PHA mixture after fermentation production by microorganisms, which tends to make industrial isolation and purification of the copolymer PHA mixture difficult.

[0027] Considering the balance between the mechanical properties of the molded article formed from the mixture and the processability and industrial handleability of the mixture, the average 3HH composition ratio of the entire copolymerized PHA mixture is preferably 10 to 22 mol%, more preferably 11 to 20 mol%, even more preferably 12 to 18 mol%, and particularly preferably 13 to 17 mol%.

[0028] (MIBK fractionation method) The copolymer PHA mixture can be fractionated into a PHA fraction (I) having a high average 3HH composition ratio and a PHA fraction (II) having a low average 3HH composition ratio by a solvent fractionation method utilizing the difference in solubility in methyl isobutyl ketone (MIBK). The higher the 3HH composition ratio of a PHA, the higher its solubility in MIBK. Therefore, it is possible to fractionate the copolymer PHA mixture into the PHA fraction (I) and the PHA fraction (II) by dissolving the entire copolymer PHA mixture in high-temperature MIBK, lowering the temperature, and precipitating the PHA component with a low 3HH composition ratio.

[0029] The specific fractionation procedure is as follows. First, approximately 100 mg of the copolymer PHA mixture is weighed into a screw-top test tube, 10 ml of MIBK is added, and the cap is closed. After that, the mixture is heated at 140°C for about 1 to 3 hours while shaking to completely dissolve the copolymer PHA mixture. After complete dissolution, the mixture is left at 25°C for 1 minute to reduce the temperature below the boiling point, and all of the dissolved solution is quickly transferred to a centrifuge tube whose weight has been measured beforehand and the cap is closed. The capped centrifuge tube is left at 25°C for an additional 15 minutes to precipitate a portion of the dissolved material. The precipitate and the dissolved solution are separated by centrifugation (9000 rpm, 5 minutes), and all of the dissolved solution is transferred to an aluminum cup whose weight has been measured beforehand. 10 ml of MIBK is added to the centrifuge tube with the precipitate remaining, mixed with a vortex mixer, centrifuged again (9000 rpm, 5 minutes), and the solution is transferred to the aluminum cup containing the dissolved solution. The aluminum cup is heated at 120°C for 30 minutes to volatilize the MIBK and precipitate the dissolved material. The precipitate remaining in the aluminum cup and the precipitate remaining in the centrifuge tube are then vacuum dried at 100°C for 6 hours. The precipitate remaining in the aluminum cup is taken as PHA fraction (I), and the precipitate remaining in the centrifuge tube is taken as PHA fraction (II). The difference between the total weight of PHA fraction (I) and PHA fraction (II) and the weight of the copolymerized PHA mixture measured initially is confirmed to be within ±3%.

[0030] (Melting behavior of copolymer PHA mixture) The copolymer PHA mixture preferably has a highest melting peak temperature of 130°C or higher in differential scanning calorimetry. By satisfying this condition, the copolymer PHA mixture can be crystallized and solidified in a short time, and the copolymer PHA mixture can have good processability. The highest melting peak temperature is preferably 130 to 165°C, more preferably 130 to 155°C.

[0031] The highest melting peak temperature exhibited by the copolymer PHA mixture is measured using a differential scanning calorimeter by weighing out about 2 mg of the copolymer PHA mixture and heating it from -30°C to 200°C at a heating rate of 10°C / min, and measuring the temperature of the melting peak on the highest temperature side in the DSC curve obtained.

[0032] The copolymer PHA mixture may have, in addition to the melting peak on the highest temperature side, another melting peak in the region on the lower temperature side than the highest temperature peak, for example, a melting peak at 100°C or lower.

[0033] (Microorganisms producing copolymer PHA mixtures) The microorganism used to produce the copolymer PHA mixture (hereinafter also referred to as "copolymer PHA mixture-producing microorganism") is not particularly limited as long as it is a microorganism capable of fermenting and producing the copolymer PHA mixture, and may be a wild-type strain that naturally accumulates PHA, a mutant strain obtained by artificially mutating such a wild-type strain, or a strain to which PHA accumulation ability has been imparted by introducing an exogenous PHA synthesis enzyme gene by genetic engineering techniques.

[0034] The copolymerized PHA mixture-producing microorganism, or when the microorganism is a transformant, the host of the transformant is not particularly limited, and preferred examples thereof include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, etc. From the viewpoints of safety and PHA productivity, more preferred are bacteria belonging to the genera Ralstonia, Cupriavidus, Aeromonas, and Wautersia, even more preferred are bacteria belonging to the genus Cupriavidus or Aeromonas, even more preferred are bacteria belonging to the genus Cupriavidus, and particularly preferred is Cupriavidus necator.

[0035] The copolymer PHA mixture-producing microorganism is preferably a microorganism having genes encoding two types of PHA synthases with different polymerization activities for 3-hydroxyhexanoyl-CoA so that two types of PHAs with different average 3HH composition ratios can be efficiently produced. 3-Hydroxyhexanoyl-CoA is a precursor of the 3HH structural unit contained in PHA. By the microorganism having genes encoding two types of PHA synthases with different polymerization activities for 3-hydroxyhexanoyl-CoA, a mixture of two types of PHAs with significantly different average 3HH composition ratios, i.e., the copolymer PHA mixture, can be produced by fermentation in the cells of the microorganism. The microorganism only needs to have at least two types of genes encoding PHA synthases with different polymerization activities for 3-hydroxyhexanoyl-CoA, and may have three or more types of the genes as long as it can produce the copolymer PHA mixture by fermentation.

[0036] The two types of PHA synthesizing enzymes having different polymerization activities for 3-hydroxyhexanoyl-CoA are not particularly limited, but the sequence identity of the amino acid sequences between the two types of PHA synthesizing enzymes is preferably 90% or less. More preferably, it is 80% or less, and even more preferably, it is 70% or less. In general, it is believed that PHA synthesizing enzymes function by forming a multimer such as a dimer. If the sequence homology of the amino acid sequences between the two types of PHA synthesizing enzymes is higher than 90%, it is possible that the two types of PHA synthesizing enzymes form a heterodimer or the like, making it impossible to produce the copolymerized PHA mixture.

[0037] A specific example of a combination of two types of PHA synthases having different polymerization activities for 3-hydroxyhexanoyl-CoA is a combination of a gene (A) encoding a PHA synthase having a higher polymerization activity for 3-hydroxyhexanoyl-CoA than the wild-type PHA synthase derived from Aeromonas caviae having the amino acid sequence set forth in SEQ ID NO: 1, and a gene (B) encoding a PHA synthase having a lower polymerization activity for 3-hydroxyhexanoyl-CoA than the wild-type polyhydroxyalkanoic acid synthase derived from Aeromonas caviae.

[0038] Examples of the gene (A) include a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas or a mutant thereof, and specifically include a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 (the amino acid sequence of a PHA synthase mutant derived from a bacterium of the genus Aeromonas). The sequence identity is preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and most preferably 99.5% or more.

[0039] The gene (B) may be a gene formed by combining a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas with a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Capriavidus, and specifically, a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence described in SEQ ID NO: 6. The gene (B) may also be a PHA synthase gene derived from a bacterium of the genus Chromobacterium or a variant thereof, and specifically, a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence described in SEQ ID NO: 4 or SEQ ID NO: 5. Alternatively, the gene (B) may also be a PHA synthase gene derived from a bacterium of the genus Bacillus or a variant thereof, and specifically, a gene encoding an amino acid sequence having 90 to 100% sequence identity to the amino acid sequences described in SEQ ID NO: 7 and SEQ ID NO: 8. The sequence identities described above for the gene (B) are preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0040] In order to efficiently produce a copolymer PHA mixture having a high average 3HH composition ratio, the copolymer PHA mixture-producing microorganism is preferably a transformed microorganism that has been transformed to increase the supply of 3-hydroxyhexanoyl-CoA to the intracellular PHA synthesis enzyme compared to a wild-type strain of the microorganism. Specifically, a transformed microorganism that has been transformed to suppress the decomposition of an intermediate metabolite having six carbon atoms in the β-oxidation of fats and oils or fatty acids is preferred. It is presumed that the suppression of the decomposition of an intermediate metabolite having six carbon atoms in the β-oxidation results in an increased supply of 3-hydroxyhexanoyl-CoA, and thus an increased average 3HH composition ratio is exhibited in the copolymer PHA mixture produced.

[0041] Examples of transformed microorganisms transformed to suppress the decomposition of intermediate metabolites having 6 carbon atoms in the β-oxidation of fats and oils or fatty acids include transformed microorganisms transformed to suppress the expression of a gene encoding a β-ketothiolase enzyme having thiolysis activity against β-ketohexanoyl-CoA, which is a β-ketoacyl-CoA having 6 carbon atoms, as described in Patent Document 7. Examples of genes encoding the β-ketothiolase enzyme include, but are not limited to, genes encoding a β-ketothiolase enzyme having an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO: 10. The sequence identity is preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

[0042] Examples of methods for suppressing the expression of a gene encoding a β-ketothiolase enzyme include a method for completely deleting the enzyme gene in a transformed microorganism, a method for inserting an entirely different gene such as a drug resistance gene into the sequence of the enzyme gene, or a method for deleting, substituting, adding, or inserting a part of the sequence of the enzyme gene (preferably a region involved in enzyme activity), etc. Examples of gene disruption techniques include homologous recombination techniques using a vector containing a gene or DNA for disruption, and techniques using transposons. Alternatively, other disruption methods include known techniques such as the CRISPR / Cas (e.g., Cas9) system for disrupting target genes and genome editing technology using TALEN (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, guide RNA (gRNA) has a sequence that can bind to a part of the base sequence of the β-ketothiolase gene to be destroyed, and plays a role in carrying Cas9 to the target. In addition, the enzyme activity can be eliminated or reduced by reducing the transcription / translation efficiency of the gene and the stability of mRNA through mutations such as deletion, substitution, addition, and insertion of the base sequence surrounding the gene.

[0043] In order to efficiently produce a copolymer PHA mixture having a high average 3HH composition ratio, the copolymer 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 3-hydroxyhexanoyl-CoA in the microbial cell. Therefore, it is presumed that the amount of 3-hydroxyhexanoyl-CoA converted is increased by the microorganism having a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity, and as a result, the average 3HH composition ratio of the copolymer PHA mixture produced is increased.

[0044] The microorganism having a gene encoding a protein exhibiting the R-specific enoyl-CoA hydratase activity may be a microorganism that inherently has the gene, or a microorganism into which an exogenous gene has been introduced by genetic engineering techniques.

[0045] Examples of the exogenous gene encoding a protein having R-specific enoyl-CoA hydratase activity include, but are not limited to, a gene derived from Aeromonas caviae encoding an R-specific enoyl-CoA hydratase having the amino acid sequence set forth in SEQ ID NO: 11, a gene derived from Capriavidus necator encoding an R-specific enoyl-CoA hydratase having the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, a Multifunctional enzyme type 2 (MFE2) gene derived from Yarrowia lipolytica encoding an enzyme having the amino acid sequence set forth in SEQ ID NO: 14, an MFE2 gene derived from Drosophila melanogaster encoding an enzyme having the amino acid sequence set forth in SEQ ID NO: 15, or a gene having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more to each of the amino acid sequences set forth in SEQ ID NOs: 11 to 15 and encoding a protein having R-specific enoyl-CoA hydratase activity.

[0046] Furthermore, in order to enhance the expression of a gene encoding a protein having R-specific enoyl-CoA hydratase activity, the expression regulatory sequence (promoter sequence and / or SD sequence) may be modified to enhance the expression of the gene, for example, as described in WO 2015 / 115619.

[0047] When a foreign gene is introduced into the copolymerized PHA mixture-producing microorganism, the introduced gene may be present on a chromosome possessed by the host microorganism, or on DNA such as a plasmid or megaplasmid. From the viewpoint of retaining the introduced gene, it is preferable that the introduced gene is present on a chromosome or megaplasmid possessed by the microorganism, and it is more preferable that the introduced gene is present on a chromosome possessed by the microorganism. In addition, when the expression level of a gene originally possessed by the host microorganism is to be increased, the expression level of the gene may be increased by substituting, deleting or adding a base sequence upstream of the gene.

[0048] Methods for site-specifically substituting or inserting any DNA into DNA possessed by a microorganism, or methods for deleting any site in DNA possessed by a microorganism, are well known to those skilled in the art and can be used when producing a transformed microorganism according to this embodiment. Representative methods include, but are not limited to, a method that utilizes the mechanism of transposon and homologous recombination (Ohman et al., J. Bacteriol., vol. 162: p. 1068 (1985)), a method based on site-specific integration caused by the mechanism of homologous recombination and its elimination by second-stage homologous recombination (Noti et al., Methods Enzymol., vol. 154, p. 197 (1987)), and a method in which the sacB gene derived from Bacillus subtilis is coexisted, and a microbial strain from which the gene has been eliminated by second-stage homologous recombination is easily isolated as a sucrose-supplemented medium-resistant strain (Schweizer, Mol. Microbiol., vol. 6, p. 1195 (1992); Lenz et al., J. Bacteriol., vol. 176, p. 4385 (1994)). The method for introducing a vector into a cell is not particularly limited, but examples thereof include the calcium chloride method, electroporation, polyethylene glycol method, and spheroplast method.

[0049] For gene cloning and gene recombination techniques, the techniques described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989 or 2001) can be used.

[0050] The promoter for expressing the introduced gene is not particularly limited. The promoter of the phaC1 gene of Caprifolius necator, the promoter of the phaP1 gene, the lac promoter derived from Escherichia coli, the lacUV5 promoter, the trc promoter, the tic promoter, the tac promoter, the artificially created lacN17 promoter having a modified base sequence derived from Escherichia coli shown in SEQ ID NO: 16, the artificially created lacN19 promoter having a modified base sequence derived from Escherichia coli shown in SEQ ID NO: 17, etc. can be used.

[0051] (Microbial Culture) By culturing the copolymerized PHA mixture-producing microorganism, the copolymerized PHA mixture can be accumulated in the microbial cells. The method for culturing the copolymerized PHA mixture-producing microorganism can be a conventional microbial culture method, and the culture may be performed in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, the method for adding the carbon source, the culture scale, the aeration and agitation conditions, the culture temperature, the culture time, and the like. It is preferable to add the carbon source to the medium continuously or intermittently.

[0052] Any carbon source can be used as a carbon source during the culture, as long as the microorganism producing the copolymerized PHA mixture can utilize it. Examples of carbon sources include, but are not limited to, sugars such as glucose, fructose, sucrose, and xylose; oils and fats such as palm oil and palm kernel oil (including palm olein, palm double olein, and palm kernel oil olein, which are low melting point fractions obtained by fractionating these oils), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and fractionated oils thereof, or refined by-products thereof; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myristic acid, and derivatives thereof, or glycerol. In addition, when the microorganism producing the copolymerized PHA mixture can utilize gases and alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources.

[0053] Among them, the carbon source preferably contains fats and oils or fatty acids. The fats and oils are preferably vegetable oils or their fractionated oils. The fats and oils or fatty acids preferably have a short chain length, and the carbon source more preferably contains a medium-chain fatty acid having 6 to 12 carbon atoms or a glyceride of the medium-chain fatty acid, and further preferably contains hexanoic acid. It is considered that when the fatty acid contained in the carbon source has 6 to 12 carbon atoms, the amount of intermediate metabolites having 6 carbon atoms produced in β-oxidation increases, and a PHA fraction having a high average 3HH composition ratio can be efficiently obtained.

[0054] In the production of the copolymerized PHA mixture, it is preferable to culture the microorganisms using a medium containing the carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Although not limited to the following, examples of the nitrogen source 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, and sodium chloride, and the like. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C, and the like.

[0055] The copolymerized PHA mixture-producing microorganism is cultured for an appropriate time to accumulate the copolymerized PHA mixture in the microbial cells, and then the copolymerized PHA mixture is recovered using a known method. There is no particular limitation on the recovery method, but industrially, recovery by separation and purification in an aqueous system with low environmental impact is preferred. For example, after the end of the culture, a cell disruption solution in which cell components other than PHA are dissolved in water can be obtained by applying mechanical shearing force or disrupting the cells using a surfactant, alkali, enzyme, or the like. The copolymerized PHA mixture can be recovered by separating the copolymerized PHA mixture from the aqueous phase by filtration or centrifugation of the cell disruption solution, and then drying. EXAMPLES

[0056] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. The overall genetic manipulation can be carried out, for example, as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, and the like used in the genetic manipulation can be purchased from commercial suppliers and used according to their instructions. The enzymes are not particularly limited as long as they can be used in genetic manipulation.

[0057] (Microbial Strain Preparation Example 1) Preparation of Copolymerized PHA Mixture-Producing Microbial Strain (1) First, a plasmid for disrupting the PHA synthase gene was prepared as follows. A DNA fragment (SEQ ID NO: 18) having the upstream and downstream base sequences of the phaC1 structural gene (PHA synthase gene) of the Capriavidus necator H16 strain was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using DNA ligase (Ligation High (Toyobo Co., Ltd.)) to prepare a plasmid vector pNS2X-sacB+phaC1UD for disrupting the PHA synthase gene.

[0058] Next, a PHA synthase gene-disrupted strain was prepared as follows using the plasmid vector pNS2X-sacB+phaC1UD for disrupting the PHA synthase gene. The E. coli S17-1 strain (ATCC47055) was transformed with the plasmid vector pNS2X-sacB+phaC1UD for disrupting the PHA synthase gene, and the resulting transformed microorganism was co-cultured with the KNK005dZ / trc-J4b / dbktB / dA1528 strain on Nutrient Agar medium (Difco) to perform conjugative transfer. 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 Capriavidus necator H16 strain have been deleted, the PHA synthase gene on the chromosome has been replaced with a modified version of the PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 2, i.e., the N149S / D171G mutant gene), the expression of the R-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, the bktB structural gene has been deleted, and the A1528 structural gene has been deleted, and can be prepared in accordance with the method described in PCT International Publication No. 2019 / 142845. The resulting culture was inoculated onto 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 kanamycin, and the strains that grew on the agar medium were selected to obtain a strain in which the plasmid was integrated into the chromosome of the KNK005dZ / trc-J4b / dbktB / dA1528 strain. After culturing this strain for two generations in Nutrient Broth medium (Difco), it was diluted and spread onto Nutrient Agar medium containing 15% sucrose, and the grown strain was obtained as a strain in which the plasmid had been lost. Furthermore, one strain that had lost the PHA synthase gene on the chromosome was isolated by PCR and DNA sequencer analysis. This gene-disrupted strain was designated KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain.

[0059] Furthermore, a plasmid for introducing the PHA synthase gene was prepared as follows. A DNA fragment (SEQ ID NO: 19) was obtained by PCR using synthetic oligo DNA, which contains the upstream and downstream base sequences of the bktB structural gene (β-ketothiolase gene) of the Capriavidus necator H16 strain, the lacN19 promoter, which is a modified Escherichia coli lac promoter, and the base sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-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-lacN19-NSDG-bktbD for introducing the PHA synthase gene.

[0060] Next, a strain into which the PHA synthase gene was introduced was prepared as follows, using the plasmid vector pNS2X-sacB+bktbU-lacN19-NSDG-bktbD for introducing the PHA synthase gene. The plasmid vector pNS2X-sacB+bktbU-lacN19-NSDG-bktb for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as above. Furthermore, by the same cultivation as above and selection with Nutrient Agar medium containing 15% sucrose, one strain was isolated in which the lacN19 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced at the position where the bktB gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lacN19-NSDG / dA1528 strain.

[0061] Furthermore, a plasmid for introducing the PHA synthase gene was prepared as follows. By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 20) was obtained that contains the upstream and downstream base sequences of the A1528 structural gene (β-ketothiolase gene) of the Capriavidus necator H16 strain, the lacN17 promoter, which is a modified lac promoter of E. coli, and the base sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 6. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-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+A1528U-lacN17-AcNSRe12-A1528D for introducing the PHA synthase gene.

[0062] Next, the plasmid vector pNS2X-sacB+A1528U-lacN17-AcNSRe12-A1528D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB::lacN19-NSDG / dA1528 strain by the same conjugation transfer method as above. Furthermore, by the same cultivation as above and selection with Nutrient Agar medium containing 15% sucrose, one strain was isolated in which the lacN17 promoter and the gene encoding the PHA synthase having the amino acid sequence shown in SEQ ID NO: 6 were introduced at the position where the A1528 gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lacN19-NSDG / dA1528::lacN17-AcNSRe12 strain (hereinafter, also referred to as copolymer PHA mixture-producing microbial strain (1)). The copolymer PHA mixture-producing microbial strain (1) is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 2 and a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 6 (i.e., a gene encoding a PHA synthase composed of a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas and a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Capriavidus) have been introduced, expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0063] (Microbial strain preparation example 2) Preparation of copolymer PHA mixture-producing microbial strain (2) First, a plasmid for introducing the PHA synthase gene was prepared as follows. A DNA fragment (SEQ ID NO: 21) was obtained by PCR using synthetic oligo DNA, which has the nucleotide sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the Capriavidus necator H16 strain, the lac promoter of Escherichia coli, and the nucleotide sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-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-lac-NSDG-bktbD for introducing the PHA synthase gene.

[0064] Next, a strain into which the PHA synthase gene was introduced was prepared as follows, using the plasmid vector pNS2X-sacB+bktbU-lac-NSDG-bktbD for introducing the PHA synthase gene. The plasmid vector pNS2X-sacB+bktbU-lac-NSDG-bktb for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as above. Furthermore, by the same cultivation as above and selection with Nutrient Agar medium containing 15% sucrose, one strain was isolated in which a lac promoter and a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2 were introduced at the position where the bktB gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528 strain.

[0065] Next, the plasmid vector pNS2X-sacB+A1528U-lacN17-AcNSRe12-A1528D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528 strain by the same conjugation transfer method as above. Furthermore, by the same cultivation as above and selection with Nutrient Agar medium containing 15% sucrose, one strain in which the lacN17 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 6 were introduced at the position where the A1528 gene originally existed on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528::lacN17-AcNSRe12 strain (hereinafter, sometimes referred to as copolymerized PHA mixture producing microorganism strain (2)). The copolymer PHA mixture-producing microbial strain (2) is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 2 and a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 6 (i.e., a gene encoding a PHA synthase composed of a combination of a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas and a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Capriavidus) have been introduced, expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0066] (Microbial Strain Preparation Example 3) Preparation of Copolymerized PHA Mixture-Producing Microbial Strain (3) First, a plasmid for expressing the PHA synthesizing enzyme gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 22) having the lacN17 promoter, which is a modified lac promoter of E. coli, was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with restriction enzymes EcoRI and MunI, and the resulting DNA fragment was ligated to a plasmid vector pCUP2 described in International Publication No. 2007 / 049716 that was cleaved with MunI, and the DNA fragment ligated in the direction in which the restriction enzyme SpeI recognition sequence of pCUP2 was located downstream of the lacN17 promoter was selected, thereby obtaining pCUP2-lacN17. Next, a DNA fragment (SEQ ID NO: 23) having the base sequence of a gene encoding a PHA synthesizing enzyme having the amino acid sequence described in SEQ ID NO: 6 and a gene encoding a PHA synthesizing enzyme having the amino acid sequence described in SEQ ID NO: 2 was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain a plasmid for expressing the PHA synthase gene, pCUP2-lacN17-AcNSRe12-NSDG.

[0067] Next, the plasmid pCUP2-lacN17-AcNSRe12-NSDG for expressing the PHA synthesis enzyme gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain to obtain the pCUP2-lacN17-AcNSRe12-NSDG / KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain (hereinafter, sometimes referred to as the copolymerized PHA mixture-producing microbial strain (3)). The introduction of the plasmid vector into the cells was carried out by electroporation as follows. A Biorad Gene Pulser was used as the gene introduction device, and a Biorad gap 0.2 cm cuvette was used. 400 μl of competent cells and 20 μl of expression vector were poured into the cuvette and set in the pulse device, and an electric pulse was applied under the conditions of capacitance 25 μF, voltage 1.5 kV, and resistance value 800 Ω. After the pulse, the bacterial solution in the cuvette was shake-cultured in Nutrient Broth medium (DIFCO) at 30° C. for 3 hours, and then cultured on a selection plate (Nutrient Agar medium (DIFCO), kanamycin 100 mg / L) at 30° C. for 2 days to obtain the grown copolymerized PHA mixture-producing microbial strain (3). The copolymer PHA mixture-producing microbial strain (3) is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 2 and a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 6 (i.e., a gene encoding a PHA synthase composed of a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas and a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Capriavidus) have been introduced, expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0068] (Microbial Strain Preparation Example 4) Preparation of Copolymerized PHA Mixture-Producing Microbial Strain (4) First, a plasmid for introducing the PHA synthase gene was prepared as follows. A DNA fragment (SEQ ID NO: 24) was obtained by PCR using synthetic oligo DNA, which has the nucleotide sequences upstream and downstream of the bktB structural gene (β-ketothiolase gene) of the Capriavidus necator H16 strain, the lac promoter of Escherichia coli, a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 6, and a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 2. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-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-lac-AcNSRe12-NSDG-bktbD for introducing the PHA synthase gene.

[0069] Next, a strain into which the PHA synthase gene was introduced was prepared as follows, using the plasmid vector pNS2X-sacB+bktbU-lac-AcNSRe12-NSDG-bktbD for introducing the PHA synthase gene. The plasmid vector pNS2X-sacB+bktbU-lac-AcNSRe12-NSDG-bktbD for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as above. Furthermore, by the same cultivation as above and selection with Nutrient Agar medium containing 15% sucrose, one strain was isolated in which a lac promoter, a gene encoding a PHA synthase having the amino acid sequence shown in SEQ ID NO:6, and a gene encoding a PHA synthase having the amino acid sequence shown in SEQ ID NO:2 were introduced at the position where the bktB gene originally existed on the chromosome. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-AcNSRe12-NSDG / dA1528 strain (hereinafter, sometimes referred to as a copolymerized PHA mixture producing microorganism strain (4)). The copolymer PHA mixture-producing microbial strain (4) is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator strain H16 have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 2 and a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 6 (i.e., a gene encoding a PHA synthase composed of a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Aeromonas and a part of a polyhydroxyalkanoic acid synthase gene derived from a microorganism of the genus Capriavidus have been introduced, expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0070] (Microbial Strain Preparation Example 5) Preparation of Copolymerized PHA Mixture-Producing Microbial Strain (5) First, a plasmid for expressing the PHA synthesizing enzyme gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 26) having the base sequence of a gene encoding a PHA synthesizing enzyme having the amino acid sequence set forth in SEQ ID NO: 7, a gene encoding a PHA synthesizing enzyme having the amino acid sequence set forth in SEQ ID NO: 8, and a gene encoding a PHA synthesizing enzyme having the amino acid sequence set forth in SEQ ID NO: 3 was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain a plasmid for expressing the PHA synthesizing enzyme gene, pCUP2-lacN17-RCYB4-NSDGST.

[0071] Next, the plasmid pCUP2-lacN17-RCYB4-NSDGST for expressing the PHA synthesis enzyme gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the electroporation method described in Example 3 of microbial strain preparation, to obtain the pCUP2-lacN17-RCYB4-NSDGST / KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain (hereinafter, sometimes referred to as the copolymerized PHA mixture-producing microbial strain (5)). The copolymer PHA mixture-producing microbial strain (5) is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO: 3 and a gene encoding a PHA synthase derived from the genus Bacillus having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8 have been introduced, expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0072] (Microbial Strain Preparation Example 6) Preparation of Copolymerized PHA Mixture-Producing Microbial Strain (6) First, a plasmid for introducing the PHA synthase gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 27) was obtained by PCR using synthetic oligo DNA, which has the upstream and downstream base sequences of the A1528 structural gene (β-ketothiolase gene) of the Capriavidus necator H16 strain, the lacN17 promoter, which is a modified lac promoter of E. coli, and the base sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 5. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-2007-259708, which was also digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare a plasmid vector pNS2X-sacB+A1528U-lacN17-CsAG-A1528D for introducing the PHA synthase gene.

[0073] Next, the plasmid vector pNS2X-sacB+A1528U-lacN17-CsAG-A1528D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528 strain by the same conjugation transfer method as above. Furthermore, by the same cultivation as above and selection with Nutrient Agar medium containing 15% sucrose, one strain in which the lacN17 promoter and the gene encoding the PHA synthase having the amino acid sequence described in SEQ ID NO: 5 were introduced at the position where the A1528 gene originally existed on the chromosome was isolated. The obtained strain was named KNK005dZ / dNSDG / trc-J4b / dbktB::lac-NSDG / dA1528::lacN17-CsAG strain (hereinafter, sometimes referred to as copolymerized PHA mixture producing microorganism strain (6)). The copolymer PHA mixture-producing microbial strain (6) is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO:2 and a gene encoding a PHA synthase derived from the genus Chromobacterium having the amino acid sequence set forth in SEQ ID NO:5 have been introduced, expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0074] (Microbial Strain Preparation Example 7) Preparation of P(3HB-co-3HH)-producing microbial strain (1) The KNK005dZ strain (hereinafter sometimes referred to as P(3HB-co-3HH)-producing microbial strain (1)) is a transformed microorganism in which a PHA synthase gene derived from the genus Aeromonas (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 2) has been introduced onto the chromosome of the Capriavidus necator H16 strain, and the phaZ1, 2, and 6 genes, which are PHA decomposition enzyme genes on the chromosome, have been deleted. This transformed microorganism can be prepared according to the method described in PCT Publication No. WO 2014 / 065253.

[0075] (Microbial Strain Preparation Example 8) Preparation of P(3HB-co-3HH)-producing microbial strain (2) First, a plasmid for introducing the PHA synthase gene was prepared as follows. By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 28) was obtained that had the upstream and downstream base sequences of the phaZ6 structural gene of Capriavidus necator H16 strain, the lacN17 promoter, which is a modified lac promoter of E. coli, and the base sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 2. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-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+phaZ6U-lacN17-NSDG-phaZ6D for introducing the PHA synthase gene. Next, the plasmid vector pNS2X-sacB+phaZ6U-lacN17-NSDG-phaZ6D for introducing the PHA synthase gene was introduced into the KNK005dZ / trc-J4b / dbktB strain by the same conjugation transfer method as above. The KNK005dZ / trc-J4b / dbktB strain is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, the PHA synthase gene on the chromosome has been replaced with a modified version of the PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 2, i.e., the N149S / D171G mutant gene), expression of the R-form-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene has been deleted, and can be prepared in accordance with the method described in PCT International Publication No. 2019 / 142845. Furthermore, by culturing in the same manner as above and selecting on Nutrient Agar medium containing 15% sucrose, one strain was isolated in which the lacN17 promoter and a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 2 were introduced into the position where the phaZ6 gene was originally located on the chromosome. The obtained strain was named KNK005dZ / trc-J4b / Z6::lacN17-NSDG / dbktB strain (hereinafter, sometimes referred to as P(3HB-co-3HH)-producing microorganism strain (2)).

[0076] (Microbial Strain Preparation Example 9) Preparation of P(3HB-co-3HH)-producing microbial strain (3) First, the plasmid vector pNS2X-sacB+phaC1UD for disrupting the PHA synthase gene was introduced into the KNK005dZ / trc-J4b / dbktB strain by the same conjugal transfer method as above. Furthermore, by culturing in the same manner as above and selecting on Nutrient Agar medium containing 15% sucrose, we isolated one strain in which the PHA synthase gene on the chromosome was deleted and named the KNK005dZ / dNSDG / trc-J4b / dbktB strain.

[0077] Next, a plasmid for introducing the PHA synthase gene was prepared as follows. A DNA fragment (SEQ ID NO: 29) was obtained by PCR using synthetic oligo DNA, which has the nucleotide sequence upstream and downstream of the phaC1 structural gene of the Capriavidus necator H16 strain and the nucleotide sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 3. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP-A-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+phaC1U-NSDGST-phaC1D for introducing the PHA synthase gene. Next, the plasmid vector pNS2X-sacB+phaC1U-NSDGST-phaC1D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB strain by the same conjugal transfer method as above. Furthermore, by culturing in the same manner as above and selecting on a Nutrient Agar medium containing 15% sucrose, one strain was isolated in which a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 3 was introduced into the position on the chromosome where the phaC1 gene originally existed. The obtained strain was named KNK005dZ / NSDGST / trc-J4b / dbktB strain.

[0078] Next, a plasmid for expressing the PHA synthase gene was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 30) having the base sequence of a gene encoding a PHA synthase having the amino acid sequence described in SEQ ID NO: 3 was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the obtained DNA fragment was ligated to a product obtained by cleaving pCUP2 described in International Publication No. 2007 / 049716 with MunI and SpeI to obtain pCUP2-NSDGST. Next, a DNA fragment (SEQ ID NO: 31) having a trp promoter was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with restriction enzyme MunI, and the obtained DNA fragment was ligated to a product obtained by cleaving pCUP2-NSDGST with MunI in such a direction that the gene encoding the PHA synthase was located downstream of the trp promoter to obtain a plasmid for expressing the PHA synthase gene, pCUP2-trp-NSDGST.

[0079] Next, the PHA synthase gene expression plasmid pCUP2-trp-NSDGST was introduced into the KNK005dZ / NSDGST / trc-J4b / dbktB strain by the electroporation method described in Example 3 of microbial strain preparation, to obtain the pCUP2-trp-NSDGST / KNK005dZ / NSDGST / trc-J4b / dbktB strain (hereinafter, sometimes referred to as P(3HB-co-3HH)-producing microbial strain (3)). The P(3HB-co-3HH)-producing microbial strain (3) is a strain in which the phaZ1, phaZ2, and phaZ6 genes on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO:3 has been introduced, expression of the R-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) has been deleted.

[0080] (Microbial Strain Preparation Example 10) Preparation of P(3HB-co-3HH)-producing microbial strain (4) First, the plasmid vector pNS2X-sacB+phaC1U-NSDGST-phaC1D for introducing the PHA synthase gene was introduced into the KNK005dZ / dNSDG / trc-J4b / dbktB / dA1528 strain by the same conjugation transfer method as described above. Furthermore, by culturing in the same manner as above and selecting on Nutrient Agar medium containing 15% sucrose, one strain was isolated in which a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 3 was introduced into the position on the chromosome where the phaC1 gene originally existed. The obtained strain was named KNK005dZ / NSDGST / trc-J4b / dbktB / dA1528 strain.

[0081] Next, the PHA synthase gene expression plasmid pCUP2-trp-NSDGST was introduced into the KNK005dZ / NSDGST / trc-J4b / dbktB / dA1528 strain by electroporation as described in Example 3 of microbial strain preparation, to obtain the pCUP2-trp-NSDGST / KNK005dZ / NSDGST / trc-J4b / dbktB / dA1528 strain (hereinafter, sometimes referred to as P(3HB-co-3HH)-producing microbial strain (4)). The P(3HB-co-3HH)-producing microbial strain (4) is a strain in which the phaZ1, phaZ2, and phaZ6 genes on the chromosome of the Capriavidus necator H16 strain have been deleted, a gene encoding a PHA synthase mutant derived from the genus Aeromonas having the amino acid sequence set forth in SEQ ID NO:3 has been introduced, expression of the R-specific enoyl-CoA hydratase gene on the chromosome has been enhanced, and the bktB structural gene (β-ketothiolase gene) and the A1528 structural gene (β-ketothiolase gene) have been deleted.

[0082] (Example 1) PHA production by copolymer PHA mixture-producing microbial strain (1) Cultivation studies were carried out using the copolymer PHA mixture-producing microbial strain (1) under the following conditions.

[0083] (Culture medium) The composition of the seed culture medium was 1 w / v% Meat-extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast-extract, 0.9 w / v% Na2HPO4·12H2O, and 0.15 w / v% KH2PO4 (pH 6.8).

[0084] The preculture medium consisted of 1.1 w / v% Na2HPO4·12H2O, 0.19 w / v% KH2PO4, 1.29 w / v% (NH4)2SO4, 0.1 w / v% MgSO4·7H2O, 2.5 w / v% palm olein oil, and 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl3·6H2O, 1 w / v% CaCl2·2H2O, 0.02 w / v% CoCl2·6H2O, 0.016 w / v% CuSO4·5H2O, and 0.012 w / v% NiCl2·6H2O dissolved in 0.1 N hydrochloric acid).

[0085] The composition of the PHA production medium was 0.385 w / v% Na2HPO4·12H2O, 0.067 w / v% KH2PO4, 0.291 w / v% (NH4)2SO4, 0.1 w / v% MgSO4·7H2O, and 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl3·6H2O, 1 w / v% CaCl2·2H2O, 0.02 w / v% CoCl2·6H2O, 0.016 w / v% CuSO4·5H2O, and 0.012 w / v% NiCl2·6H2O dissolved in 0.1 N hydrochloric acid).

[0086] (Method for measuring the percentage of accumulated PHA) The ratio of PHA accumulation to the dry bacterial cells was measured as follows. The bacterial cells were collected from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain the dry bacterial cells, and their weight was measured. 100 ml of chloroform was added to 1 g of the obtained dry bacterial cells, and the mixture was stirred at room temperature for one day and night to extract the PHA (copolymerized PHA mixture) in the bacterial cells. After filtering the bacterial cell residue, it was concentrated in an evaporator until the total volume was 30 ml, after which 90 ml of hexane was gradually added and the mixture was left for one hour with slow stirring. The precipitated PHA was filtered and then vacuum-dried at 50°C for three hours. The weight of the dried PHA was measured, and the ratio of the PHA accumulation to the dry bacterial cell weight was calculated.

[0087] (Method for measuring weight ratio of PHA fractions (I) and (II)) The weight ratio of PHA fractions (I) and (II) in the copolymer PHA mixture was measured as follows. First, the dry PHA was fractionated into PHA fraction (I) and PHA fraction (II) by the MIBK fractionation method, and each fraction was weighed. Next, the weight ratio of each fraction to the total weight of PHA fraction (I) and PHA fraction (II) was calculated.

[0088] (Method for measuring average 3HH composition ratio) The average 3HH composition ratio of each of the copolymerized PHA mixture, PHA fraction (I), and PHA fraction (II) was measured as follows. 1 ml of sulfuric acid-methanol mixture (15:85) and 1 ml of chloroform were added to about 20 mg of the dried copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II), and the mixture was sealed and heated at 100°C for 140 minutes to obtain methyl esters of PHA decomposition products. After cooling, 0.5 ml of deionized water was added to the mixture and mixed well, and the mixture was left until the aqueous layer and the organic layer were separated. The monomer unit composition of the PHA decomposition products in the separated organic layer was then analyzed by capillary gas chromatography. The gas chromatograph used was Shimadzu GC-17A, and the capillary column used was GL Science NEUTRA BOND-1 (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 100 kPa, and 1 μl of the sample was injected. The temperature was increased from an initial temperature of 50 to 200° C. at a rate of 8° C. / min, and then increased from 200 to 290° C. at a rate of 30° C. / min. The average 3HH composition ratio of the copolymerized PHA mixture, PHA fraction (I), or PHA fraction (II) was calculated from the peaks obtained by the analysis under the above conditions.

[0089] (Method of measuring peak melting temperature of copolymer PHA mixture) Using a differential scanning calorimeter (DSC8500, manufactured by PerkinElmer), approximately 2 mg of the copolymerized PHA mixture was weighed out, and the temperature of the melting peak with a melting enthalpy of 0.5 J / g or more was determined in the DSC curve obtained when the mixture was heated from -30°C to 200°C at a heating rate of 10°C / min.

[0090] (Processability evaluation of copolymer PHA mixture and pellet production) 4.5 g of the copolymerized PHA mixture, 0.045 g of pentaerythritol (manufactured by Mitsubishi Chemical: NeuRizer P) as additives, 0.0225 g of behenic acid amide (manufactured by Nippon Fine Chemicals: BNT-22H), and 0.0225 g of erucic acid amide (manufactured by Nippon Fine Chemicals: Neutron-S) were charged into a small kneader (manufactured by DSM: DSM Xplore 5 Model 2005) and kneaded for 5 minutes under conditions of a barrel temperature of 170 ° C and a screw rotation speed of 100 rpm. After the kneading was completed, the molten strand-shaped resin composition was discharged from the die and immediately charged into a water bath heated to 60 ° C, and the time to crystallize and solidify was measured. If it solidified within 100 seconds, the processability was evaluated as good (○). Thereafter, the strands which had crystallized and solidified in a water bath were cut with nippers to obtain resin composition pellets.

[0091] (Evaluation of tear strength) A PET film (thickness 50 μm) with one side release-treated was placed on a 2 mm thick SUS plate (30 cm × 35 cm) with the release surface facing away from the SUS plate, and 1.3 g of resin composition pellets were placed on the PET film. Furthermore, a 70 μm shim plate was placed as a spacer to surround the resin composition pellets. Thereafter, the resin composition pellets were covered with a plate similar to the SUS plate so as to sandwich the resin composition pellets, and placed on the heating press plate of a press machine (manufactured by Shinto Metal Industries Co., Ltd.: compression molding machine NSF-50) heated to 160 ° C., and preheated for 5 minutes. After preheating, the pressure was gradually increased to 5 MPa over a period of 2 minutes, and the pressure was maintained for 2 minutes. After pressing was completed, the film was cooled to room temperature on a cooling plate cooled to about 20 ° C., and a film with a thickness of about 50 μm was obtained. This film was aged for 1 week in an environment of room temperature 23 ° C. and humidity 50%, and used as a film sample. The value measured using a light-load tear tester (No. 2037 special specification machine, manufactured by Kumagai Riki Kogyo Co., Ltd.) having functions and structure conforming to the standard Elmendorf tear tester specified in JIS P-8116 was divided by the film thickness to obtain the Elmendorf tear strength of the film sample.

[0092] (PHA production culture) PHA production culture was carried out as follows. First, a glycerol stock (50 μl) of the copolymer PHA mixture-producing microbial strain (1) was inoculated into a seed medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture liquid was inoculated at 1.0 v / v% into a 3 L jar fermenter (Marubishi Bioengineering MDL-300 type) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 30°C, a stirring speed of 500 rpm, and aeration of 1.8 L / min, and the culture was carried out for 28 hours while controlling the pH between 6.7 and 6.8 to carry out preculture. A 14% aqueous solution of ammonium hydroxide was used for pH control.

[0093] Next, the preculture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (Marubishi Bioengineering MDS-U50 type) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 33°C, an agitation speed of 420 rpm, and an aeration rate of 2.1 L / min, and the pH was controlled between 6.7 and 6.8. A 25% aqueous solution of ammonium hydroxide was used for pH control. The carbon source was added intermittently. Palm olein oil was used as the carbon source. The culture was continued until the ratio of the PHA accumulation amount to the dry cell mass reached 80% or more. The ratio of the PHA accumulation amount to the dry cell mass, the average 3HH composition ratio of the copolymer PHA mixture, the PHA fraction (I), or the PHA fraction (II), the weight ratio of the PHA fractions (I) and (II), the melting peak temperature and melting enthalpy, the processability, and the Elmendorf tear strength were measured as described above. The results are shown in Table 1.

[0094] (Example 2) PHA production by copolymer PHA mixture-producing microbial strain (2) Cultivation studies were carried out using the copolymer PHA mixture-producing microbial strain (2) under the same conditions as in Example 1. The ratio of PHA accumulation to the dry cells, the average 3HH composition ratio of the copolymer PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the peak melting temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1.

[0095] (Example 3) PHA production by copolymer PHA mixture-producing microbial strain (3) Cultivation studies were carried out using the copolymer PHA mixture-producing microbial strain (3) under the same conditions as in Example 1. The ratio of PHA accumulation to the dry cells, the average 3HH composition ratio of the copolymer PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the peak melting temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1.

[0096] (Example 4) PHA production by copolymer PHA mixture-producing microbial strain (4) Cultivation studies were carried out using the copolymer PHA mixture-producing microbial strain (4) under the same conditions as in Example 1. The ratio of PHA accumulation to the dry cells, the average 3HH composition ratio of the copolymer PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the peak melting temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1.

[0097] (Example 5) PHA production by copolymer PHA mixture-producing microbial strain (5) A culture study was carried out using the copolymer PHA mixture-producing microbial strain (5) under the same conditions as in Example 1. The ratio of PHA accumulation to the dry cell mass, the average 3HH composition ratio of the copolymer PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the peak melting temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1.

[0098] (Example 6) PHA production by copolymer PHA mixture-producing microbial strain (6) A culture study was carried out using the copolymer PHA mixture-producing microbial strain (6) under the same conditions as in Example 1. The ratio of PHA accumulation to the dry cell mass, the average 3HH composition ratio of the copolymer PHA mixture, PHA fraction (I), or PHA fraction (II), the weight ratio of PHA fractions (I) and (II), the peak melting temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1.

[0099] (Comparative Example 1) PHA production by P(3HB-co-3HH)-producing microbial strain (1) A culture study was carried out using the P(3HB-co-3HH) producing microbial strain (1) under the same conditions as in Example 1. The ratio of the amount of accumulated PHA to the dry cell mass, the average 3HH composition ratio of P(3HB-co-3HH), the MIBK soluble fraction, or the MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1. In addition, in each analysis, the PHA accumulated by the P(3HB-co-3HH) producing microbial strain (1) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (1) was P(3HB-co-3HH. The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0100] (Comparative Example 2) PHA production by P(3HB-co-3HH)-producing microbial strain (2) A culture study was carried out using the P(3HB-co-3HH) producing microbial strain (2) under the same conditions as in Example 1. The ratio of the amount of accumulated PHA to the dry cell mass, the average 3HH composition ratio of P(3HB-co-3HH), the MIBK soluble fraction, or the MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1. In addition, in each analysis, the PHA accumulated by the P(3HB-co-3HH) producing microbial strain (2) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (2) was P(3HB-co-3HH. The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0101] (Comparative Example 3) PHA production by P(3HB-co-3HH)-producing microbial strain (3) A culture study was carried out using the P(3HB-co-3HH) producing microbial strain (3) under the same conditions as in Example 1. The ratio of the amount of accumulated PHA to the dry cell mass, the average 3HH composition ratio of P(3HB-co-3HH), the MIBK soluble fraction, or the MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1. In addition, in each analysis, the PHA accumulated by the P(3HB-co-3HH) producing microbial strain (3) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (3) was P(3HB-co-3HH. The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0102] (Comparative Example 4) PHA production by P(3HB-co-3HH)-producing microbial strain (4) A culture study was carried out using the P(3HB-co-3HH) producing microbial strain (4) under the same conditions as in Example 1. The ratio of the amount of accumulated PHA to the dry cell mass, the average 3HH composition ratio of P(3HB-co-3HH), the MIBK soluble fraction, or the MIBK insoluble fraction, the weight ratio of the MIBK soluble fraction and the MIBK insoluble fraction, the melting peak temperature and melting enthalpy, the processability, and the Elmendorf tear strength are shown in Table 1. In addition, in each analysis, the PHA accumulated by the P(3HB-co-3HH) producing microbial strain (4) was used instead of the copolymerized PHA mixture. The PHA accumulated by the P(3HB-co-3HH) producing microbial strain (4) was P(3HB-co-3HH. The MIBK soluble fraction and the MIBK insoluble fraction refer to fractions obtained by the same method as the PHA fraction (I) or the PHA fraction (II), respectively.

[0103] [Table 1]

[0104] From Table 1, the copolymer PHA mixtures obtained in Examples 1 to 6 had good processability, high Elmendorf tear strength, and excellent mechanical properties. On the other hand, in Comparative Example 1, in which the obtained copolymer PHA mixture did not contain PHA fraction (I), the Elmendorf tear strength was low. In Comparative Example 2, in which the average 3HH composition ratio of the PHA fraction (I) was not 20 mol% or more, the processability was poor and the Elmendorf tear strength was also low. In Comparative Examples 3 and 4, in which the average 3HH composition ratio of the copolymer PHA mixture was not 22 mol% or less and did not contain PHA fraction (II), the processability was poor.

Claims

1. 1. A method for producing a copolymerized polyhydroxyalkanoic acid mixture, comprising the steps of: The method includes culturing a microorganism that produces the copolymerized polyhydroxyalkanoic acid mixture, The microorganism is It is a transformant of Capriavidus necator, The present invention has a gene (A) encoding a polyhydroxyalkanoic acid synthase having an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, and a gene (B) encoding a polyhydroxyalkanoic acid synthase having an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, The copolymerized polyhydroxyalkanoic acid mixture is A polyhydroxyalkanoic acid fraction (I) containing a copolymerized polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit and having an average composition ratio of 3-hydroxyhexanoic acid of 20 mol % or more; and The present invention comprises a polyhydroxyalkanoic acid fraction (II) which contains a polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and has an average composition ratio of 3-hydroxyhexanoic acid of 0 mol % or more and 15 mol % or less, The copolymerized polyhydroxyalkanoic acid mixture has an average composition ratio of 3-hydroxyhexanoic acid of 22 mol % or less.

2. 2. The method according to claim 1, wherein the weight ratio of the polyhydroxyalkanoic acid fraction (I) in the copolymerized polyhydroxyalkanoic acid mixture is 10 to 90%.

3. 3. The method according to claim 1, wherein the copolymerized polyhydroxyalkanoic acid mixture has an average composition ratio of 3-hydroxyhexanoic acid of 10 to 22 mol %.

4. The method according to any one of claims 1 to 3, wherein the gene (A) is a gene encoding an amino acid sequence having 99.5 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:

3.

5. The method according to any one of claims 1 to 4, wherein the microorganism is a transformed microorganism that has been transformed so as to increase the supply of 3-hydroxyhexanoyl-CoA to intracellular polyhydroxyalkanoic acid synthase, as compared to a wild-type strain of the microorganism.

6. The method according to claim 5 , wherein the transformed microorganism is transformed so as to inhibit decomposition of an intermediate metabolite having six carbon atoms in the β-oxidation of fats and oils or fatty acids.

7. The method according to claim 6, wherein the transformed microorganism is transformed so that expression of a gene encoding a β-ketothiolase enzyme having thiolysis activity against β-ketohexanoyl-CoA, which is a β-ketoacyl-CoA having six carbon atoms, is suppressed.

8. The method according to claim 7, wherein the β-ketothiolase enzyme has an amino acid sequence that shows 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:

10.

9. The method according to any one of claims 1 to 8, wherein the microorganism has a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity.

10. The method according to any one of claims 1 to 9, wherein a carbon source containing an oil or a fatty acid is added in the culture step.

11. The method according to claim 10, wherein the carbon source containing an oil or a fatty acid is a carbon source containing a medium-chain fatty acid having 6 to 12 carbon atoms or a glyceride of the medium-chain fatty acid.

12. The method according to claim 11 , wherein the medium chain fatty acid is hexanoic acid.

13. A transformed microorganism that produces a copolymerized polyhydroxyalkanoic acid mixture, comprising: The transformed microorganism is It is a transformant of Capriavidus necator, The present invention has a gene (A) encoding a polyhydroxyalkanoic acid synthase having an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, and a gene (B) encoding a polyhydroxyalkanoic acid synthase having an amino acid sequence having 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, The transformed microorganism is transformed so as to increase the supply of 3-hydroxyhexanoyl-CoA to intracellular polyhydroxyalkanoic acid synthase, as compared with a wild-type strain of the transformed microorganism; The copolymerized polyhydroxyalkanoic acid mixture is A polyhydroxyalkanoic acid fraction (I) containing a copolymerized polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and a 3-hydroxyhexanoic acid structural unit and having an average composition ratio of 3-hydroxyhexanoic acid of 20 mol % or more; and The present invention comprises a polyhydroxyalkanoic acid fraction (II) which contains a polyhydroxyalkanoic acid having a 3-hydroxybutyric acid structural unit and has an average composition ratio of 3-hydroxyhexanoic acid of 0 mol % or more and 15 mol % or less, The copolymerized polyhydroxyalkanoic acid mixture has an average composition ratio of 3-hydroxyhexanoic acid of 22 mol % or less.

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