Polyhydroxyalkanoate synthase variant, gene and transformant thereof, and method for producing polyhydroxyalkanoate

Abstract

This polyhydroxyalkanoate synthase variant exhibits 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, contains an amino acid sequence having at least one mutation selected from mutation (a) and mutation (b), and has activity for synthesizing a copolymerized polyhydroxyalkanoate containing a 3-hydroxyalkanoic acid monomer unit having 8 carbon atoms. Mutation (a): a mutation in which proline at position 245 from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than proline. Mutation (b): a mutation in which isoleucine at position 252 from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than isoleucine.

Description

Mutant polyhydroxyalkanoate synthase, its gene and transformant, and method for producing polyhydroxyalkanoate

[0001] The present invention relates to a mutant polyhydroxyalkanoate synthase, a gene encoding the enzyme, a transformant having the gene, and a method for producing polyhydroxyalkanoate using the transformant.

[0002] Polyhydroxyalkanoate (PHA) is a thermoplastic polyester produced and stored as an energy storage substance within the cells of many microbial species. Produced by microorganisms from various natural carbon sources, PHA is an environmentally friendly plastic that is completely biodegradable by microorganisms in soil and water.

[0003] Polyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyrate (3-hydroxybutyrate; hereinafter abbreviated as "3HB"), is known as a PHA. However, PHB is highly crystalline, and due to its high degree of crystallinity, it is hard and brittle, and also has the problem of poor melt processability.

[0004] As a PHA with improved brittleness and melt-processability compared to PHB, a copolymer polyester Poly (3HB-co-3HH) (hereinafter abbreviated as "PHBH") of 3HB and 3-hydroxyhexanoate (3-hydroxyhexanoate; hereinafter abbreviated as "3HH") has been reported. PHBH is a copolymer that has a lower degree of crystallinity compared to PHB due to having 3HH as a monomer unit, and has flexible and soft physical properties.

[0005] One reported method for producing PHBH involves fermentation using a transformant in which PHA synthase derived from Aeromonas caviae has been introduced into the soil bacterium Cupriavidus necator as a host. However, research is underway to increase the 3HH ratio in PHBH in order to enhance its flexibility.

[0006] Non-patent documents 1 and 2 explore methods for introducing mutations into PHA synthase to increase the 3HH ratio in PHBH. Specifically, Non-patent document 1 reports that by introducing mutations into PHA synthase derived from Aeromonas caviar, such as the substitution of asparagine at position 149 with serine, or the substitution of aspartic acid at position 171 with glycine, the activity of PHA synthase and substrate specificity for 3HH-CoA are improved, making it possible to produce PHBH with a 3HH ratio of up to 18 mol%.

[0007] Furthermore, Non-Patent Document 2 reports that PHA synthase (hereinafter abbreviated as "NSDG") with these two mutations duplicated can produce PHBH with an even higher 3HH ratio.

[0008] Non-patent document 3 reports that by introducing a substitution of the 389th serine to threonine in NSDG, substrate specificity to 3HH-CoA is improved, and PHBH with an even higher 3HH ratio can be produced.

[0009] T. Kichise, S. Taguchi, Y. Doi, Appl. Environ. Microbiol. , 68, pp. 2411-2419 (2002) T. Tsuge, S. Watanabe, D. Shimada, H. Abe, Y. Doi, S. Taguchi, FEMS Microbiol. Lett. , 277, pp. 217-222 (2007) K. Harada, S. Kobayashi, K. Oshima, S. Yoshida, T. Tsuge & S. Sato, Front. Bioeng. Biotechnol. , Volume 9 (2021)

[0010] Generally, it is known that the crystallinity and glass transition temperature (Tg) of PHA decrease as the polymerization ratio of monomer units with a large number of carbon atoms increases. Therefore, studies are being conducted to increase the 3HO composition of polyester Poly (3HB-co-3HH-co-3HO) (hereinafter abbreviated as "PHBHO") obtained by copolymerizing PHBH with 3-hydroxyoctanoate (3-hydroxyoctanoate; hereinafter abbreviated as "3HO").

[0011] However, PHA copolymers produced by culturing transformants into which the PHA synthase gene derived from Aeromonas caviar has been introduced have an extremely low 3HO ratio. Therefore, there is a need to construct a PHA synthase library that can produce PHA copolymers with a higher 3HO ratio.

[0012] Therefore, the present invention aims to provide a mutant PHA synthase that enables the production of a PHA copolymer with a higher 3HO ratio, a gene encoding the enzyme, a transformant having the gene, and a method for producing PHA using the transformant.

[0013] To solve the above problems, the inventors conducted extensive research and, as a result, developed PhaC, a wild-type PHA synthase derived from Aeromonas caviar, composed of the amino acid sequence shown in Sequence ID No. 1. Ａｃ In this process, we discovered that a PHA copolymer with a higher 3HO ratio can be produced by introducing a mutation to at least one amino acid among the 245th amino acid from the N-terminus and the 252nd amino acid, thus completing the present invention.

[0014] In other words, the present invention relates to a mutant polyhydroxyalkanoate synthase that exhibits 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1 and includes an amino acid sequence having at least one of the following mutations (a) to (b), and has the activity to synthesize copolymerized polyhydroxyalkanoate containing a carbon-8 3-hydroxyalkanoate monomer unit. The present invention also relates to a gene encoding the mutant polyhydroxyalkanoate synthase, or a transformant having said gene. Furthermore, the present invention relates to a method for producing copolymerized polyhydroxyalkanoate, comprising the steps of culturing the transformant in the presence of a carbon source, and recovering copolymerized polyhydroxyalkanoate containing a carbon-8 3-hydroxyalkanoate monomer unit from the transformant.

[0015] According to the present invention, it is possible to provide a mutant PHA synthase that enables the production of a PHA copolymer with a higher 3HO ratio, a gene encoding the enzyme, and a transformant having the gene. Furthermore, by culturing the transformant, it becomes possible to ferment-produce a PHA copolymer with a higher 3HO ratio.

[0016] Embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described below, and various modifications are possible within the scope defined in the claims. Furthermore, the components described below can be arbitrarily combined, and such combinations may also constitute an embodiment of the present invention. (Mutant PHA synthase) The mutant PHA synthase according to this disclosure exhibits 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and includes an amino acid sequence having at least one of the following mutations (a) to (b). The present disclosure also provides a gene encoding the mutant PHA synthase (hereinafter abbreviated as "mutant PHA synthase gene").

[0017] Sequence ID 1 is wild-type PHA synthase PhaC derived from Aeromonas caviar. Ａｃ The amino acid sequence is shown. By introducing at least one of the mutations (a) to (b) into the amino acid sequence shown in Sequence ID No. 1, wild-type PHA synthase PhaC Ａｃ Compared to that, it is possible to produce PHA copolymers with a higher 3HO ratio.

[0018] The amino acid sequence of the mutant PHA synthase according to this disclosure exhibits 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. Within the range that satisfies this sequence identity, the mutant PHA synthase according to this disclosure may have mutations that do not fall under either (a) or (b). The sequence identity is preferably 92% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0019] The mutant PHA synthase relating to this disclosure may be formed by binding with a heterologous protein having a different function to form a fusion protein. In this case, the amino acid sequence of the heterologous protein is not considered when calculating the sequence identity described above.

[0020] The nucleotide sequence of the mutant PHA synthase gene relating to this disclosure is not particularly limited insofar as it is a nucleotide sequence that encodes the amino acid sequence constituting the mutant PHA synthase relating to this disclosure.

[0021] Next, the mutations (a) and (b) in the amino acid sequence of the mutant PHA synthase relating to this disclosure will be described. The amino acid sequence may contain either one of mutations (a) and (b), or it may contain both of these mutations.

[0022] Mutation (a): A mutation in which the 245th proline from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than proline. In mutation (a), the substituted amino acid can be selected considering the 3HO ratio of the PHA produced. Specific examples include alanine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, asparagine, cysteine, glutamine, serine, threonine, aspartic acid, glutamic acid, arginine, histidine, lysine, and glycine. To produce a PHA copolymer with a higher 3HO ratio, a mutation in which the 245th proline is replaced with serine is particularly preferred.

[0023] Mutation (b): A mutation in which the isoleucine at position 252 from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is replaced with an amino acid other than isoleucine. In mutation (b), the substituted amino acid can be selected considering the 3HO ratio of the PHA produced. Specific examples include alanine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, asparagine, cysteine, glutamine, serine, threonine, aspartic acid, glutamic acid, arginine, histidine, lysine, glycine, and proline. To produce a PHA copolymer with a higher 3HO ratio, a mutation in which the 252nd isoleucine is replaced with alanine, cysteine, or leucine is preferred, and a mutation in which it is replaced with alanine or leucine is more preferred. Furthermore, leucine is particularly preferred because it can produce a PHA copolymer with a high 3HH ratio in addition to a high 3HO ratio.

[0024] The amino acid sequence of the mutant PHA synthase according to this disclosure preferably further includes, in addition to the mutations described above, a mutation in which the asparagine at position 149 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine, and / or a mutation in which the aspartic acid at position 171 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine, in order to further increase the 3HH ratio and 3HO ratio of the PHA copolymer.

[0025] Furthermore, in order to further increase the 3HH ratio and 3HO ratio of the PHA copolymer, the amino acid sequence of the mutant PHA synthase according to this disclosure preferably includes, in addition to the mutations described above, a mutation in which the serine at position 389 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than serine. The amino acid other than serine is not particularly limited and can be selected considering the 3HH ratio and 3HO ratio of the PHA copolymer produced, but specific examples include alanine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, tyrosine, asparagine, cysteine, glutamine, threonine, aspartic acid, glutamic acid, arginine, histidine, lysine, glycine, and proline. Among these, cysteine, isoleucine, threonine, or valine are preferred from the viewpoint of obtaining a PHA copolymer with a higher 3HH ratio and 3HO ratio.

[0026] The mutant PHA synthase according to this disclosure has the activity to synthesize copolymer PHA containing 3HO (3-hydroxyalkanoic acid with 8 carbon atoms) monomer units. In a preferred embodiment, the composition ratio of 3HO in the copolymer PHA synthesized by the mutant PHA synthase according to this disclosure can reach 0.4 mol% or more. In a more preferred embodiment, the composition ratio of 3HO can reach 1.0 mol% or more, 1.5 mol% or more, 2.0 mol% or more, 2.5 mol% or more, or 3.0 mol% or more. The upper limit of the composition ratio of 3HO is not particularly limited, but may be, for example, 10 mol% or less, or 5 mol% or less.

[0027] The hydroxyalkanoic acid monomer units other than 3HO included in the copolymer PHA are not particularly limited, and examples include 2-hydroxyalkanoic acid, 3-hydroxyalkanoic acid, and 4-hydroxyalkanoic acid having 4 to 16 carbon atoms. There may be one or more hydroxyalkanoic acids other than 3HO.

[0028] In particular, as the hydroxyalkanoic acid other than 3HO, it is preferable to contain 3-hydroxybutyric acid. Further, in addition to 3-hydroxybutyric acid, it is more preferable to contain 3-hydroxyhexanoic acid. That is, the copolymerized PHA synthesized by the mutant PHA synthase according to the present disclosure is particularly preferably P(3HB-co-3HH-co-3HO), which is a copolymer of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, and 3-hydroxyoctanoic acid. Note that the inclusion of other hydroxyalkanoic acid monomer units in this terpolymer is not excluded.

[0029] The mutant PHA synthase according to the present disclosure can synthesize a copolymerized PHA having a relatively high composition ratio of 3HH (3-hydroxyhexanoic acid) monomer units. In this case, the composition ratio of 3HH in the copolymerized PHA is not particularly limited, but may be, for example, about 1 mol% or more and about 30 mol%. The lower limit may be 3 mol% or more, 5 mol% or more, 8 mol% or more, 10 mol% or more, 12 mol% or more, or 15 mol% or more.

[0030] The type of the produced copolymerized PHA can be appropriately selected depending on the type of the PHA synthase gene possessed by the used microorganism or separately introduced, the type of the gene of the metabolic system involved in PHA synthesis, the carbon source used for culturing, and other culturing conditions.

[0031] (Transformant producing copolymerized PHA containing 3HO units) The transformant according to the present disclosure is a transformant having the mutant PHA synthase gene according to the present disclosure, and is produced by introducing the gene into a host microorganism.

[0032] There are no particular limitations on the host of the transformant according to this disclosure, and any microorganism such as fungi (molds, mushrooms, yeasts, etc.), bacteria, and archaea can be used, but bacteria are preferred. Preferred examples of such bacteria include those belonging to the genera Ralstonia, Cupriavidus, Woutersia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, and Halomonas. From the viewpoint of safety and productivity, it is more preferable to use bacteria belonging to the genera Ralstonia, Capriavidus, Aeromonas, or Woutersia, even more preferably bacteria belonging to the genera Capriavidus or Aeromonas, even more preferably bacteria belonging to the genera Capriavidus, particularly preferably Capriavidus necator, and most preferably Capriavidus necator H16 strain.

[0033] In producing the transformants according to this disclosure, any method can be used to introduce the mutant PHA synthase gene according to this disclosure into a host microorganism. For example, the mutant PHA synthase gene according to this disclosure may be introduced onto DNA such as chromosomes, plasmids, or megaplasmides possessed by the host microorganism using known genetic recombination techniques, or a plasmid vector or artificial chromosome into which the gene has been introduced may be introduced into the host microorganism. However, from the viewpoint of retaining the introduced gene, a method of introducing the gene onto chromosomes or megaplasmides possessed by the microorganism is preferred, and a method of introducing the gene onto chromosomes possessed by the microorganism is more preferred.

[0034] Methods for site-specific substitution or insertion of any base sequence on the DNA possessed by a microorganism, or methods for deleting any base sequence in the DNA possessed by a microorganism, are widely known to those skilled in the art and can be used in producing the transformants according to the present disclosure. Although not particularly limited, representative methods include those utilizing the mechanism of transposons and homologous recombination (Ohman et al., J. Bacteriol., vol. 162: p. 1068 (1985)), methods based on the principle of site-specific integration by the mechanism of homologous recombination and excision by a second-stage homologous recombination (Noti et al., Methods Enzymol., vol. 154, p. 197 (1987)), a method of co-existing the sacB gene derived from Bacillus subtilis and easily isolating a microbial strain in which a gene has been excised by a second-stage homologous recombination as a sucrose-added medium-resistant strain (Schweizer, Mol. Microbiol., vol. 6, p. 1195 (1992); Lenz et al., J. Bacteriol., vol. 176, p. 4385 (1994)), etc. Also, the method for introducing a vector into a microorganism is not particularly limited, and examples include the calcium chloride method, the electroporation method, the polyethylene glycol method, the spheroplast method, etc.

[0035] Regarding 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), etc. can be utilized.

[0036] When introducing the mutant PHA synthase gene according to this disclosure, the gene can be linked to any regulatory expression sequence. In this specification, the regulatory expression sequence is described as a sequence consisting of a promoter and a Shine-Dalgano sequence. Examples of such regulatory expression sequences include the regulatory expression sequence of the phaC1 gene (SEQ ID NO: 2) or the regulatory expression sequence of the phaP1 gene (SEQ ID NO: 3) of Capriavidus nekator. Alternatively, the lac promoter (SEQ ID NO: 4) or trp promoter (SEQ ID NO: 5) derived from Escherichia coli, or artificially created lacUV5 promoter (SEQ ID NO: 6), trc promoter (SEQ ID NO: 7), tic promoter (SEQ ID NO: 8), tac promoter (SEQ ID NO: 9), lacN17 promoter (SEQ ID NO: 10), etc., can be linked to the SD sequence (SEQ ID NO: 11) derived from the Capriavidus nekator H16 strain and used as a regulatory expression sequence.

[0037] A transformed microorganism according to a preferred embodiment may be one into which a gene encoding a protein having R-specific enoyl-CoA-hydratase activity that recognizes enoyl-CoA having 8 or more carbon atoms as a substrate has been introduced, or into which the expression of said gene has been enhanced. This increases the amount of 3HA monomers having 8 or more carbon atoms produced, and can further increase the composition ratio of the produced 3HO monomer units.

[0038] In this specification, "protein having R-specific enoyl-CoA hydratase activity" refers to a protein having enzymatic activity that produces (R)-3-hydroxyacyl-CoA, which is a monomer of PHA, using enoyl-CoA, an intermediate in the β-oxidation system of fatty acids, as a substrate. It is believed that when this protein uses enoyl-CoA with 8 or more carbon atoms as a substrate, the amount converted to (R)-3-hydroxyacyl-CoA with 8 or more carbon atoms increases, and as a result, the composition ratio of 3HO monomer units in copolymerized PHA becomes higher.

[0039] Proteins possessing R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA with 8 or more carbon atoms as a substrate are not particularly limited, but examples include bacterial R-specific enoyl-CoA hydratase (PhaJ) and eukaryotic multifunctional enzyme type 2 (MFE2).

[0040] The origin of PhaJ is not particularly limited, but examples include bacteria of the genus Pseudomonas and actinomycetes. On the other hand, PhaJ derived from the genus Capriavidus has low activity against enoyl-CoA with 8 or more carbon atoms. The origin of MFE2 is not particularly limited, but examples include Drosophylla melanogaster and Yarowia liporitica.

[0041] More specifically, the protein having R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA having 8 or more carbon atoms as a substrate is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 12, 13, or 14, or a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, 13, or 14. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0042] A gene encoding a protein having R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA with 8 or more carbon atoms as a substrate may be introduced into a transformed microorganism, or the transformed microorganism may be transformed in such a way that the expression of the gene is enhanced. The introduction of the gene can be carried out by the method described above. To enhance the expression of the gene, for example, the expression regulatory sequence (promoter sequence and / or SD sequence) can be modified to enhance the expression of the gene, as described in International Publication No. 2015 / 115619.

[0043] In this specification, enhanced gene expression refers to a state in which the transcription amount of the target gene or the expression amount of the polypeptide encoded by the target gene is increased compared to a strain in which the expression of the target gene is not enhanced. The amount of increase is not particularly limited, but it should be more than 1x compared to a strain in which the expression of the target gene is not enhanced, preferably 1.1x or more, more preferably 1.2x or more, even more preferably 1.5x or more, and even more preferably 2x or more.

[0044] In the preferred embodiment, the transformed microorganism may be transformed such that the expression of the gene encoding β-ketothiolase is suppressed. The β-ketothiolase has thiolysis activity against β-ketoacyl-CoA having 8 or fewer carbon atoms. By suppressing the expression of the β-ketothiolase gene, the enzymatic activity of β-ketothiolase can be eliminated or reduced. This suppresses the degradation of β-ketoacyl-CoA having 8 or fewer carbon atoms, and allows for more efficient production of copolymerized PHA containing 3HO monomer. The expression of only one β-ketothiolase gene may be suppressed, or there may be two or more. Furthermore, the transformed microorganism in the preferred embodiment may include both suppression of β-ketothiolase gene expression and introduction or enhancement of expression of a gene encoding a protein having the R-isomer-specific enoyl-CoA-hydratase activity described above.

[0045] In this specification, "β-ketothiolase" refers to an enzyme that catalyzes the reaction in which β-ketoacyl-CoA undergoes thiolysis (thiol cleavage) in the presence of coenzyme A during β-oxidation of fatty acids, producing a fatty acid acyl-CoA shortened by two carbon atoms and acetyl-CoA.

[0046] The β-ketothiolase gene whose expression is suppressed may be any gene encoding a β-ketothiolase having thiolysis activity for β-ketoacyl-CoA with 8 or fewer carbon atoms. This β-ketothiolase may simultaneously have thiolysis activity for β-ketoacyl-CoA with 9 or more carbon atoms in addition to thiolysis activity for β-ketoacyl-CoA with 8 or fewer carbon atoms. For example, it may have thiolysis activity for β-ketoacyl-CoA with 4 to 8 carbon atoms, or 4 to 18 carbon atoms, or 6 to 20 carbon atoms, but is not limited to these.

[0047] The gene encoding β-ketothiolase is not particularly limited, but examples include the bktB gene and the A1528 gene. Specifically, examples include a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 15 or 16, and a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 15 or 16. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0048] To suppress the expression of the gene encoding β-ketothiolase, for example, methods include completely deleting the enzyme gene in a transformed microorganism, inserting a completely different gene such as a drug resistance gene into the sequence of the enzyme gene, or deleting, substituting, adding, or inserting a part of the sequence of the enzyme gene (preferably a region involved in enzyme activity). Gene disruption operations include, for example, homologous recombination techniques using vectors containing disruption genes or disruption DNA, and techniques utilizing transposons. Alternatively, known technologies such as the CRISPR / Cas (e.g., Cas9) system or TALEN genome editing technology for disrupting target genes can be used (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732; Bogdanove and Voytas, Science, 333:1843-1846, 2011; Jinek, et al., Science, 337:816-821, 2012; Shalem, et al., Science, 343:84-87, 2014; Wang, et al., Science, 343:80-84, 2014). For example, in the CRISPR / Cas9 system, the guide RNA (gRNA) has a sequence that can bind to a portion of the base sequence of the β-ketothiolase gene to be disrupted, and plays a role in transporting Cas9 to the target. In addition, mutations such as deletions, substitutions, additions, and insertions of the base sequence around the gene can reduce the transcription and translation efficiency of the gene and the stability of the mRNA, thereby eliminating or reducing the activity of the enzyme.

[0049] (Method for producing PHA) PHA can be produced by culturing the transformant according to the present disclosure, causing the transformant to produce PHA, and then recovering the obtained PHA.

[0050] In the production of PHA, it is preferable to culture the transformed organisms in a culture medium containing a carbon source, a nitrogen source (a nutrient source other than a carbon source), inorganic salts, and other organic nutrients.

[0051] The carbon source is not particularly limited as long as it is a carbon source that the transformant according to this disclosure can utilize, and any carbon source can be used. Specifically, examples include sugars such as glucose, fructose, sucrose, and xylose; oils and fats such as palm oil and palm kernel oil (including low-melting-point fractions obtained by fractionating these, such as palm olein, palm double olein, and palm kernel oil olein), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, and their fractionated oils, or their refining by-products; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, myristic acid, and their derivatives, or glycerol. Furthermore, if the transformed microorganism can utilize gases or alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources. Among these, the use of vegetable oil as a carbon source is preferred.

[0052] Examples of nitrogen sources include ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, and yeast extract.

[0053] Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.

[0054] Other organic nutrients include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; and vitamins such as vitamin B1, vitamin B12, and vitamin C.

[0055] The conditions for culturing the transformants according to this disclosure, such as culture temperature, culture time, culture pH, and culture medium, may be those commonly used for culturing host microorganisms, such as those of the genera Ralstonia, Capriavidus, Woutersia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, and Halomonas, and are not particularly limited.

[0056] In the present invention, the recovery of PHA from the microbial cells after culturing the transformants is not particularly limited and can be carried out by known methods. As an example, PHA can be recovered by the following method: After culturing is complete, the microbial cells are separated from the culture medium using a centrifuge or the like, and the microbial cells are washed with distilled water, methanol, etc., and dried. PHA is extracted from these dried microbial cells using an organic solvent such as chloroform. From this organic solvent solution containing PHA, microbial components are removed by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the PHA. Furthermore, the supernatant is removed by filtration or centrifugation, and the PHA is recovered by drying.

[0057] The composition ratio (mol%) of monomer units such as 3HO units and 3HH units contained in the obtained PHA can be analyzed, for example, by gas chromatography or nuclear magnetic resonance spectroscopy.

[0058] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to these items. [Item 1] A mutant polyhydroxyalkanoate synthase having synthetic activity for copolymer polyhydroxyalkanoates containing a carbon-8 3-hydroxyalkanoate monomer unit, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having at least one of the following mutations (a) to (b). Mutation (a): A mutation in which the 245th proline from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than proline. Mutation (b): A mutation in which the 252nd isoleucine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than isoleucine. [Item 2] The mutant polyhydroxyalkanoate synthase according to Item 1, wherein in mutation (a), the amino acid other than proline is serine. [Item 3] The mutant polyhydroxyalkanoate synthase according to Item 1 or 2, wherein in mutation (b), the amino acid other than isoleucine is alanine, cysteine, or leucine. [Item 4] A mutant polyhydroxyalkanoate synthase according to any one of items 1 to 3, further comprising a mutation in which the 389th serine from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is replaced with an amino acid other than serine. [Item 5] A mutant polyhydroxyalkanoate synthase according to item 4, wherein the amino acid other than serine is cysteine, isoleucine, threonine, or valine. [Item 6] A mutant polyhydroxyalkanoate synthase according to any one of items 1 to 5, further comprising a mutation in which the 149th asparagine from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is replaced with serine. [Item 7] A mutant polyhydroxyalkanoate synthase according to any one of items 1 to 6, further comprising a mutation in which the 171st aspartic acid from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is replaced with glycine. [Item 8] A gene encoding a mutant polyhydroxyalkanoate synthase according to any one of items 1 to 7. [Item 9] A transformant having the gene according to item 8. [Item 10] A transformant described in Item 9, whose host is a bacterium.[Item 11] The transformant according to Item 10, wherein the eubacteria is a bacterium belonging to the genus Capriavidus. [Item 12] A method for producing copolymerized polyhydroxyalkanoic acid, comprising the steps of: culturing the transformant according to any one of Items 9 to 11 in the presence of a carbon source; and recovering copolymerized polyhydroxyalkanoic acid containing carbon 8 3-hydroxyalkanoic acid monomer units from the transformant. [Item 13] The method for producing copolymerized polyhydroxyalkanoic acid according to Item 12, wherein the composition ratio of the carbon 8 3-hydroxyalkanoic acid monomer units in the copolymerized polyhydroxyalkanoic acid is 1.0 mol% or more.

[0059] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0060] The genetic manipulation described below can be carried out by referring to the description in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Furthermore, the enzymes, cloning hosts, etc., used in the genetic manipulation can be purchased from market suppliers and used according to their instructions. Note that the enzymes are not particularly limited as long as they can be used for genetic manipulation.

[0061] The "KNK005dZ strain" used in the following manufacturing examples is a transformed microorganism also known as the KNK005ΔphaZ1,2,6 strain. In this transformed microorganism, the phaC1 gene on the chromosome of the Cupriavidus necator H16 strain is replaced with a PHA polymerase gene mutant (NSDG) derived from Aeromonas caviar, and the phaZ1,2,6 genes, which are PHA-degrading enzyme genes on the chromosome, are deleted. This transformed microorganism can be produced in accordance with the method described in International Publication No. 2014 / 065253.

[0062] (Manufacturing Example 1) Preparation of KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp strain First, an NSDG disruption plasmid was prepared. The preparation was carried out as follows: Using the genomic DNA of the KNK005dZ strain as a template, a DNA fragment was prepared by linking the base sequences upstream and downstream of the NSDG gene by PCR (SEQ ID NO: 17). This DNA fragment was digested with the restriction enzyme SmiI, and the obtained DNA fragment was linked with the vector pNS2X-sacB described in Japanese Patent Publication No. 2007-259708, which was also digested with SmiI, using DNA ligase to prepare the NSDG disruption plasmid vector pNS2X-sacB+NSDGUD.

[0063] Next, the KNK005dZ / dNSDG strain was prepared using the NSDG disruption plasmid vector pNS2X-sacB+NSDGUD as follows.

[0064] Escherichia coli strain S17-1 (ATCC47055) was transformed with pNS2X-sacB + NSDGUD, and the resulting transformed microorganisms were mixed cultured with strain KNK005dZ on Nutrient Agar (Difco) medium for conjugation transfer.

[0065] The obtained cultured cells were seeded onto Simmons agar medium containing 250 mg / L kanamycin (2 g / L sodium citrate, 5 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 1 g / L ammonium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 15 g / L agar, pH 6.8). Strains that grew on the agar medium were selected to obtain strains in which the plasmid was incorporated into the chromosome of the KNK005dZ strain. After culturing this strain for two generations in Nutrient Broth medium, it was diluted and spread onto Nutrient Agar medium containing 15% sucrose, and the resulting strains were obtained as strains from which the plasmid had been removed. Furthermore, one strain in which the NSDG gene on the chromosome was removed was isolated by PCR analysis or sequencing analysis. The obtained strain was named KNK005dZ / dNSDG.

[0066] Plasmids for disrupting the phaJ4a gene were prepared. The preparation was carried out as follows: Using the genomic DNA of strain KNK005dZ as a template, a DNA fragment was prepared by ligating the nucleotide sequences upstream and downstream of the phaJ4a structural gene by PCR (SEQ ID NO: 18). This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare the phaJ4a disruption plasmid vector pNS2X-sacB+phaJ4aUD.

[0067] Using the plasmid vector pNS2X-sacB+phaJ4aUD for phaJ4a gene disruption, the phaJ4a gene disruption strain KNK005dZ / dNSDG / dphaJ4a was prepared using KNK005dZ / dNSDG as the parent strain in the same manner as described above.

[0068] Plasmids for disrupting the bktB gene were prepared. The preparation was carried out as follows: Using the genomic DNA of strain KNK005dZ as a template, a DNA fragment was prepared by ligating the nucleotide sequences upstream and downstream of the bktB structural gene using PCR (SEQ ID NO: 19). This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare the bktB gene disruption plasmid vector pNS2X-sacB+bktBUD.

[0069] Next, using the bktB gene disruption plasmid vector pNS2X-sacB+bktBUD, the bktB gene disruption strain KNK005dZ / dNSDG / dphaJ4a / dbktB was prepared using the same method as described above, with KNK005dZ / dNSDG / dphaJ4a as the parent strain.

[0070] Plasmids for disrupting the A1528 gene were prepared. The preparation was carried out as follows: Using the genomic DNA of strain KNK005dZ as a template, a DNA fragment was prepared by ligating the nucleotide sequences upstream and downstream of the A1528 structural gene by PCR (SEQ ID NO: 20). This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare the A1528 gene disruption plasmid vector pNS2X-sacB+A1528UD.

[0071] Next, using the A1528 gene disruption plasmid vector pNS2X-sacB+A1528UD, the A1528 gene disruption strain KNK005dZ / dNSDG / dphaJ4a / dbktB / dA1528 was prepared using the same method as described above, with KNK005dZ / dNSDG / dphaJ4a / dbktB as the parent strain.

[0072] Plasmids for introducing the phaJ4pp gene were prepared. The preparation was carried out as follows: By PCR using an artificially synthesized gene as a template, a DNA fragment (SEQ ID NO: 21) was obtained containing the nucleotide sequences upstream and downstream of the phaJ4a structural gene, a trc promoter having the nucleotide sequence described in SEQ ID NO: 7, and a nucleotide sequence containing the gene encoding phaJ4pp having the amino acid sequence described in SEQ ID NO: 12. This DNA fragment was digested with the restriction enzyme SmiI, and the obtained DNA fragment was ligated with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare the phaJ4pp gene introduction plasmid vector pNS2X-sacB+phaJ4aU-trc-phaJ4pp-phaJ4aD.

[0073] Next, using the plasmid vector pNS2X-sacB+phaJ4aU-trc-phaJ4pp-phaJ4aD for phaJ4pp gene transfer, chromosomal DNA modification was performed using KNK005dZ / dNSDG / dphaJ4a / dbktB as the parent strain in the same manner as the gene disruption described above, to create the phaJ4pp gene insertion strain KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp.

[0074] (Production Example 2) pCUP2-Ptrp-phaC Ａｃ Using synthesized oligoDNA, etc., a DNA fragment represented by SEQ ID NO: 22, with EcoR1 and Mun1 cleavage sites at its ends, was amplified by PCR using the trp promoter (SEQ ID NO: 5) as a template. The obtained DNA fragment was digested using EcoR1 and Mun1, and the DNA fragment obtained by digesting the pCUP2 vector described in Japanese Patent Publication No. 2007-259708 with Mun1 was ligated with DNA ligase to obtain pCUP2-Ptrp. Using synthetic oligoDNA, etc., a DNA fragment represented by SEQ ID NO: 23 was amplified using a base sequence containing the gene encoding SEQ ID NO: 1 as a template. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp with Mun1 were ligated using NEBuilderHiFiDNAAssembleMasterMix (New England Biolab) to obtain pCUP2-Ptrp-phaC Ａｃ Obtained: pCUP2-Ptrp-phaC Ａｃ Under the trp promoter, phaC Ａｃ This is a plasmid that expresses [a specific gene / substance].

[0075] (Production Example 3) Preparation of pCUP2-Ptrp-NSDG A DNA fragment having the base sequence shown in SEQ ID NO: 24 was amplified by PCR using synthetic oligoDNA, etc. The obtained DNA fragment was ligated with a DNA fragment obtained by digesting the pCUP2 vector with MunI and SpeI using the In-fusion HD Cloning Kit (Takara Bio) to obtain pCUP2-Ptrp-NSDG. pCUP2-Ptrp-NSDG is a plasmid that expresses NSDG under the trp promoter.

[0076] NSDG is a mutant PHA synthase consisting of the amino acid sequence shown in SEQ ID NO: 25. It is a mutant PHA synthase in which two types of mutations have been introduced into the amino acid sequence shown in SEQ ID NO: 1, namely the substitution of asparagine at position 149 from the N-terminus to serine, and the substitution of aspartic acid at position 171 to glycine.

[0077] (Production Example 4) Preparation of pCUP2-Ptrp-NSDG-S389T Using pCUP2-Ptrp-NSDG prepared in Production Example 3 as a template, a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 26 was amplified by PCR using synthetic oligo DNA or the like. This was cloned into the pCUP2 vector in the same manner as in Production Example 3 to obtain pCUP2-Ptrp-S389T. pCUP2-Ptrp-NSDG-S389T is a plasmid that expresses NSDG-S389T under the trp promoter.

[0078] NSDG-S389T is a mutant PHA synthase in which the 389th serine from the N-terminus is substituted with threonine with respect to the amino acid sequence represented by SEQ ID NO: 25.

[0079] (Production Example 5) Preparation of pCUP2-Ptrp-phaC Ａｃ -P245S Using pCUP2-Ptrp-phaC prepared in Production Example 2 as a template, DNA fragments having the nucleotide sequences represented by SEQ ID NO: 27 and SEQ ID NO: 28 were amplified by PCR using synthetic oligo DNA or the like. The obtained DNA fragments were ligated using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) with the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 to obtain pCUP2-Ptrp-phaC Ａｃ -P245S. pCUP2-Ptrp-phaC Ａｃ -P245S is a plasmid that expresses phaC Ａｃ -P245S under the trp promoter. Ａｃ phaC

[0080] -P245S is a mutant PHA synthase in which the 245th proline from the N-terminus is substituted with serine with respect to the amino acid sequence represented by SEQ ID NO: 1. Ａｃ

[0081] (Production Example 6) Preparation of pCUP2-Ptrp-phaC Ａｃ -I252A Using pCUP2-Ptrp-phaC prepared in Production Example 2 as a template, ＡｃUsing this as a template, DNA fragments having the base sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30 were amplified by PCR using synthetic oligoDNA, etc. The obtained fragments were cloned in the same manner as in Production Example 5 and pCUP2-Ptrp-phaC Ａｃ - Obtained I252A. pCUP2-Ptrp-phaC Ａｃ -I252A is phaC under the trp promoter. Ａｃ - This plasmid expresses I252A.

[0082] phaC Ａｃ -I252A is a mutant PHA synthase in which the isoleucine at position 252 from the N-terminus is replaced with alanine in the amino acid sequence shown in Sequence ID No. 1.

[0083] (Production Example 7) pCUP2-Ptrp-phaC Ａｃ - pCUP2-Ptrp-phaC fabricated in manufacturing example 2 of I252L Ａｃ Using this as a template, DNA fragments having the base sequences shown in SEQ ID NOs. 31 and 32 were amplified by PCR using synthetic oligoDNA, etc. The obtained fragments were cloned in the same manner as in Production Example 5, and pCUP2-Ptrp-phaC Ａｃ - Obtained I252L. pCUP2-Ptrp-phaC Ａｃ -I252L is phaC under the trp promoter. Ａｃ - This plasmid expresses I252L.

[0084] phaC Ａｃ -I252L is a mutant PHA synthase in which the isoleucine at position 252 from the N-terminus is replaced with leucine in the amino acid sequence shown in Sequence ID No. 1.

[0085] (Production Example 8) Preparation of pCUP2-Ptrp-NSDG-P245S An artificially synthesized DNA fragment (SEQ ID NO: 33) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssembleMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-P245S. pCUP2-Ptrp-NSDG-P245S is a plasmid that expresses NSDG-P245S under the trp promoter.

[0086] NSDG-P245S is a mutant PHA synthase in which the 245th proline from the N-terminus is replaced with serine in the amino acid sequence shown in Sequence ID No. 25.

[0087] (Production Example 9) Preparation of pCUP2-Ptrp-NSDG-I252A An artificially synthesized DNA fragment (SEQ ID NO: 36) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssemblelyMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-I252A. pCUP2-Ptrp-NSDG-I252A is a plasmid that expresses NSDG-I252A under the trp promoter.

[0088] NSDG-I252A is a mutant PHA synthase in which the isoleucine at position 252 from the N-terminus is replaced with alanine in the amino acid sequence shown in Sequence ID No. 25.

[0089] (Production Example 10) Preparation of pCUP2-Ptrp-NSDG-I252C An artificially synthesized DNA fragment (SEQ ID NO: 37) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssemblelyMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-I252C. pCUP2-Ptrp-NSDG-I252C is a plasmid that expresses NSDG-I252C under the trp promoter.

[0090] NSDG-I252C is a mutant PHA synthase in which the isoleucine at position 252 from the N-terminus is replaced with cysteine, compared to the amino acid sequence shown in Sequence ID No. 25.

[0091] (Production Example 11) Preparation of pCUP2-Ptrp-NSDG-I252L An artificially synthesized DNA fragment (SEQ ID NO: 38) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssemblelyMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-I252L. pCUP2-Ptrp-NSDG-I252L is a plasmid that expresses NSDG-I252L under the trp promoter.

[0092] NSDG-I252L is a mutant PHA synthase in which the isoleucine at position 252 from the N-terminus is replaced with leucine in the amino acid sequence shown in Sequence ID No. 25.

[0093] (Production Example 12) Preparation of pCUP2-Ptrp-NSDG-P245S-S389T An artificially synthesized DNA fragment (SEQ ID NO: 39) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssemblelyMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-P245S-S389T. pCUP2-Ptrp-NSDG-P245S-S389T is a plasmid that expresses NSDG-P245S-S389T under the trp promoter.

[0094] NSDG-P245S-S389T is a mutant PHA synthase in which the 245th proline from the N-terminus is replaced with serine and the 389th serine is replaced with threonine, compared to the amino acid sequence shown in Sequence ID No. 25.

[0095] (Production Example 13) Preparation of pCUP2-Ptrp-NSDG-I252A-S389T An artificially synthesized DNA fragment (SEQ ID NO: 40) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssemblelyMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-I252A-S389T. pCUP2-Ptrp-NSDG-I252A-S389T is a plasmid that expresses NSDG-I252A-S389T under the trp promoter.

[0096] NSDG-I252A-S389T is a mutant PHA synthase in which the amino acid sequence shown in Sequence ID No. 25 has been modified in which the isoleucine at position 252 from the N-terminus is replaced with alanine, and the serine at position 389 is replaced with threonine.

[0097] (Production Example 14) Preparation of pCUP2-Ptrp-NSDG-I252C-S389T An artificially synthesized DNA fragment (SEQ ID NO: 41) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssembleMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-I252C-S389T. pCUP2-Ptrp-NSDG-I252C-S389T is a plasmid that expresses NSDG-I252C-S389T under the trp promoter.

[0098] NSDG-I252C-S389T is a mutant PHA synthase in which the amino acid sequence shown in Sequence ID No. 25 has been modified in which the isoleucine at position 252 from the N-terminus is replaced with cysteine, and the serine at position 389 is replaced with threonine.

[0099] (Production Example 15) Preparation of pCUP2-Ptrp-NSDG-I252L-S389T An artificially synthesized DNA fragment (SEQ ID NO: 42) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssembleMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-I252L-S389T. pCUP2-Ptrp-NSDG-I252L-S389T is a plasmid that expresses NSDG-I252L-S389T under the trp promoter.

[0100] NSDG-I252L-S389T is a mutant PHA synthase in which the amino acid sequence shown in Sequence ID No. 25 has been modified in which the isoleucine at position 252 from the N-terminus is replaced with leucine, and the serine at position 389 is replaced with threonine.

[0101] (Production Example 16) Preparation of pCUP2-Ptrp-NSDG-P245S-I252A An artificially synthesized DNA fragment (SEQ ID NO: 43) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssemblelyMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-P245S-I252A. pCUP2-Ptrp-NSDG-P245S-I252A is a plasmid that expresses NSDG-P245S-I252A under the trp promoter.

[0102] NSDG-P245S-I252A is a mutant PHA synthase in which the 245th proline from the N-terminus is replaced with serine and the 252nd isoleucine is replaced with alanine, compared to the amino acid sequence shown in Sequence ID No. 25.

[0103] (Production Example 17) Preparation of pCUP2-Ptrp-NSDG-P245S-I252C An artificially synthesized DNA fragment (SEQ ID NO: 44) was used as a template, and the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssembleMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-P245S-I252C. pCUP2-Ptrp-NSDG-P245S-I252C is a plasmid that expresses NSDG-P245S-I252C under the trp promoter.

[0104] NSDG-P245S-I252C is a mutant PHA synthase in which the 245th proline from the N-terminus is replaced with serine and the 252nd isoleucine is replaced with cysteine, compared to the amino acid sequence shown in Sequence ID No. 25.

[0105] (Production Example 18) Preparation of pCUP2-Ptrp-NSDG-P245S-I252C-S389T Using an artificially synthesized DNA fragment (SEQ ID NO: 45) as a template, the DNA fragment was amplified by PCR using primers SEQ ID NO: 34 and SEQ ID NO: 35. The obtained DNA fragment and the DNA fragment obtained by digesting pCUP2-Ptrp prepared in Production Example 2 with Mun1 were ligated using NEBuilderHiFiDNAAssembleMasterMix (New England Biolab) to obtain pCUP2-Ptrp-NSDG-P245S-I252C-S389T. pCUP2-Ptrp-NSDG-P245S-I252C-S389T is a plasmid that expresses NSDG-P245S-I252C-S389T under the trp promoter.

[0106] NSDG-P245S-I252C-S389T is a mutant PHA synthase in which the amino acid sequence shown in Sequence ID No. 25 has been modified in which the 245th proline from the N-terminus is replaced with serine, the 252nd isoleucine is replaced with cysteine, and the 389th serine is replaced with threonine.

[0107] (Production Example 19) Introduction of each plasmid into the KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp strain First, the KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp strain prepared in Production Example 1 was cultured overnight in Nutrient Broth medium (DIFCO). 0.5 mL of the resulting culture was inoculated into 100 mL of Nutrient Broth medium and cultured at 30°C for 3 hours. The resulting culture was rapidly cooled on ice, the cells were collected and thoroughly washed with ice-cooled distilled water, and then the obtained cells were suspended in 2 mL of distilled water. The bacterial cell suspension was mixed with the plasmid solutions prepared in Production Examples 2-18, injected into a cuvette, and electroporation was performed. Electroporation was carried out using a MicroPulser electroporator (Bio-Rad) under conditions of voltage 1.5 kV, resistance 800 Ω, and current 25 μF. After electroporation, the bacterial cell suspension was collected, 5 mL of Nutrient Broth medium was added, and the mixture was incubated at 30°C for 3 hours. The resulting culture medium was spread onto Nutrient Agar medium containing 100 mg / L kanamycin sulfate. After incubation at 30°C for 2 days, each strain into which the plasmid had been introduced was obtained from the resulting colonies.

[0108] (Comparative Example 1) KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp / pCUP2-Ptrp-phaC Ａｃ The composition of the PHA-producing strain culture medium was 1% (w / v) meat extract, 1% (w / v) Bacto-Trypton, 0.2% (w / v) yeast extract, 0.9% (w / v) disodium hydrogen phosphate dodecahydrate, 0.15% (w / v) potassium dihydrogen phosphate, 50 μg / L kanamycin, and pH 6.8.

[0109] The PHA production medium used for PHA production consisted of 1.1% (w / v) disodium hydrogen phosphate dodecahydrate, 0.19% (w / v) potassium dihydrogen phosphate, 1.29% (w / v) ammonium sulfate, 0.1% (w / v) magnesium sulfate heptahydrate, 0.5% (v / v) trace metal salt solution (1.6% (w / v) iron(II) chloride hexahydrate, 1% (w / v) calcium chloride dihydrate, 0.02% (w / v) cobalt chloride hexahydrate, 0.016% (w / v) copper sulfate pentahydrate, and 0.012% (w / v) nickel chloride hexahydrate dissolved in 0.1N hydrochloric acid), and 50 μg / L kanamycin. Palm oil was used as the carbon source at a concentration of 1.5% (w / v).

[0110] The PHA flask production culture method was carried out as follows: First, 50 μl of the strain's glycerol stock was inoculated into 5 ml of seed medium and cultured for 16 hours, and the resulting culture was used as the seed stock. Next, 0.5 ml of the seed stock culture was inoculated into a 500 ml Sakaguchi flask containing 50 ml of PHA production medium. The operating conditions were a culture temperature of 30°C and a stirring speed of 130 rpm, and the culture was carried out for 72 hours. After the culture was completed, the bacterial cells were collected from the culture, washed with ethanol, vacuum dried, and the dry bacterial weight was measured.

[0111] (PHA production and monomer composition analysis) The amount of PHA produced and the monomer composition were analyzed by gas chromatography. Approximately 20 mg of dried bacterial cells recovered after the culture was completed were mixed with 1 ml of sulfuric acid-methanol mixture (15:85) and 1 ml of chloroform, sealed tightly, and heated at 100°C for 140 minutes to methyl esterify the PHA contained in the dried bacterial cells. After cooling, 0.5 ml of pure water was added and allowed to stand for about 30 minutes to separate into two layers. The lower chloroform layer was then collected, filtered, and the PHA was analyzed by capillary gas chromatography to quantify 3HB (4 carbon atoms), 3HH (6 carbon atoms), and 3HO (8 carbon atoms), and the ratio of each monomer composition was calculated. In addition, the amount of PHA contained in the dried bacterial cells and the PHA content (wt%) were calculated from the polymerization amount of each monomer. A Shimadzu Nexus GC-2030 gas chromatograph was used, and a Frontier Labs Ultra ALLOYU A1 (MS / HT)-15M-0.25F capillary column (column length 15 m, column inner diameter 0.25 mm, liquid film thickness 0.25 μm) was used. N was used as the carrier gas. ２ Using a column inlet pressure of 43.8 kPa, 1 μl of sample was injected. The temperature conditions were as follows: an initial temperature of 50°C was maintained for 2 minutes, then the temperature was increased from 50°C to 275°C at a rate of 22.5°C / min, and then maintained at 275°C for 10 minutes. The results of the analysis under the above conditions, including the amount of PHA produced (dried intracellular PHA content (wt%)) and monomer composition ratio, are shown in Tables 1 and 2.

[0112] (Comparative Example 2) PHA production using the KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp / pCUP2-Ptrp-NSDG strain. The strain was cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1, and are shown in Table 2.

[0113] (Comparative Example 3) PHA production using strain KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp / pCUP2-Ptrp-NSDG-S389T was performed using the same method as in Comparative Example 1, and the amount of PHA produced and the monomer composition ratio were calculated using the same method as in Comparative Example 1, and are shown in Table 2.

[0114] (Examples 1-14) PHA production using each plasmid-derived strain. Each plasmid-derived strain prepared in Production Example 19 was cultured in the same manner as in Comparative Example 1, and the PHA production amount and monomer composition ratio were calculated in the same manner as in Comparative Example 1. The results are shown in Tables 1 and 2.

[0115]

[0116]

[0117] <Discussion> Based on the results in Table 1, wild-type PHA synthase PhaC has the amino acid sequence shown in Sequence ID No. 1. Ａｃ In contrast, the mutant PHA synthases of Examples 1 to 3, which had mutations in P245S, I252A, or I252L, showed an increased 3HO ratio compared to the wild-type PHA synthase of Comparative Example 1, which did not have these mutations. From these results, it can be seen that mutant PHA synthases having mutations in the 245th or 252nd amino acid from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 are useful for producing PHA copolymers with a high 3HO ratio.

[0118] Table 2 shows the results for mutant PHA synthases in which mutations were introduced at the 245th and / or 252nd amino acid, in addition to mutations at the 149th and 171st amino acids from the N-terminus of the amino acid sequence shown in Sequence ID No. 1. Comparing Examples 4-7 with Comparative Example 2, an increase in the 3HO ratio is observed. This indicates that by introducing mutations at the 245th or 252nd amino acid in NSDG, it is possible to produce PHA with a high 3HO ratio.

[0119] Examples 8 to 11 show that the combination of the 245th or 252nd mutation with the S389T mutation is effective in improving the 3HO ratio. On the other hand, Comparative Example 3 shows that introducing the S389T mutation alone into NSDG does not improve the 3HO ratio compared to Comparative Example 2.

[0120] Examples 12 to 14 show that introducing mutations at both positions 245 and 252, and combining them with the S389T mutation, is effective in improving the 3HO ratio.

[0121] From these results, it is clear that a mutant PHA synthase, in which a mutation is introduced at the 245th and / or 252nd amino acids from the N-terminus of the amino acid sequence shown in Sequence ID No. 1, enables the production of PHA copolymers with a higher 3HO ratio.

Claims

1. A mutant polyhydroxyalkanoate synthase that exhibits 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and contains an amino acid sequence having at least one of the following mutations (a) to (b), and has the activity to synthesize copolymerized polyhydroxyalkanoates containing a carbon-8 3-hydroxyalkanoate monomer unit. Mutation (a): A mutation in which the 245th proline from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than proline. Mutation (b): A mutation in which the 252nd isoleucine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than isoleucine.

2. The mutant polyhydroxyalkanoate synthase according to claim 1, wherein in mutation (a), the amino acid other than proline is serine.

3. The mutant polyhydroxyalkanoate synthase according to claim 1, wherein in mutation (b), the amino acid other than isoleucine is alanine, cysteine, or leucine.

4. A mutant polyhydroxyalkanoate synthase according to any one of claims 1 to 3, further comprising a mutation in which the 389th serine molecule from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is replaced with an amino acid other than serine.

5. The mutant polyhydroxyalkanoate synthase according to claim 4, wherein the amino acid other than serine is cysteine, isoleucine, threonine, or valine.

6. A mutant polyhydroxyalkanoate synthase according to any one of claims 1 to 3, further comprising a mutation in which the asparagine at position 149 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine.

7. A mutant polyhydroxyalkanoate synthase according to any one of claims 1 to 3, further comprising a mutation in which the aspartic acid at position 171 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine.

8. A gene encoding a mutant polyhydroxyalkanoate synthase according to any one of claims 1 to 3.

9. A transformant having the gene described in claim 8.

10. The transformant according to claim 9, wherein the host is a bacterium.

11. The transformant according to claim 10, wherein the eubacterium is a bacterium belonging to the genus Capriavidus.

12. A method for producing copolymerized polyhydroxyalkanoic acid, comprising the steps of: culturing the transformant described in claim 9 in the presence of a carbon source; and recovering copolymerized polyhydroxyalkanoic acid containing a 3-hydroxyalkanoic acid monomer unit having 8 carbon atoms from the transformant.

13. The method for producing copolymerized polyhydroxyalkanoic acid according to claim 12, wherein the composition ratio of the carbon-8 3-hydroxyalkanoic acid monomer units in the copolymerized polyhydroxyalkanoic acid is 1.0 mol% or more.

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