Mutant polyhydroxyalkanoic acid synthase, gene thereof, transformed microorganism, and method for producing polyhydroxyalkanoic acid

Abstract

Provided is a mutant polyhydroxyalkanoic acid synthase comprising an amino acid sequence which has at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and in which at least one among 11th, 23rd, 74th, 176th, 224th, 241st, 327th, 360th, 400th, 419th, 452nd, 484th, 485th, 509th, and 511th amino acids from the N-terminal is substituted with a specific amino acid, wherein the mutant polyhydroxyalkanoic acid synthase has a synthetic activity of a copolymerized polyhydroxyalkanoic acid including a 3-hydroxyalkanoic acid monomer unit having 8 or more carbon atoms.

Description

Mutant polyhydroxyalkanoic acid synthase, its gene, transformed microorganism, and method for producing polyhydroxyalkanoic acid

[0001] The present invention relates to a mutant polyhydroxyalkanoic acid synthase, a gene thereof, a transformed microorganism, and a method for producing polyhydroxyalkanoic acid.

[0002] Polyhydroxyalkanoates (hereinafter referred to as PHAs) are polyesters that accumulate intracellularly as energy storage materials in microorganisms. Because PHAs are completely biodegradable by microorganisms in soil and water, they have recently attracted attention as environmentally friendly plastics that can replace conventional petroleum-derived plastics.

[0003] Known monomer units constituting PHA include, for example, 3-hydroxybutyric acid (abbreviation: 3HB), 3-hydroxyvaleric acid (abbreviation: 3HV), 3-hydroxyhexanoic acid (abbreviation: 3HH), 3-hydroxyoctanoic acid (abbreviation: 3HO), 3-hydroxydecanoic acid (abbreviation: 3HD), 3-hydroxydodecanoic acid (abbreviation: 3HDD), etc. Currently, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (abbreviation: PHBH), a copolymer PHA composed of 3HB units and 3HH units, has been put to practical use.

[0004] However, PHAs currently produced industrially from 3-hydroxyalkanoic acid monomer units having 4 to 6 carbon atoms have a glass transition temperature (Tg) of around 0°C, and their mechanical properties tend to deteriorate in low-temperature environments. Therefore, there is a need for the development of PHAs with sufficiently low Tg.

[0005] Known PHAs with low Tg include those containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms (hereinafter also referred to as medium-chain 3HA). In general, the Tg of a PHA tends to decrease as the composition ratio of monomer units with a large number of carbon atoms increases.

[0006] In Non-Patent Document 1, PHA synthases (PhaCs) are classified into four classes, Class 1, Class 2, Class 3, and Class 4, depending on the substrate specificity and subunit structure of the enzyme. According to this document, PhaCs classified into Class 1, Class 3, and Class 4 have polymerization activity for 3-hydroxyalkanoic acids having 3 to 5 carbon atoms, while Class 2 PhaCs have polymerization activity for 3-hydroxyalkanoic acids having 6 to 14 carbon atoms.

[0007] Conventionally, known methods for producing copolymerized PHAs containing 3HA monomer units having 8 or more carbon atoms include culturing Pseudomonas bacteria or culturing transformed microorganisms into which a gene encoding Class 2 PhaC derived from Pseudomonas bacteria or a mutant thereof has been introduced.

[0008] For example, Non-Patent Documents 2 and 3 describe the production of copolymerized PHAs containing three medium-chain-length HA monomer units by culturing transformed microorganisms into which genes encoding Pseudomonas sp. H9-derived PhaC (PhaC1H9), Pseudomonas putida Gpo1-derived PhaC (PhaC1po), and Pseudomonas mendocina-derived PhaC (PhaC1pm) were introduced as Class 2 PhaCs derived from the genus Pseudomonas.

[0009] Furthermore, Patent Documents 1 and 2 describe the production of a copolymerized PHA containing three medium-chain length HA monomer units by culturing a transformant into which a gene encoding a mutant of Class 2 PhaC (PhaC1ps) derived from Pseudomonas sp. 61-3 strain was introduced.

[0010] Although different from the Class 2 PhaC derived from Pseudomonas bacteria described above, Patent Document 3 describes the production of a copolymer PHA containing a 3-hydroxyhexanoate unit having six carbon atoms by culturing a transformant into which a gene for CO9 synthase or D12 synthase, which is a PhaC of unknown class derived from the actinomycete Rhodococcus aetherivorans I24, has been introduced.

[0011] The literature describes that these synthases can polymerize substrates having 7 to 8 carbon atoms, but there is no description that copolymerized PHAs containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms were produced.

[0012] JP 2007-125004 A International Publication No. 2003 / 100055 JP 2013-510572 A

[0013] Rehm, Bernd H. A. , Biochem. J. (2003) 376, 15-33 Liu, Chung-Hsien, et al. , Enzyme and Microbial Technology, 143 (2021) 109719 Hein, S. , et al. , Appl Microbiol Biotechnol (2002) 58:229-236

[0014] In the transformed microorganisms into which the above-mentioned Class 2 PhaC derived from Pseudomonas bacteria or its mutants, or the genes encoding the CO9 synthase or D12 synthase derived from the actinomycete Rhodococcus aetherivorans have been introduced, the composition ratio of the medium-chain length 3HA monomer unit in the copolymerized PHA produced is not high, and the productivity of the copolymerized PHA may also be insufficient, which has been limited in terms of realizing industrial production. For this reason, there is a demand for the construction of a PHA synthase that can produce copolymerized PHA with a high composition ratio of medium-chain length 3HA monomer units with good productivity.

[0015] In view of the above-mentioned current situation, the present invention aims to provide a polyhydroxyalkanoic acid synthase that enables improved productivity of polyhydroxyalkanoic acid while increasing the composition ratio of medium-chain length 3HA monomer units in polyhydroxyalkanoic acid, a gene encoding the enzyme, a transformed microorganism having the gene, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.

[0016] As a result of extensive research conducted by the present inventors to solve the above-mentioned problems, they found that a transformed microorganism into which a gene encoding the PHA synthase PhaCmc consisting of the amino acid sequence shown in SEQ ID NO: 1 derived from Mycobacterium cookie has been introduced can produce copolymerized PHA containing a high proportion of medium-chain length three HA monomer units with good productivity compared to conventionally known transformed microorganisms.

[0017] Based on this finding, further investigation was conducted and it was found that by introducing a mutation into at least one of the amino acids at positions 11, 23, 74, 176, 224, 241, 327, 360, 400, 419, 452, 484, 485, 509, and 511 from the N-terminus in the amino acid sequence shown in SEQ ID NO: 1, it is possible to improve the composition ratio of medium-chain length 3HA monomer units in copolymerized PHA while simultaneously improving PHA productivity compared to wild-type PhaCmc, which led to the completion of the present invention.

[0018] Specifically, the present invention relates to a mutant polyhydroxyalkanoate synthase that has an amino acid sequence that shows 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and has one or more of the following mutations (a) to (o), and that has the activity of synthesizing a copolymerized polyhydroxyalkanoate containing 3-hydroxyalkanoate monomer units having 8 or more carbon atoms. Mutation (a): A mutation in which the alanine at position 11 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine or methionine. Mutation (b): A mutation in which the lysine at position 23 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine, asparagine, or tryptophan. Mutation (c): A mutation in which the arginine at position 74 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine. Mutation (d): A mutation in which the methionine at position 176 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine. Mutation (e): A mutation in which the proline at position 224 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine. Mutation (f): A mutation in which the isoleucine at position 241 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine or serine. Mutation (g): A mutation in which the threonine at position 327 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine. Mutation (h): A mutation in which the aspartic acid at position 360 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with cysteine. Mutation (i): A mutation in which the asparagine at position 400 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine or alanine. Mutation (j): A mutation in which the leucine at position 419 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamine. Mutation (k): A mutation in which the alanine at position 452 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with aspartic acid. Mutation (l): A mutation in which the alanine at position 484 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine. Mutation (m): A mutation in which the alanine at the 485th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine ​​or serine. Mutation (n): A mutation in which the glutamine at the 509th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with asparagine.Mutation (o): A mutation in which the leucine at position 511 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with tyrosine or methionine. The present invention also relates to a gene encoding the mutant polyhydroxyalkanoate synthase, and to a transformed microorganism capable of producing a copolymerized polyhydroxyalkanoate containing a 3-hydroxyalkanoate monomer unit having 8 or more carbon atoms, the transformed microorganism having the gene. The present invention further relates to a method for producing a copolymerized polyhydroxyalkanoate, the method comprising the steps of culturing the transformed microorganism in the presence of a carbon source, and recovering from the transformed microorganism the copolymerized polyhydroxyalkanoate containing a 3-hydroxyalkanoate monomer unit having 8 or more carbon atoms.

[0019] According to the present invention, there are provided a polyhydroxyalkanoic acid synthase that enables improved productivity of polyhydroxyalkanoic acid while increasing the composition ratio of medium-chain-length 3HA monomer units in polyhydroxyalkanoic acid, a gene encoding the enzyme, and a transformed microorganism harboring the gene. By culturing the transformed microorganism, it is possible to produce a copolymerized polyhydroxyalkanoic acid containing a high proportion of medium-chain-length 3HA monomer units through fermentation with high productivity. The resulting copolymerized polyhydroxyalkanoic acid has the advantage that it can have better mechanical properties in low-temperature environments than a polyhydroxyalkanoic acid composed solely of 3-hydroxyalkanoic acid monomer units having 7 or fewer carbon atoms or a polyhydroxyalkanoic acid having a low composition ratio of medium-chain-length 3HA monomer units.

[0020] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments. (Mutant PHA synthase) A mutant PHA synthase (synthase) according to this embodiment has an amino acid sequence that shows 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and has one or more mutations selected from the group consisting of (a) to (o) described below.

[0021] SEQ ID NO: 1 shows the amino acid sequence of wild-type PhaC (hereinafter also referred to as wild-type PhaCmc) derived from Mycobacterium cookie. By introducing specific amino acid mutations into the amino acid sequence of this wild-type PhaCmc, the composition ratio of medium-chain length 3HA monomer units in the produced copolymerized PHA can be improved compared to wild-type PhaCmc, and further, the productivity of the copolymerized PHA can be improved.

[0022] The amino acid sequence of the mutant PHA synthase of this embodiment has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 1, but the sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0023] The amino acid sequence of the mutant PHA synthase according to this embodiment has one or more mutations selected from the following (a) to (o). The amino acid sequence may contain only one of the mutations selected from the following (a) to (o), or may contain two or more of these mutations. Mutation (a): A mutation in which the alanine at position 11 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine or methionine. Mutation (b): A mutation in which the lysine at position 23 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine, asparagine, or tryptophan. Mutation (c): A mutation in which the arginine at position 74 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine. Mutation (d): A mutation in which the methionine at position 176 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine. Mutation (e): A mutation in which the proline at position 224 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine. Mutation (f): A mutation in which the isoleucine at position 241 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine or serine. Mutation (g): A mutation in which the threonine at position 327 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine. Mutation (h): A mutation in which the aspartic acid at position 360 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with cysteine. Mutation (i): A mutation in which the asparagine at position 400 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine or alanine. Mutation (j): A mutation in which the leucine at position 419 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamine. Mutation (k): A mutation in which the alanine at position 452 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with aspartic acid. Mutation (l): A mutation in which the alanine at position 484 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine. Mutation (m): A mutation in which the alanine at the 485th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine ​​or serine. Mutation (n): A mutation in which the glutamine at the 509th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with asparagine.Mutation (o): A mutation in which leucine at the 511th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine or methionine.

[0024] Among the above-mentioned mutations, preferred mutations that can particularly increase the productivity of copolymerized PHA are the mutation in (b) where tyrosine or tryptophan is substituted, the mutation in (f) where serine is substituted, and the mutation in (g).

[0025] Of the above-mentioned mutations, the mutations (e), (i), (k), and (n) are preferred as those that can particularly increase the composition ratio of medium-chain length 3HA monomer units in the copolymerized PHA produced.

[0026] The amino acid sequence of the mutant PHA synthase according to this embodiment may have a combination of two or more of the mutations (a) to (o) above, or may have a combination of the mutations (a) to (o) above and another mutation. Specific examples of such combinations include the following (A) to (D). (A): Having the mutation (d) above and a mutation in which glutamine at the 508th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine. (B): Having the mutation (d) above and a mutation in which aspartic acid at the 126th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine, and a mutation in which alanine at the 422nd position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with proline. (C): Having the mutation (e) above and a mutation in (m) in which an amino acid is substituted with cysteine. (D): Having the mutation (g), (k), and a mutation in (m) in which an amino acid is substituted with cysteine.

[0027] The mutant PHA synthase of this embodiment is an enzyme that exhibits the activity of synthesizing a copolymerized PHA containing three medium-chain-length HA monomer units. The copolymerized PHA refers to a copolymer of medium-chain-length 3HA and another hydroxyalkanoic acid copolymerizable with the medium-chain-length 3HA. The copolymerized PHA has three medium-chain-length 3HA monomer units, which can improve its mechanical properties in low-temperature environments.

[0028] The mutant PHA synthase according to this embodiment may have the activity of synthesizing a copolymerized PHA having a higher composition ratio of medium-chain length 3HA monomer units compared to wild-type PhaCmc (i.e., a PHA synthase consisting of the amino acid sequence shown in SEQ ID NO: 1). According to a preferred embodiment, the composition ratio of medium-chain length 3HA monomer units in the copolymerized PHA synthesized by the mutant PHA synthase according to this embodiment is preferably 1.1 times or more, more preferably 1.3 times or more, even more preferably 1.5 times or more, and particularly preferably 1.7 times or more, relative to the same ratio in the copolymerized PHA synthesized by wild-type PhaCmc. The above description can also be applied to a comparison between a transformed microorganism having a mutant PHA synthase according to this embodiment and a transformed microorganism having wild-type PhaCmc, as described below.

[0029] When comparing the composition ratio of medium-chain length 3HA monomer units in copolymerized PHA produced between a mutant PHA synthase and a wild-type PhaCmc, the gene encoding the target enzyme is introduced into the same host using the same method, and the resulting transformed strain is cultured under the same culture conditions, and the composition ratios in the resulting copolymerized PHA are compared.

[0030] Furthermore, the mutant PHA synthase according to this embodiment can produce copolymerized PHAs with higher productivity than wild-type PhaCmc. According to a preferred embodiment, the productivity of copolymerized PHA produced by the mutant PHA synthase according to this embodiment is preferably 1.1 times or more, more preferably 1.3 times or more, and particularly preferably 1.5 times or more, the productivity of copolymerized PHA produced by wild-type PhaCmc. The above description can also be applied to a comparison between a transformed microorganism having a mutant PHA synthase according to this embodiment and a transformed microorganism having wild-type PhaCmc, which will be described later.

[0031] When comparing the productivity of copolymerized PHA between a mutant PHA synthase and a wild-type PhaCmc, the gene encoding the target enzyme is introduced into the same host using the same method, and the amount of copolymerized PHA produced when the resulting transformed strain is cultured under the same culture conditions is compared.

[0032] The upper limit of the number of carbon atoms contained in the medium chain length 3HA is not particularly limited, but may be, for example, 14 or less, or 12 or less.

[0033] Specific examples of medium chain length 3HA include 3-hydroxyoctanoic acid (8 carbon atoms, abbreviation: 3HO), 3-hydroxynonanoic acid (9 carbon atoms), 3-hydroxydecanoic acid (10 carbon atoms, abbreviation: 3HD), 3-hydroxyundecanoic acid (11 carbon atoms), 3-hydroxydodecanoic acid (12 carbon atoms, abbreviation: 3HDD), 3-hydroxytetradecanoic acid (14 carbon atoms), etc. The copolymerized PHA may contain only one type of these medium chain length 3HAs, or may contain two or more types.

[0034] The copolymerized PHA preferably contains at least one medium chain length 3HA selected from the group consisting of 3HO, 3HD, and 3HDD. The medium chain length 3HA may contain only 3HO and / or 3HD, or may contain only 3HO. In particular, the medium chain length 3HA preferably contains at least 3HO.

[0035] The other hydroxyalkanoic acid copolymerized with the medium-chain-length 3HA is not particularly limited, and may be a 3-hydroxyalkanoic acid having 7 or less carbon atoms, such as 3-hydroxybutyric acid (4 carbon atoms, abbreviated as 3HB), 3-hydroxyvaleric acid (5 carbon atoms), 3-hydroxyhexanoic acid (6 carbon atoms, abbreviated as 3HH), and 3-hydroxyheptanoic acid (7 carbon atoms). The other hydroxyalkanoic acid may also be a hydroxyalkanoic acid other than 3-hydroxyalkanoic acid, such as 2-hydroxyalkanoic acid, 4-hydroxyalkanoic acid, 5-hydroxyalkanoic acid, and 6-hydroxyalkanoic acid. The copolymerized PHA may contain only one of these other hydroxyalkanoic acids, or may contain two or more of them.

[0036] The copolymerized PHA may contain 3HB and / or 3HH as other hydroxyalkanoic acids, or may contain 3HB and 3HH. When both 3HB and 3HH are contained, the content ratio (3HB:3HH) is not particularly limited, but may be, for example, about 3:1 to 1:30, or 2:1 to 1:10 on a molar basis.

[0037] In particular, the copolymer PHA may contain at least one medium-chain-length 3HA selected from the group consisting of 3HO, 3HD, and 3HDD, and 3HB and / or 3HH, or may contain at least one medium-chain-length 3HA selected from the group consisting of 3HO, 3HD, and 3HDD, and 3HB and 3HH.

[0038] The total content of the medium chain length 3HA monomer units in the copolymerized PHA may be 1 mol% or more. However, from the viewpoint of improving the mechanical properties of the copolymerized PHA in a low-temperature environment, it is preferably 16 mol% or more, more preferably 20 mol% or more, and particularly preferably 25 mol% or more. It may also be 30 mol% or more, or 40 mol% or more. The upper limit is not particularly limited, but may be, for example, 70 mol% or less, 60 mol% or less, or 50 mol% or less.

[0039] (Gene) According to one aspect of the present invention, there is provided a gene encoding the mutant PHA synthase described above (hereinafter abbreviated as "mutant PHA synthase gene"). The base sequence encoding the gene is not particularly limited as long as it is a base sequence encoding the amino acid sequence of the mutant PHA synthase according to the present disclosure.

[0040] According to one aspect of the present invention, there is provided a transformed microorganism capable of producing a copolymerized PHA containing three medium-chain-length HA monomer units, the transformed microorganism having a gene encoding a mutant PHA synthase according to the present disclosure. The transformed microorganism is produced by introducing the gene into a host microorganism.

[0041] (Transformed Microorganism) The host for the transformed microorganism according to the present disclosure is not particularly limited, and examples thereof include the genus Cupriavidus such as Cupriavidus necator, the genus Alcaligenes such as Alcaligenes latus, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas resinovorans, and Pseudomonas oleovorans. Examples of the bacterium include the genus Pseudomonas such as Bacillus megaterium, the genus Azotobacter, the genus Nocardia, the genus Aeromonas such as Aeromonas caviae and Aeromonas hydrophila, the genus Ralstonia, the genus Wautersia, the genus Comamonas, and the genus Halomonas.

[0042] Further, gram-negative bacteria such as those of the genus Esherichia, gram-positive bacteria such as those of the genus Bacillus, and yeasts such as those of the genus Saccharomyces, Yarrowia, and Candida may also be used.

[0043] As the host, bacteria are preferred because they are capable of accumulating a large amount of PHA, bacteria belonging to the genus Cupriavidus are more preferred, and Cupriavidus necator is particularly preferred.

[0044] The number of mutant PHA synthase genes to be introduced may be one or more. When multiple mutant PHA synthase genes are introduced, they may be the same gene or different genes.

[0045] The method for introducing a mutant PHA synthase gene into a host is not particularly limited, and examples thereof include a method for directly inserting or substituting a target gene onto a chromosome of the host, a method for directly inserting or substituting a target gene onto a megaplasmid carried by the host, or a method for placing a target gene on a vector such as a plasmid, phage, or phagemid and introducing the gene into the host. Two or more of these methods may be used in combination.

[0046] Considering the stability of the introduced gene, a method of directly inserting or substituting the target gene onto the host chromosome or onto a megaplasmid possessed by the host is preferred, and a method of directly inserting or substituting the target gene onto the host chromosome is more preferred. To ensure expression of the introduced gene, it is preferable to introduce the target gene so that it is located downstream of a "gene expression regulatory sequence" originally possessed by the host, or so that it is located downstream of an exogenous "gene expression regulatory sequence." The "gene expression regulatory sequence" may be a DNA sequence containing a nucleotide sequence (e.g., a promoter sequence) that controls the transcription level of the gene and / or a nucleotide sequence (e.g., a Shine-Dalgarno sequence) that regulates the translation level of messenger RNA transcribed from the gene. The "gene expression regulatory sequence" may be any naturally occurring nucleotide sequence, or an artificially constructed or modified nucleotide sequence.

[0047] Introduction of a foreign gene can be carried out by methods well known to those skilled in the art. Representative methods include a method utilizing the mechanism of transposon and homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)), a method based on site-specific integration caused by the mechanism of homologous recombination and elimination by second-stage homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)), and a method in which the sacB gene derived from Bacillus subtilis is coexisted, and a microbial strain in which the gene has been eliminated by second-stage homologous recombination is easily isolated as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195-1204 (1992), Lenz et al. al., J. Bacteriol., 176:4385-4393 (1994)), or a method of introducing the vector using a plasmid vector can be used.

[0048] Introduction of a foreign gene into a microorganism using a plasmid vector can be carried out by a commonly used method, such as the calcium chloride method, electroporation, polyethylene glycol method, spheroplast method, etc. The plasmid vector may be prepared by ligating a DNA fragment having the base sequence of the foreign gene with a plasmid vector such as pCUP2.

[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) and the like can be used.

[0050] The promoter for expressing the introduced gene is not particularly limited. Examples of promoters that can be used include the phaC1 gene promoter and phaP1 gene promoter of Capriavidus necator, the trp promoter, lac promoter, lacUV5 promoter, trc promoter, tic promoter, and tac promoter derived from Escherichia coli, as well as the lacN17 promoter having an artificially engineered modified nucleotide sequence derived from Escherichia coli, and the lacN19 promoter having an artificially engineered modified nucleotide sequence derived from Escherichia coli.

[0051] In a preferred embodiment, the transformed microorganism 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 one that has been transformed so that expression of the gene is enhanced. This increases the amount of 3HA monomers having 8 or more carbon atoms produced, and further increases the content of 3HA monomer units having 8 or more carbon atoms in the produced copolymerized PHA.

[0052] As used herein, a "protein having R-specific enoyl-CoA hydratase activity" refers to a protein having the enzymatic activity to produce (R)-3-hydroxyacyl-CoA, a PHA monomer, using enoyl-CoA, an intermediate in the β-oxidation system of fatty acids, as a substrate. When the protein uses enoyl-CoA having 8 or more carbon atoms as a substrate, the amount converted to (R)-3-hydroxyacyl-CoA having 8 or more carbon atoms is increased, and as a result, the content of 3HA monomer units having 8 or more carbon atoms in the copolymerized PHA is thought to be increased.

[0053] Examples of proteins having R-specific enoyl-CoA hydratase activity that recognizes enoyl-CoA having 8 or more carbon atoms as a substrate include, but are not limited to, bacterial R-specific enoyl-CoA hydratase (PhaJ) and eukaryotic multifunctional enzyme type 2 (MFE2).

[0054] 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 toward enoyl-CoA having 8 or more carbon atoms. The origin of MFE2 is not particularly limited, but examples include Drosophila melanogaster and Yarrowia lipolytica.

[0055] 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: 2 or a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0056] 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 may be introduced into a transformed microorganism, or the transformed microorganism may be transformed so that expression of the gene is enhanced. Introduction of the gene can be carried out by the method described above. To enhance expression of the gene, the expression regulatory sequence (promoter sequence and / or SD sequence) can be modified to enhance expression of the gene, as described in WO 2015 / 115619, for example.

[0057] As used herein, "enhanced gene expression" refers to a state in which the transcription level of a gene of interest or the expression level of a polypeptide encoded by the gene of interest is increased compared to a strain in which expression of the gene of interest is not enhanced. The amount of increase is not particularly limited, but it is sufficient if it is more than 1-fold, preferably 1.1-fold or more, more preferably 1.2-fold or more, even more preferably 1.5-fold or more, and even more preferably 2-fold or more, compared to a strain in which expression of the gene of interest is not enhanced.

[0058] A transformed microorganism according to a preferred embodiment may be transformed so that the expression of a gene encoding a β-ketothiolase is suppressed. The β-ketothiolase has thiolysis activity for β-ketoacyl-CoA having 8 or fewer carbon atoms. By suppressing the expression of the β-ketothiolase gene, the enzymatic activity of the β-ketothiolase can be eliminated or reduced. This suppresses the degradation of β-ketoacyl-CoA having 8 or fewer carbon atoms, enabling more efficient production of copolymerized PHA containing a medium-chain-length 3HA monomer. The expression of only one β-ketothiolase gene may be suppressed, or two or more β-ketothiolase genes may be suppressed. Furthermore, a transformed microorganism according to a preferred embodiment may be one in which the expression of a β-ketothiolase gene is suppressed and in which the expression of a gene encoding a protein having the R-specific enoyl-CoA hydratase activity described above is introduced or enhanced.

[0059] As used herein, the term "β-ketothiolase" refers to an enzyme that catalyzes the reaction in the β-oxidation of fatty acids, in which β-ketoacyl-CoA undergoes thiolysis (thiol cleavage) in the presence of coenzyme A to produce fatty acyl-CoA that is two carbon atoms shorter and acetyl-CoA.

[0060] The β-ketothiolase gene whose expression is inhibited may be a gene encoding a β-ketothiolase having thiolysis activity for a β-ketoacyl-CoA having 8 or less carbon atoms. The β-ketothiolase may have thiolysis activity for a β-ketoacyl-CoA having 9 or more carbon atoms, in addition to thiolysis activity for a β-ketoacyl-CoA having 8 or less carbon atoms. For example, the β-ketothiolase may have thiolysis activity for a β-ketoacyl-CoA having 4 to 8 carbon atoms, or may have thiolysis activity for a β-ketoacyl-CoA having 4 to 18 carbon atoms, or may have thiolysis activity for a β-ketoacyl-CoA having 6 to 20 carbon atoms, but is not limited to these.

[0061] The gene encoding the β-ketothiolase is not particularly limited, and examples thereof include the bktB gene and the A1528 gene. Specific examples include genes encoding proteins consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4, and genes encoding proteins consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 or 4. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0062] Examples of methods for suppressing the expression of a gene encoding β-ketothiolase include a method of completely deleting the enzyme gene in a transformed microorganism, a method of inserting an entirely different gene such as a drug resistance gene into the sequence of the enzyme gene, or a method of deleting, substituting, adding, or inserting a part of the sequence of the enzyme gene (preferably a region involved in enzymatic activity).Gene disruption techniques include, for example, 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 techniques 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 portion of the β-ketothiolase gene sequence to be disrupted, and serves to transport Cas9 to the target. Furthermore, mutations such as deletion, substitution, addition, and insertion of the nucleotide sequence surrounding the gene can also be used to eliminate or reduce the enzyme activity by reducing the transcription and translation efficiency of the gene and mRNA stability.

[0063] (Culturing of Microorganisms) The transformed microorganisms can be cultured in the presence of a carbon source to allow the copolymerized PHA to accumulate within the microbial cells. The transformed microorganisms can be cultured according to conventional microbial culture methods, provided that the culture is carried out in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, carbon source addition method, culture scale, aeration and agitation conditions, culture temperature, culture time, etc. The carbon source is preferably added to the medium continuously or intermittently.

[0064] Any carbon source can be used as a carbon source during cultivation as long as it can be utilized by the transformed microorganism. Examples 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 their low-melting-point fractions, 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, as well as their fractionated oils and refined by-products; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myrinsic acid, as well as their derivatives, and 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, carbon sources containing oils and fats (particularly vegetable oils) or free fatty acids are preferred. The number of carbon atoms in the constituent fatty acids of the fat or oil or the free fatty acids is not particularly limited, but from the viewpoint of productivity of copolymerized PHA containing a medium-chain-length 3HA monomer, it is preferably 8 or more, more preferably 10 or more, and even more preferably 12 or more.

[0065] In culturing the transformed microorganism, it is preferable to use 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. Examples of nitrogen sources include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, and yeast extract. Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.

[0066] After culturing the transformed microorganism for an appropriate period of time to accumulate the copolymerized PHA within the microbial cells, the copolymerized PHA can be recovered using a known method. The recovery method is not particularly limited, but industrially, recovery by separation and purification in an aqueous system with low environmental impact is preferred. For example, after completion of the culture, cells can be disrupted by applying mechanical shearing force or using a surfactant, alkali, enzyme, or the like to obtain a cell lysate in which cellular components other than the copolymerized PHA are dissolved in water. The copolymerized PHA can be recovered by separating the copolymerized PHA from the aqueous phase by filtration or centrifugation of the cell lysate and then drying it.

[0067] The following items list preferred aspects of the present disclosure, but the present invention is not limited to them. [Item 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 one or more of the following mutations (a) to (o), and that has the activity of synthesizing a copolymerized polyhydroxyalkanoate containing 3-hydroxyalkanoate monomer units having 8 or more carbon atoms. Mutation (a): A mutation in which the alanine at position 11 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine or methionine. Mutation (b): A mutation in which the lysine at position 23 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine, asparagine, or tryptophan. Mutation (c): A mutation in which the arginine at position 74 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine. Mutation (d): A mutation in which the methionine at position 176 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine. Mutation (e): A mutation in which the proline at position 224 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine. Mutation (f): A mutation in which the isoleucine at position 241 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine or serine. Mutation (g): A mutation in which the threonine at position 327 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine. Mutation (h): A mutation in which the aspartic acid at position 360 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with cysteine. Mutation (i): A mutation in which the asparagine at position 400 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with serine or alanine. Mutation (j): A mutation in which the leucine at position 419 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamine. Mutation (k): A mutation in which the alanine at position 452 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with aspartic acid. Mutation (l): A mutation in which the alanine at the 484th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine. Mutation (m): A mutation in which the alanine at the 485th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine ​​or serine.Mutation (n): A mutation in which glutamine at the 509th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with asparagine. Mutation (o): A mutation in which leucine at the 511th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine or methionine. [Item 2] The mutant polyhydroxyalkanoate synthase according to Item 1, comprising one or more mutations selected from the group consisting of a mutation in which tyrosine or tryptophan is substituted in (b), a mutation in which serine is substituted in (f), and (g). [Item 3] The mutant polyhydroxyalkanoate synthase according to Item 1 or 2, comprising one or more mutations selected from the group consisting of (e), (i), (k), and (n). [Item 4] The mutant polyhydroxyalkanoate synthase according to any one of Items 1 to 3, which satisfies any one of the following (A) to (D): (A): Having (d) above and a mutation in which glutamine at position 508 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine. (B): Having (d) above and a mutation in which aspartic acid at position 126 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine, and a mutation in which alanine at position 422 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with proline. (C): Having (e) above and a mutation in (m) in which an amino acid is substituted with cysteine. (D): Having (g), (k), and a mutation in (m) in which an amino acid is substituted with cysteine. [Item 5] A gene encoding the mutant polyhydroxyalkanoate synthase according to any one of Items 1 to 4. [Item 6] A transformed microorganism capable of producing a copolymerized polyhydroxyalkanoate containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms, the transformed microorganism having the gene according to Item 5. [Item 7] The transformed microorganism according to Item 6, into which a gene encoding a protein having R-specific enoyl-CoA hydratase activity toward an enoyl-CoA substrate having 8 or more carbon atoms has been introduced, or which has been transformed so that expression of the gene is enhanced.[Item 8] The transformed microorganism according to Item 7, wherein the gene encoding the protein having R-specific enoyl-CoA hydratase activity is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2. [Item 9] The transformed microorganism according to any of Items 6 to 8, which has been transformed so that expression of a gene encoding a β-ketothiolase is suppressed. [Item 10] The transformed microorganism according to Item 9, wherein the gene encoding the β-ketothiolase is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4, or a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 or 4. [Item 11] The transformed microorganism according to any of Items 6 to 10, wherein the host of the transformed microorganism belongs to the genus Capriavidus. [Item 12] The transformed microorganism according to Item 11, wherein the host of the transformed microorganism is Capriavidus necator. [Item 13] A method for producing a copolymerized polyhydroxyalkanoic acid, comprising the steps of: culturing the transformed microorganism according to any one of Items 6 to 12 in the presence of a carbon source; and recovering from the transformed microorganism a copolymerized polyhydroxyalkanoic acid containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms. [Item 14] The method for producing a copolymerized polyhydroxyalkanoic acid according to Item 13, wherein the total content of the 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms in the copolymerized polyhydroxyalkanoic acid is 1 mol % or more.

[0068] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0069] (Breeding of strains) The genetic manipulation in this example can be carried out by the method described in Molecular Cloning (Cold Spring Harbor Laboratory Press, 1989). The enzymes, cloning hosts, and other enzymes used in the genetic manipulation can be purchased from commercial suppliers and used according to their instructions. The enzymes used in this example are not particularly limited as long as they can be used in genetic manipulation.

[0070] The "KNK005dZ strain" used in the following production examples is a transformed microorganism also known as the KNK005ΔphaZ1,2,6 strain, in which the phaC1 gene on the chromosome of Cupriavidus necator H16 strain has been replaced with a PHA synthase gene mutant (NSDG) derived from Aeromonas caviae, and the phaZ1,2,6 gene, a PHA degrading enzyme gene on the chromosome, has been deleted. This transformed microorganism can be produced according to the method described in WO 2014 / 065253.

[0071] (Production Example 1) Preparation of KNK005dZ / dNSDG Strain First, a plasmid for disrupting NSDG was prepared. This was done as follows. A DNA fragment linking the nucleotide sequences upstream and downstream of the NSDG gene was prepared by PCR using the genomic DNA of the KNK005dZ strain as a template (SEQ ID NO: 5). This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was ligated with DNA ligase to the vector pNS2X-sacB described in JP 2007-259708 A, which had also been digested with SmiII, to prepare the NSDG disruption plasmid vector pNS2X-sacB+NSDGUD.

[0072] Next, the KNK005dZ / dNSDG strain was constructed as follows using the NSDG disruption plasmid vector pNS2X-sacB+NSDGUD.

[0073] Escherichia coli strain S17-1 (ATCC47055) was transformed with pNS2X-sacB+NSDGUD, and the resulting transformed microorganism was mixed and cultured with strain KNK005dZ on Nutrient Agar (Difco) medium to carry out conjugative transfer.

[0074] The resulting cultured cells were inoculated onto Simmons agar medium containing 250 mg / L of kanamycin (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8), and strains grown on the agar medium were selected to obtain a strain in which the plasmid had been integrated into the chromosome of the KNK005dZ strain. This strain was cultured for two generations in Nutrient Broth medium, then diluted and spread onto Nutrient Agar medium containing 15% sucrose, and the grown strain was identified as a strain in which the plasmid had been removed. Furthermore, one strain in which the NSDG gene on the chromosome had been removed was isolated by PCR analysis or sequence analysis. The resulting strain was named KNK005dZ / dNSDG.

[0075] (Production Example 2) Preparation of KNK005dZ / dNSDG / dphaJ4a strain First, a plasmid for disrupting the phaJ4a gene was prepared. This was done as follows. PCR was performed using the genomic DNA of the KNK005dZ strain as a template to prepare a DNA fragment linking the upstream and downstream base sequences of the phaJ4a structural gene (SEQ ID NO: 6). 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 SmiII, using DNA ligase to prepare the plasmid vector pNS2X-sacB+phaJ4aUD for disrupting the phaJ4a gene.

[0076] Using the plasmid vector pNS2X-sacB+phaJ4aUD for disrupting the phaJ4a gene, the phaJ4a gene-disrupted strain KNK005dZ / dNSDG / dphaJ4a was prepared in the same manner as above, using KNK005dZ / dNSDG obtained in Production Example 1 as a parent strain.

[0077] (Production Example 3) Preparation of KNK005dZ / dNSDG / dphaJ4a / dbktB strain First, a plasmid for disrupting the bktB gene was prepared. This was done as follows. PCR was performed using the genomic DNA of the KNK005dZ strain as a template to prepare a DNA fragment linking the nucleotide sequences upstream and downstream of the bktB structural gene (SEQ ID NO: 7). 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 plasmid vector pNS2X-sacB+bktBUD for disrupting the bktB gene.

[0078] Next, using the bktB gene disruption plasmid vector pNS2X-sacB+bktBUD, the bktB gene disruption strain KNK005dZ / dNSDG / dphaJ4a obtained in Production Example 2 was used as a parent strain in the same manner as above to create the bktB gene disruption strain KNK005dZ / dNSDG / dphaJ4a / dbktB.

[0079] (Production Example 4) Preparation of KNK005dZ / dNSDG / dphaJ4a / dbktB / dA1528 strain First, a plasmid for disrupting the A1528 gene was prepared. This was done as follows. PCR was performed using the genomic DNA of the KNK005dZ strain as a template to prepare a DNA fragment linking the upstream and downstream base sequences of the A1528 structural gene (SEQ ID NO: 8). 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 plasmid vector pNS2X-sacB+A1528UD for disrupting the A1528 gene.

[0080] Next, using the A1528 gene disruption plasmid vector pNS2X-sacB+A1528UD, the A1528 gene disruption strain KNK005dZ / dNSDG / dphaJ4a / dbktB obtained in Production Example 3 was used as a parent strain in the same manner as above. The A1528 gene disruption strain KNK005dZ / dNSDG / dphaJ4a / dbktB / dA1528 was prepared.

[0081] (Production Example 5) Preparation of KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp strain First, a plasmid for introducing the phaJ4pp gene was prepared. This was done as follows. PCR using an artificially synthesized gene as a template yielded a DNA fragment (SEQ ID NO: 10) containing a base sequence including the base sequences upstream and downstream of the phaJ4a structural gene, the trc promoter having the base sequence set forth in SEQ ID NO: 9, and a gene encoding phaJ4pp having the amino acid sequence set forth in SEQ ID NO: 2. 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 plasmid vector pNS2X-sacB+phaJ4aU-trc-phaJ4pp-phaJ4aD for introducing the phaJ4pp gene.

[0082] Using the plasmid vector pNS2X-sacB+phaJ4aU-trc-phaJ4pp-phaJ4aD for introducing the phaJ4pp gene, chromosomal DNA was modified in the same manner as in the gene disruption described above using KNK005dZ / dNSDG / dphaJ4a / dbktB / dA1528 obtained in Production Example 4 as a parent strain, to prepare the phaJ4pp gene-inserted strain KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp.

[0083] (Production Example 6) Preparation of pCUP2-trc-phaCmc plasmid In order to introduce the gene encoding the PhaCmc enzyme derived from Mycobacterium cookiei into a host for evaluating PHA production, a plasmid pCUP2-trc-phaCmc for introducing the wild-type phaCmc gene was prepared.

[0084] First, an artificially synthesized DNA fragment (SEQ ID NO: 11) containing a nucleotide sequence encoding the amino acid sequence of PhaCmc shown in SEQ ID NO: 1 was digested with restriction enzymes MunI and SpeI. This DNA fragment was ligated using DNA ligase to the pCUP2 vector described in JP 2007-259708 A that had been digested with MunI and SpeI to prepare the pCUP2-phaCmc plasmid.

[0085] Next, PCR was performed using the trc promoter and pCUP2-trc-A2365 described in Japanese Patent No. 7425783 as a template, and the DNAs shown in SEQ ID NO: 12 and SEQ ID NO: 13 as primers, and the resulting DNA fragment was digested with EcoRI and MunI. This DNA fragment was ligated using DNA ligase to pCUP2-phaCmc digested with MunI to prepare pCUP2-trc-phaCmc, in which the phaCmc gene sequence was ligated so as to be located downstream of the trc promoter.

[0086] (Preparation Example 7) Preparation of plasmid for pCUP2-trc-phaCmcA11R gene introduction The pCUP2-trc-phaCmc prepared in Preparation Example 6 was treated with MunI and SpeI to obtain a pCUP2-trc vector fragment, which was then ligated to the nucleotide sequence shown in SEQ ID NO: 14 using NEBuilder (New England Biolabs) to prepare the pCUP2-trc-phaCmcA11R plasmid.

[0087] PhaCmcA11R is a mutant PHA synthase in which the alanine at the 11th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine.

[0088] (Production Example 8) Preparation of plasmid for introducing pCUP2-trc-phaCmcA11M gene A pCUP2-trc-phaCmcA11M plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 15 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcA11M gene.

[0089] PhaCmcA11M is a mutant PHA synthase in which the alanine at the 11th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with methionine.

[0090] (Production Example 9) Preparation of plasmid for introducing pCUP2-trc-phaCmcK23Y gene A pCUP2-trc-phaCmcK23Y plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 16 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcK23Y gene.

[0091] PhaCmcK23Y is a mutant PHA synthase in which the lysine at the 23rd position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 has been substituted with tyrosine.

[0092] (Production Example 10) Preparation of plasmid for introducing pCUP2-trc-phaCmcK23N gene A pCUP2-trc-phaCmcK23N plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 17 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcK23N gene.

[0093] PhaCmcK23N is a mutant PHA synthase in which the lysine at the 23rd position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 has been substituted with asparagine.

[0094] (Production Example 11) Preparation of plasmid for pCUP2-trc-phaCmcK23W gene introduction A pCUP2-trc-phaCmcK23W plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 18 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcK23W gene.

[0095] PhaCmcK23W is a mutant PHA synthase in which the 23rd lysine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 has been substituted with tryptophan.

[0096] (Production Example 12) Preparation of plasmid for pCUP2-trc-phaCmcR74V gene introduction A pCUP2-trc-phaCmcR74V plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 19 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcR74V gene.

[0097] PhaCmcR74V is a mutant PHA synthase in which arginine at the 74th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine.

[0098] (Production Example 13) Preparation of plasmid for pCUP2-trc-phaCmcM176T gene introduction A pCUP2-trc-phaCmcM176T plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 20 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcM176T gene.

[0099] PhaCmcM176T is a mutant PHA synthase in which methionine at the 176th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine.

[0100] (Production Example 14) Preparation of plasmid for pCUP2-trc-phaCmcM176T / Q508R gene introduction A pCUP2-trc-phaCmcM176T / Q508R plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 21 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcM176T / Q508R gene.

[0101] PhaCmcM176T / Q508R is a mutant PHA synthase in which the methionine at the 176th position from the N-terminus is replaced with threonine and the glutamine at the 508th position from the N-terminus is replaced with arginine in the amino acid sequence shown in SEQ ID NO: 1.

[0102] (Production Example 15) Preparation of plasmid for pCUP2-trc-phaCmcD126G / M176T / A422P gene introduction A pCUP2-trc-phaCmcD126G / M176T / A422P plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 22 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the phaCmcA11R gene introduction plasmid pCUP2-trc-phaCmcA11R was replaced with the phaCmcD126G / M176T / A422P gene.

[0103] PhaCmcD126G / M176T / A422P is a mutant PHA synthase in which, relative to the amino acid sequence shown in SEQ ID NO: 1, the aspartic acid at the 126th position from the N-terminus is replaced with glycine, the methionine at the 176th position from the N-terminus is replaced with threonine, and the alanine at the 422nd position from the N-terminus is replaced with proline.

[0104] (Production Example 16) Preparation of plasmid for pCUP2-trc-phaCmcP224T gene introduction A pCUP2-trc-phaCmcP224T plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 23 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcP224T gene.

[0105] PhaCmcP224T is a mutant PHA synthase in which the proline at the 224th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 has been replaced with threonine.

[0106] (Production Example 17) Preparation of plasmid for pCUP2-trc-phaCmcP224T / A485C gene introduction A pCUP2-trc-phaCmcP224T / A485C plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 24 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcP224T / A485C gene.

[0107] PhaCmcP224T / A485C is a mutant PHA synthase in which the proline at the 224th position from the N-terminus is replaced with threonine and the alanine at the 485th position from the N-terminus is replaced with cysteine ​​in the amino acid sequence shown in SEQ ID NO: 1.

[0108] (Preparation Example 18) Preparation of plasmid for pCUP2-trc-phaCmcI241T gene introduction A pCUP2-trc-phaCmcI241T plasmid was prepared in the same manner as in Preparation Example 7, except that the base sequence shown in SEQ ID NO: 25 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcI241T gene.

[0109] PhaCmcI241T is a mutant PHA synthase in which the isoleucine at the 241st position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine.

[0110] (Preparation Example 19) Preparation of plasmid for introducing pCUP2-trc-phaCmcI241S gene A pCUP2-trc-phaCmcI241S plasmid was prepared in the same manner as in Preparation Example 7, except that the base sequence shown in SEQ ID NO: 26 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcI241S gene.

[0111] PhaCmcI241S is a mutant PHA synthase in which the isoleucine at the 241st position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with serine.

[0112] (Production Example 20) Preparation of plasmid for introducing pCUP2-trc-phaCmcT327S gene A pCUP2-trc-phaCmcT327S plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 27 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcT327S gene.

[0113] PhaCmcT327S is a mutant PHA synthase in which the threonine at the 327th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with serine.

[0114] (Production Example 21) Preparation of plasmid for introducing pCUP2-trc-phaCmcD360C gene A pCUP2-trc-phaCmcD360C plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 28 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcD360C gene.

[0115] PhaCmcD360C is a mutant PHA synthase in which the aspartic acid at the 360th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine.

[0116] (Production Example 22) Preparation of plasmid for introducing pCUP2-trc-phaCmcN400S gene A pCUP2-trc-phaCmcN400S plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 29 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcN400S gene.

[0117] PhaCmcN400S is a mutant PHA synthase in which the asparagine at the 400th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with serine.

[0118] (Production Example 23) Preparation of plasmid for introducing pCUP2-trc-phaCmcN400A gene A pCUP2-trc-phaCmcN400A plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 30 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcN400A gene.

[0119] PhaCmcN400A is a mutant PHA synthase in which the asparagine at the 400th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine.

[0120] (Production Example 24) Preparation of plasmid for introducing pCUP2-trc-phaCmcL419Q gene A pCUP2-trc-phaCmcL419Q plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 31 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcL419Q gene.

[0121] PhaCmcL419Q is a mutant PHA synthase in which the 419th leucine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamine.

[0122] (Production Example 25) Preparation of plasmid for introducing pCUP2-trc-phaCmcA452D gene A pCUP2-trc-phaCmcA452D plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 32 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcA452D gene.

[0123] PhaCmcA452D is a mutant PHA synthase in which the alanine at the 452nd position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid.

[0124] (Production Example 26) Preparation of plasmid for pCUP2-trc-phaCmcT327S / A452D / A485C gene introduction A pCUP2-trc-phaCmcT327S / A452D / A485C plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 33 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the phaCmcA11R gene introduction plasmid pCUP2-trc-phaCmcA11R was replaced with the phaCmcT327S / A452D / A485C gene.

[0125] PhaCmcT327S / A452D / A485C is a mutant PHA synthase in which, relative to the amino acid sequence shown in SEQ ID NO: 1, the threonine at the 327th position from the N-terminus is replaced with serine, the alanine at the 452nd position from the N-terminus is replaced with aspartic acid, and the alanine at the 485th position from the N-terminus is replaced with cysteine.

[0126] (Production Example 27) Preparation of plasmid for pCUP2-trc-phaCmcA484T gene introduction A pCUP2-trc-phaCmcA484T plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 34 was used instead of the base sequence shown in SEQ ID NO: 14, in which the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcA484T gene.

[0127] PhaCmcA484T is a mutant PHA synthase in which the alanine at the 484th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine.

[0128] (Preparation Example 28) Preparation of plasmid for introducing pCUP2-trc-phaCmcA485C gene A pCUP2-trc-phaCmcA485C plasmid was prepared in the same manner as in Preparation Example 7, except that the base sequence shown in SEQ ID NO: 35 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcA485C gene.

[0129] PhaCmcA485C is a mutant PHA synthase in which the alanine at the 485th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine.

[0130] (Preparation Example 29) Preparation of plasmid for pCUP2-trc-phaCmcA485S gene introduction A pCUP2-trc-phaCmcA485S plasmid was prepared in the same manner as in Preparation Example 7, except that the base sequence shown in SEQ ID NO: 36 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcA485S gene.

[0131] PhaCmcA485S is a mutant PHA synthase in which the alanine at the 485th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with serine.

[0132] (Production Example 30) Preparation of plasmid for introducing pCUP2-trc-phaCmcQ509N gene A pCUP2-trc-phaCmcQ509N plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 37 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcQ509N gene.

[0133] PhaCmcQ509N is a mutant PHA synthase in which glutamine at the 509th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with asparagine.

[0134] (Production Example 31) Preparation of plasmid for pCUP2-trc-phaCmcL511Y gene introduction A pCUP2-trc-phaCmcL511Y plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 38 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for phaCmcA11R gene introduction was replaced with the phaCmcL511Y gene.

[0135] PhaCmcL511Y is a mutant PHA synthase in which the leucine at the 511th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine.

[0136] (Production Example 32) Preparation of plasmid for introducing pCUP2-trc-phaCmcL511M gene A pCUP2-trc-phaCmcL511M plasmid was prepared in the same manner as in Production Example 7, except that the base sequence shown in SEQ ID NO: 39 was used instead of the base sequence shown in SEQ ID NO: 14. In this case, the phaCmcA11R gene in the plasmid pCUP2-trc-phaCmcA11R for introducing the phaCmcA11R gene was replaced with the phaCmcL511M gene.

[0137] PhaCmcL511M is a mutant PHA synthase in which the leucine at the 511th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 has been substituted with methionine.

[0138] (Production Example 33) Preparation of a strain containing a plasmid for introducing the wild-type phaCmc gene, using KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp as a parent strain. For the purpose of preparing a production test strain, a strain containing the plasmid for introducing the wild-type phaCmc gene prepared in Production Example 6, using KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp as a parent strain, was prepared by the following procedure.

[0139] First, the host (KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp) was cultured overnight in 5 ml of Nutrient Broth medium. The resulting culture was quickly cooled on ice, and the cells were collected and thoroughly washed with ice-cold distilled water. The resulting cells were then suspended in 2 ml of distilled water. 1 ml of the cell solution was mixed with the plasmid solution and injected into a cuvette for electroporation. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) at a voltage of 1.5 kV, a resistance of 800 Ω, and a current of 25 μF. After electroporation, the cell solution was collected, 1 ml of Nutrient Broth medium was added, and the mixture was cultured at 30 ° C for 3 hours. The resulting culture medium was applied to a Nutrient Agar medium containing 100 mg / L kanamycin sulfate, and cultured at 30° C. for 2 days, and a strain into which the plasmid had been introduced was obtained from the resulting colonies.

[0140] (Production Example 34) Preparation of strains incorporating plasmids for introducing mutant phaCmc genes using KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp as a parent strain In a similar manner to Production Example 33, strains were prepared by using KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp prepared in Production Example 5 as a parent strain and introducing each of the plasmids for introducing mutant phaCmc genes prepared in Production Examples 7 to 32.

[0141] Comparative Example 1: PHA production using wild-type PhaCmc The PHA production of the strain obtained by introducing a plasmid for introducing the wild-type phaCmc gene into KNK005dZ / dNSDG / dbktB / dA1528 / dphaJ4a::trc-phaJ4pp, prepared in Production Example 33, was evaluated by the following method.

[0142] The composition of the seed 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, pH 6.8.

[0143] 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 0.1 N hydrochloric acid), and 50 μg / L kanamycin. Palm kernel oil was used as the carbon source at a concentration of 1.0% (w / v).

[0144] The PHA flask production culture method was performed as follows. First, 50 μl of glycerol stock of the strain was inoculated into 5 ml of seed medium and cultured for 16 hours, and the resulting culture solution was used as the seed medium. Next, 0.5 ml of the seed medium culture solution 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 performed for 72 hours. After the culture was completed, the bacterial cells were recovered from the culture solution, washed with ethanol, and vacuum dried, and the dry bacterial weight was measured.

[0145] (Analysis of PHA Production Amount and Monomer Composition) The production amount and monomer composition of the resulting PHA were analyzed by gas chromatography. Approximately 20 mg of dried cells recovered after the completion of the culture were added with 1 ml of a sulfuric acid-methanol mixture (15:85) and 1 ml of chloroform, and the mixture was sealed and heated at 100°C for 140 minutes to methyl-esterify the PHA contained in the dried cells. After cooling, 0.5 ml of pure water was added and the mixture was allowed to stand for approximately 30 minutes to separate into two layers. The lower chloroform layer was then collected and passed through a filter. The PHA was then analyzed by capillary gas chromatography, and 3HB (carbon number 4), 3HH (carbon number 6), 3HO (carbon number 8), 3HD (carbon number 10), and 3HDD (carbon number 12) were quantified, and the compositional ratio of each monomer was calculated. Furthermore, the amount of PHA contained in the dried cells and the PHA content (wt%) were calculated from the amount of polymerization of each monomer. Finally, the PHA production amount (g / L) in the culture medium was calculated from the dry cell weight (DCW) and PHA content (wt%) of the culture medium. The gas chromatograph was a Shimadzu GC-2014AF / SPL, and the capillary column was a Frontier Labs Ultra ALLOYUA1 (MS / HT)-15M-0.25F (column length 15 m, column inner diameter 0.25 mm, liquid film thickness 0.25 μm). He was used as the carrier gas, the column inlet pressure was 43.8 kPa, and 1 μl of sample was injected. The temperature conditions were as follows: the initial temperature was held at 50 ° C for 2 minutes, then the temperature was increased from 50 ° C to 275 ° C at a rate of 22.5 ° C / min, and then 275 ° C was held for 10 minutes. The PHA production amount and monomer composition ratio obtained as a result of analysis under the above conditions are shown in Table 1.

[0146]

[0147] The wild-type phaCmc gene-transfected strain evaluated in Comparative Example 1 produced a copolymerized PHA containing medium-chain-length 3HA monomers (3HO, 3HD, and 3HDD). The PHA production amount was 0.56 g / L. The proportion of medium-chain-length 3HA monomers (the total proportion of 3HO, 3HD, and 3HDD) in the produced copolymerized PHA was 15.6 mol%.

[0148] (Examples 1 to 26) PHA production using mutant PhaCmc The strains prepared in Production Example 34 into which the plasmids for introducing each mutant phaCmc gene had been introduced were 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 obtained PHA production amount and monomer composition ratio are shown in Table 1.

[0149] All of the strains of Examples 1 to 26 into which the mutant phaCmc gene was introduced produced copolymerized PHA containing medium-chain-length 3HA monomers (3HO, 3HD, and 3HDD). These strains produced higher PHA production (g / L) and a higher proportion of medium-chain-length 3HA monomers (the total proportion of 3HO, 3HD, and 3HDD) than the wild-type phaCmc gene-introduced strain of Comparative Example 1.

[0150] In particular, the phaCmcK23Y gene-introduced strain of Example 3, the phaCmcK23W gene-introduced strain of Example 5, the phaCmcI241S gene-introduced strain of Example 13, and the phaCmcT327S gene-introduced strain of Example 14 had PHA production (g / L) of 0.84 g / L or more, which was more than 1.5-fold higher than the wild-type phaCmc gene-introduced strain of Comparative Example 1.

[0151] Furthermore, the phaCmcP224T gene-introduced strain of Example 10, the phaCmcN400S gene-introduced strain of Example 16, the phaCmcN400A gene-introduced strain of Example 17, the phaCmcA452D gene-introduced strain of Example 19, and the phaCmcQ509N gene-introduced strain of Example 24 had a proportion of medium-chain-length 3HA monomers (total proportion of 3HO, 3HD, and 3HDD) of 26.5 mol% or more, which was more than 1.7-fold higher than the wild-type phaCmc gene-introduced strain of Comparative Example 1.

Claims

1. A mutant polyhydroxyalkanoate synthase that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and contains an amino acid sequence having one or more of the following mutations (a) to (o), and that has the activity of synthesizing a copolymerized polyhydroxyalkanoate containing 3-hydroxyalkanoate monomer units having 8 or more carbon atoms: Mutation (a): A mutation in which the alanine at the 11th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with arginine or methionine. Mutation (b): A mutation in which the lysine at the 23rd position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine, asparagine, or tryptophan. Mutation (c): A mutation in which arginine at the 74th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with valine. Mutation (d): A mutation in which methionine at the 176th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine. Mutation (e): A mutation in which the proline at the 224th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine. Mutation (f): A mutation in which the isoleucine at the 241st position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with threonine or serine. Mutation (g): A mutation in which threonine at the 327th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with serine. Mutation (h): A mutation in which the aspartic acid at the 360th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine. Mutation (i): A mutation in which the asparagine at the 400th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with serine or alanine. Mutation (j): A mutation in which the leucine at the 419th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glutamine. Mutation (k): A mutation in which alanine at the 452nd position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with aspartic acid. Mutation (1): A mutation in which alanine at the 484th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with threonine. Mutation (m): A mutation in which alanine at the 485th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine ​​or serine. Mutation (n): A mutation in which glutamine at the 509th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with asparagine. Mutation (o): A mutation in which leucine at the 511th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with tyrosine or methionine.

2. A mutant polyhydroxyalkanoic acid synthase according to claim 1, which comprises one or more mutations selected from the group consisting of a mutation in which (b) is substituted with tyrosine or tryptophan, a mutation in (f) is substituted with serine, and (g).

3. A mutant polyhydroxyalkanoic acid synthase according to claim 1, comprising one or more mutations selected from the group consisting of (e), (i), (k), and (n).

4. The mutant polyhydroxyalkanoate synthase according to claim 1, which satisfies any one of the following (A) to (D): (A): The mutant polyhydroxyalkanoate synthase has (d) and a mutation in which glutamine at position 508 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with arginine. (B): The mutant polyhydroxyalkanoate synthase has (d) and a mutation in which aspartic acid at position 126 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with glycine, and a mutation in which alanine at position 422 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with proline. (C): The mutant polyhydroxyalkanoate synthase has (e) and a mutation in which an amino acid at position 422 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine. (D): The mutant polyhydroxyalkanoate synthase has (g), (k), and a mutation in which an amino acid at position 422 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is substituted with cysteine.

5. A gene encoding the mutant polyhydroxyalkanoate synthase according to any one of claims 1 to 4.

6. A transformed microorganism capable of producing a copolymerized polyhydroxyalkanoic acid containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms, said transformed microorganism having the gene according to claim 5.

7. The transformed microorganism according to claim 6, into which a gene encoding a protein having R-specific enoyl-CoA hydratase activity for an enoyl-CoA substrate having 8 or more carbon atoms has been introduced, or which has been transformed so that expression of said gene is enhanced.

8. The transformed microorganism according to claim 7, wherein the gene encoding the protein having R-specific enoyl-CoA hydratase activity is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:2, or a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

2.

9. The transformed microorganism according to claim 6, which has been transformed so that expression of a gene encoding β-ketothiolase is repressed.

10. The transformed microorganism according to claim 9, wherein the gene encoding the β-ketothiolase is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 or 4, or a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3 or 4.

11. The transformed microorganism of claim 6, wherein the host of the transformed microorganism belongs to the genus Capriavidus.

12. The transformed microorganism of claim 11, wherein the host of the transformed microorganism is Capriavidus necator.

13. A method for producing a copolymerized polyhydroxyalkanoic acid, comprising the steps of: culturing the transformed microorganism according to claim 6 in the presence of a carbon source; and recovering from the transformed microorganism a copolymerized polyhydroxyalkanoic acid containing 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms.

14. The method for producing a copolymerized polyhydroxyalkanoic acid according to claim 13, wherein the total content of 3-hydroxyalkanoic acid monomer units having 8 or more carbon atoms in the copolymerized polyhydroxyalkanoic acid is 1 mol % or more.

Images