MUTANT ACETOACETYL CoA REDUCTASE, GENE AND TRANSFORMANT FOR SAME, AND METHOD FOR PRODUCING POLYHYDROXY ALKANOIC ACID

Abstract

Provided is a mutant acetoacetyl CoA reductase comprising an amino acid sequence that has not less than 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and that has mutation (a) and / or mutation (b). Mutation (a): In the amino acid sequence represented by SEQ ID NO: 1, valine at position 95 from the N-terminus is substituted with an amino acid other than valine. Mutation (b): In the amino acid sequence represented by SEQ ID NO: 1, isoleucine at position 202 from the N-terminus is substituted with an amino acid other than isoleucine.

Description

Mutant acetoacetyl CoA reductase, the gene and transformant thereof, and method for producing polyhydroxyalkanoate

[0001] The present invention relates to a mutant acetoacetyl CoA reductase, a gene encoding the enzyme, a transformant comprising the gene, and a method for producing polyhydroxyalkanoate using the transformant.

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

[0003] As a PHA, poly-3-hydroxybutyrate (hereinafter abbreviated as "PHB"), which is a homopolymer of 3-hydroxybutyrate (hereinafter abbreviated as "3HB"), is known. However, PHB has high crystallinity and high degree of crystallinity, so it is hard and brittle, and has the problems of low melt processability.

[0004] As a PHA with improved brittleness and melt processability over PHB, the copolyester poly(3HB-co-3HH) (hereinafter abbreviated as "P3HB3HH") of 3HB and 3-hydroxyhexanoate (hereinafter abbreviated as "3HH") has been reported. By having 3HH as a monomer unit, P3HB3HH has lower crystallinity than PHB, and is a copolymer having ductile and soft physical properties.

[0005] As a production method for P3HB3HH, fermentative production using a transformant introduced with PHA synthase (PhaC) derived from Aeromonas caviae using Cupriavidus necator, a soil bacterium, as the host has been reported. In order to increase the flexibility of P3HB3HH, studies on increasing the proportion of 3HH in P3HB3HH have been conducted.

[0006] PHA is biosynthesized from carbon sources such as fatty acids via the β-oxidation cycle. In this β-oxidation cycle, enzymes such as PHA synthase (PhaC), acetoacetyl-CoA reductase (PhaB), and R-specific enoyl-CoA hydratase are known to be involved.

[0007] Patent Document 1 describes that by introducing acetoacetyl-CoA reductase (PhaB) derived from a different species of organism than the genus Capriavidus into Capriavidus nekator, it is possible to produce P3HB3HH with an improved 3HH ratio.

[0008] Patent Document 2 describes that the production of P3HB3HH can be improved by introducing mutations into the 141st, 12th, 194th, 42nd, or 55th amino acids in the amino acid sequence of acetoacetyl-CoA reductase (PhaB).

[0009] Special table 2024-515917 publication Special table 2024-516050 publication

[0010] There are limited technologies that can adjust the 3HH ratio of PHA copolymers produced by culturing transformants to a high level. On the other hand, for industrial production of PHA copolymers, good PHA productivity is required. Therefore, even if a technology can adjust the 3HH ratio to a high level, if it reduces PHA productivity as a result, it cannot be used for industrial production of PHA.

[0011] Therefore, the present invention aims to provide a mutant acetoacetyl-CoA reductase derived from the genus Capriavidus that enables the production of a PHA copolymer with a high 3HH ratio while maintaining PHA productivity, a gene encoding the enzyme, a transformant having the gene, and a method for producing PHA using the transformant.

[0012] As a result of diligent research to solve the above problems, the present inventors have discovered that by introducing a mutation to at least one amino acid among the 95th amino acid and the 202nd amino acid from the N-terminus in wild-type acetoacetyl CoA reductase (PhaB1) derived from Capriavidas nekator, which is composed of the amino acid sequence shown in Sequence ID No. 1, it is possible to produce a PHA copolymer with a higher 3HH ratio while maintaining PHA productivity, thus completing the present invention.

[0013] In other words, the present invention relates to a mutant acetoacetyl-CoA reductase that exhibits 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and includes an amino acid sequence having one or both of the following mutations (a) to (b): Mutation (a): A mutation in which the 95th valine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than valine. Mutation (b): A mutation in which the 202nd 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. The present invention also relates to a method for producing polyhydroxyalkanoic acid, comprising the steps of: a gene encoding the mutant acetoacetyl-CoA reductase; a transformant having the gene; a step of culturing the transformant in the presence of a carbon source; and a step of recovering polyhydroxyalkanoic acid from the transformant.

[0014] According to the present invention, a mutant acetoacetyl-CoA reductase derived from the genus Capriavidus, a gene encoding the enzyme, and a transformant having the gene can be provided, which enable the production of PHA copolymers with a high 3HH ratio while maintaining PHA productivity. Furthermore, by culturing the transformant, it becomes possible to ferment-produce PHA copolymers with a higher 3HH ratio with good productivity.

[0015] 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 configurations described below can be combined in any way, and such combinations may also constitute an embodiment of the present invention.

[0016] (Mutant Acetoacetyl CoA Reductase) The mutant acetoacetyl CoA reductase relating 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 one or both of the following mutations (a) and (b). The disclosure also provides a gene encoding the mutant acetoacetyl CoA reductase (hereinafter abbreviated as "mutant acetoacetyl CoA reductase gene" or "mutant gene").

[0017] Acetoacetyl-CoA reductase (PhaB) is present in many microorganisms that can naturally accumulate PHA and is an enzyme involved in the biosynthesis of (R)-3-hydroxyacyl-CoA, the most common PHA biosynthesis substrate. This enzyme catalyzes the reaction in which 3-ketoacyl-CoA is reduced to (R)-3-hydroxyacyl-CoA in the presence of the coenzyme NADPH. The mutant acetoacetyl-CoA reductase according to this disclosure has the enzymatic function described above.

[0018] Sequence ID 1 shows the amino acid sequence of wild-type acetoacetyl-CoA reductase derived from Capriavidas necator. By introducing at least one mutation (a) and (b) into the amino acid sequence shown in Sequence ID 1, it is possible to produce a PHA copolymer with a higher 3HH ratio while maintaining PHA productivity compared to the wild-type acetoacetyl-CoA reductase.

[0019] The amino acid sequence of the mutant acetoacetyl-CoA reductase 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 acetoacetyl-CoA reductase according to this disclosure may further 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.

[0020] Although Patent Document 1 discloses acetoacetyl-CoA reductase derived from a species other than the genus Capriavidus, the sequence identity between its amino acid sequence and the amino acid sequence shown in Sequence ID No. 1 of this disclosure is extremely low, at approximately 50-60%.

[0021] The mutant acetoacetyl-CoA reductase relating to this disclosure may be formed by binding to 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.

[0022] The nucleotide sequence of the acetoacetyl-CoA reductase gene relating to this disclosure is not particularly limited as long as it is a nucleotide sequence that encodes the amino acid sequence constituting the acetoacetyl-CoA reductase relating to this disclosure.

[0023] Next, the mutations (a) and (b) in the amino acid sequence of the mutant acetoacetyl-CoA reductase 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.

[0024] Mutation (a): A mutation in which the 95th valine molecule from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is replaced with an amino acid other than valine. In mutation (a), the substituted amino acid can be selected considering the productivity of PHA and the 3HH ratio of the PHA produced. Specific examples include alanine, isoleucine, leucine, methionine, 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 3HH ratio while maintaining PHA productivity, a mutation in which the 95th valine molecule is replaced with leucine is particularly preferred.

[0025] Mutation (b): In mutation (b), the isoleucine at position 202 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than isoleucine. The substituted amino acid can be selected considering the productivity of PHA and the 3HH 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 3HH ratio while maintaining PHA productivity, a mutation in which the isoleucine at position 202 is replaced with valine is particularly preferred.

[0026] According to a preferred embodiment of the present disclosure, the mutant acetoacetyl-CoA reductase exhibits 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and includes an amino acid sequence having both a mutation in which valine at position 95 is replaced by leucine and a mutation in which isoleucine at position 202 is replaced by valine.

[0027] (Transformant) The transformant according to this disclosure is a transformant having the mutant acetoacetyl-CoA reductase gene according to this disclosure, and is produced by introducing the gene into a host microorganism.

[0028] 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.

[0029] In producing the transformants according to this disclosure, any method can be used to introduce the mutant acetoacetyl-CoA reductase gene according to this disclosure into a host microorganism. For example, the mutant acetoacetyl-CoA reductase 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, the method of introducing the gene onto chromosomes or megaplasmides possessed by the microorganism is preferred, and the method of introducing the gene onto chromosomes possessed by the microorganism is more preferred.

[0030] Methods for site-specific substitution or insertion of arbitrary base sequences into the DNA of microorganisms, or for deletion of arbitrary base sequences in the DNA of microorganisms, are widely known to those skilled in the art and can be used when producing transformants according to this disclosure. While 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 site-specific integration and subsequent removal by homologous recombination (Noti et al., Methods Enzymol., vol. 154, p. 197 (1987)), and Bacillus One example is a method in which the sacB gene derived from subtilis is co-introduced, and the resulting microbial strain, in which the gene is lost by a second-stage homologous recombination, is easily isolated as a sucrose-tolerant medium (Schweizer, Mol. Microbiol., vol. 6, p. 1195 (1992); Lenz et al., J. Bacteriol., vol. 176, p. 4385 (1994)). Furthermore, there are no particular limitations on the method of introducing the vector into the microorganism, but examples include the calcium chloride method, electroporation method, polyethylene glycol method, and spheroplast method.

[0031] Furthermore, regarding gene cloning and genetic engineering 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 used.

[0032] When introducing the mutant acetoacetyl-CoA reductase 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 necator. 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 necator H16 strain and used as a regulatory expression sequence.

[0033] The transformed microorganisms relating to this disclosure may have only the mutant gene relating to this disclosure as the acetoacetyl-CoA reductase (PhaB) gene, or they may also have an acetoacetyl-CoA reductase gene that does not correspond to the mutant gene relating to this disclosure. Such acetoacetyl-CoA reductase genes may be those inherently present in the host, foreign genes, or genes in which mutations have been introduced into the amino acid sequence.

[0034] The transformed microorganism according to this disclosure has a gene encoding PHA synthase (PhaC). PHA synthase catalyzes the reaction that synthesizes PHA using (R)-3-hydroxyacyl-CoA as a substrate. As a result, the transformed microorganism according to this disclosure can produce PHA.

[0035] The PHA synthase gene may be one that is naturally present in the host, or it may be an exogenous gene. The PHA synthase gene is not particularly limited and may include PHA synthase genes derived from Aeromonas cavie, Aeromonas hydrophylla, Pseudomonas SP 61-3, or Capriavidus nekator, a chimeric PHA synthase gene combining two or more of the above-mentioned PHA synthase genes, or a gene encoding a protein containing an amino acid sequence that exhibits 90% or more sequence identity with respect to the amino acid sequences of each of the aforementioned PHA synthases. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more.

[0036] The transformed microorganisms relating to this disclosure may have one or more PHA synthase genes. Furthermore, if they have multiple PHA synthase genes, they may be the same gene or different genes.

[0037] The transformed microorganisms according to this disclosure may have a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity. R-specific enoyl-CoA hydratase has the function of converting hexenoyl-CoA to (R)-3-hydroxyhexanoyl-CoA. Having this gene increases the supply of (R)-3-hydroxyhexanoyl-CoA and can increase the 3HH ratio in the copolymerized PHA produced. However, the transformed microorganisms according to this disclosure may not have a gene encoding a protein exhibiting R-specific enoyl-CoA hydratase activity.

[0038] Genes encoding proteins with R-specific enoyl-CoA hydratase activity include the phaJ4a gene, the phaJ4b gene, and the MFE2 gene.

[0039] The gene encoding the protein exhibiting R-specific enoyl-CoA hydratase activity may be a gene inherent to the host or an exogenous gene. Examples of gene sources include Capriavidus necatol, Aeromonas caviar, Yarowia liporitica, and Drosophylla melanogaster.

[0040] The gene encoding a protein having R-specific enoyl-CoA hydratase activity 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, the gene expression regulatory sequence (promoter sequence and / or SD sequence) can be modified to enhance the expression of the gene, for example, as described in International Publication No. 2015 / 115619.

[0041] 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.

[0042] The transformed microorganisms according to this disclosure may be transformed in such a way 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 3HH. The expression of only one β-ketothiolase gene may be suppressed, or there may be two or more.

[0043] Furthermore, the transformed microorganism according to the present disclosure may comprise both suppression of expression of a β-ketothiolase gene and introduction or enhanced expression of a gene encoding a protein having the R-form-specific enoyl-CoA hydratase activity described above.

[0044] As used herein, the term "β-ketothiolase" refers to an enzyme that catalyzes a 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 shortened by two carbons and acetyl-CoA.

[0045] The β-ketothiolase gene whose expression is to be suppressed only needs to be a gene encoding a β-ketothiolase having thiolysis activity against β-ketoacyl-CoA with 8 or less carbon atoms. Said β-ketothiolase may simultaneously have thiolysis activity against β-ketoacyl-CoA with 9 or more carbon atoms in addition to thiolysis activity against β-ketoacyl-CoA with 8 or less carbon atoms. For example, it may have thiolysis activity against β-ketoacyl-CoA with 4 to 8 carbon atoms, may have thiolysis activity against β-ketoacyl-CoA with 4 to 18 carbon atoms, or may have thiolysis activity against β-ketoacyl-CoA with 6 to 20 carbon atoms, but is not limited to the above.

[0046] The gene encoding the β-ketothiolase is not particularly limited, and examples thereof include the bktB gene and the A1528 gene. Specific examples include a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 13, and a gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 13. Said sequence identity is preferably 95% or more, more preferably 97% or more, and still more preferably 99% or more.

[0047] To suppress the expression of a gene encoding β-ketothiolase, for example, mention may be made of a method of completely deleting the enzyme gene in a transformed microorganism, a method of inserting a completely different gene such as a drug resistance gene into the internal sequence of the enzyme gene, and a method of deleting, substituting, adding or inserting a part of the sequence of the enzyme gene (preferably a region involved in enzyme activity). Gene disruption procedures include, for example, homologous recombination techniques using a vector containing a disruption gene or disruption DNA, techniques using transposons, and the like. Alternatively, as another disruption method, known techniques such as CRISPR / Cas (e.g., Cas9) system for disrupting a target gene and genome editing technology using TALEN (Y. Wang et al., ACS Synth Biol. 2016, 5(7): 721-732; Bogdanove and Voytas, Science, 333: 1843-1846, 2011; Jinek, et al., Science, 337: 816-821, 2012; Shalem, et al., Science, 343: 84-87, 2014; Wang, et al., Science, 343: 80-84, 2014) can be mentioned. For example, in the CRISPR / Cas9 system, a guide RNA (gRNA) has a sequence capable of binding to a part of the nucleotide sequence of the β-ketothiolase gene to be disrupted, and has a role of delivering Cas9 to the target. In addition, the enzyme activity can also be eliminated or reduced by reducing gene transcription / translation efficiency and mRNA stability through mutations such as deletion, substitution, addition, and insertion of nucleotide sequences around the relevant gene.

[0048] (Method for producing PHA) By culturing the transformant according to the present disclosure, PHA can be produced by causing the transformant to produce PHA and recovering the obtained PHA.

[0049] In PHA production, it is preferable to culture the transformant in a medium containing a carbon source, a nitrogen source which is a nutrient other than the carbon source, inorganic salts, and other organic nutrients.

[0050] 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.

[0051] Examples of nitrogen sources include ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, and yeast extract.

[0052] Examples of inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.

[0053] 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.

[0054] 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.

[0055] In this disclosure, 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, 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.

[0056] As another example, bacterial cells can be separated from the culture medium using a centrifuge or similar device, and then washed with distilled water, methanol, etc. Subsequently, the washed sample is mixed with a sodium lauryl sulfate (SDS) solution, the cell membrane is destroyed by ultrasonic disruption, and the bacterial components and PHA are separated using a centrifuge or similar device. The PHA can then be recovered by drying.

[0057] The PHA produced by the transformant according to this disclosure is a copolymer PHA containing at least a 3-hydroxyhexanoic acid (also known as "3HH") monomer unit. The hydroxyalkanoic acid monomer units other than 3HH contained in the copolymer PHA are not particularly limited and include, for example, 2-hydroxyalkanoic acid having 4 to 16 carbon atoms, 3-hydroxyalkanoic acid having 4 to 16 carbon atoms, 4-hydroxyalkanoic acid having 4 to 16 carbon atoms, etc. There may be one or more hydroxyalkanoic acids other than 3HH.

[0058] In particular, it is preferable that the hydroxyalkanoic acid other than 3HH includes 3-hydroxybutyric acid. That is, it is especially preferable that the copolymer PHA produced by the transformant according to this disclosure is P(3HB-co-3HH), which is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. It should be noted that this copolymer may further contain other hydroxyalkanoic acid monomer units.

[0059] A transformant into which the mutant acetoacetyl-CoA reductase according to this disclosure has been introduced can produce copolymerized PHA with a high proportion of 3HH units. In this case, the proportion of 3HH in the copolymerized PHA is not particularly limited, but may be, for example, 1 mol% to 30 mol%. The lower limit may be 3 mol% or more, 5 mol% or more, 8 mol% or more, 10 mol% or more, or 12 mol% or more. The upper limit may be 25 mol% or less, or 20 mol% or less.

[0060] The monomer composition of the copolymerized PHA produced can be appropriately selected depending on the type of PHA synthase gene possessed by the microorganism used or separately introduced, the type of genes in the metabolic pathway involved in PHA synthesis, the carbon source used for cultivation, and other culture conditions.

[0061] The 3HH ratio (mol%) in the produced copolymer PHA can be analyzed, for example, by gas chromatography or nuclear magnetic resonance spectroscopy.

[0062] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to these items. [Item 1] A mutant acetoacetyl CoA reductase having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and comprising an amino acid sequence having one or both of the following mutations (a) to (b). Mutation (a): A mutation in which the 95th valine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than valine. Mutation (b): A mutation in which the 202nd 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 acetoacetyl CoA reductase according to Item 1, having mutation (a) and the amino acid other than valine being leucine. [Item 3] The mutant acetoacetyl CoA reductase according to Item 1, having mutation (b) and the amino acid other than isoleucine being valine. [Item 4] A mutant acetoacetyl-CoA reductase according to Item 1, having both mutation (a) and mutation (b), wherein the amino acid other than valine in mutation (a) is leucine, and the amino acid other than isoleucine in mutation (b) is valine. [Item 5] A gene encoding the mutant acetoacetyl-CoA reductase according to any one of Items 1 to 4. [Item 6] A transformant having the gene according to Item 5. [Item 7] A transformant according to Item 6, wherein the host is a bacterium. [Item 8] A transformant according to Item 7, wherein the bacterium is a bacterium belonging to the genus Capriavidus. [Item 9] A method for producing polyhydroxyalkanoic acid, comprising the steps of culturing the transformant according to any one of Items 6 to 8 in the presence of a carbon source, and recovering polyhydroxyalkanoic acid from the transformant. [Item 10] A method for producing polyhydroxyalkanoic acid according to Item 9, wherein the polyhydroxyalkanoic acid contains 3-hydroxyhexanoic acid as a monomer unit. [Item 11] The method for producing polyhydroxyalkanoic acid according to Item 10, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0063] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0064] 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.

[0065] 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.

[0066] (Manufacturing Example 1) Preparation of KNK005dZ / dphaJ4a / dphaB1 / dbktB / dA1528 strain To eliminate the influence of (R)-enoyl-CoA hydratase, which is involved in the supply of 3HB monomer and 3HH monomer, a plasmid for disrupting the phaJ4a gene was prepared. The preparation was carried out as follows: A DNA fragment (SEQ ID NO: 14) was prepared by PCR using the genomic DNA of the KNK005dZ strain as a template, by linking the base sequences upstream and downstream of the phaJ4a structural gene. This DNA fragment was digested with the restriction enzyme SmiI, and the obtained DNA fragment was linked with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare the phaJ4a gene disruption plasmid vector pNS2X-sacB+phaJ4aUD.

[0067] Escherichia coli S17-1 strain (ATCC47055) was transformed with the phaJ4a gene disruption plasmid vector pNS2X-sacB+phaJ4aUD. The resulting transformed microorganisms were then mixed cultured with strain KNK005dZ on Nutrient Agar (Difco) medium for conjugation transfer.

[0068] 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 phaJ4a gene on the chromosome was removed was isolated by PCR analysis or sequencing analysis. The obtained strain was named KNK005dZ / dphaJ4a.

[0069] Plasmids for disrupting the phaB1 gene were prepared. The preparation was carried out as follows: A DNA fragment (SEQ ID NO: 15) was prepared by PCR using the genomic DNA of strain KNK005dZ as a template, by ligating the nucleotide sequences upstream and downstream of the phaB1 structural gene. 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 phaB1 gene disruption plasmid vector pNS2X-sacB+phaB1UD.

[0070] Next, using the phaB1 gene disruption plasmid vector pNS2X-sacB+phaB1UD, the phaB1 gene disruption strain KNK005dZ / dphaJ4a / dphaB1 was prepared using the same method as described above, with KNK005dZ / dphaJ4a as the parent strain.

[0071] Plasmids for disrupting the bktB gene were prepared. The preparation was carried out as follows: A DNA fragment (SEQ ID NO: 16) was prepared by linking the nucleotide sequences upstream and downstream of the bktB structural gene using PCR with the genomic DNA of strain KNK005dZ as a template. This DNA fragment was digested with the restriction enzyme SmiI, and the resulting DNA fragment was linked with pNS2X-sacB, which had also been digested with SmiI, using DNA ligase to prepare the bktB gene disruption plasmid vector pNS2X-sacB+bktBUD.

[0072] Next, using the bktB gene disruption plasmid vector pNS2X-sacB+bktBUD, the bktB gene disruption strain KNK005dZ / dphaJ4a / dphaB1 / dbktB was prepared using the same method as described above, with KNK005dZ / dphaJ4a / dphaB1 as the parent strain.

[0073] Plasmids for disrupting the A1528 gene were prepared. The preparation was carried out as follows: A DNA fragment (SEQ ID NO: 17) was prepared by PCR using the genomic DNA of strain KNK005dZ as a template, by ligating the nucleotide sequences upstream and downstream of the A1528 structural gene. 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.

[0074] Next, using the A1528 gene disruption plasmid vector pNS2X-sacB+A1528UD, the A1528 gene disruption strain KNK005dZ / dphaJ4a / dphaB1 / dbktB was used as the parent strain, and the same method as described above was used to create the A1528 gene disruption strain KNK005dZ / dphaJ4a / dphaB1 / dbktB / dA1528.

[0075] (Production Example 2) Preparation of pCUP2-Ptrc-PhaB1 First, a DNA fragment (SEQ ID NO: 18) containing the base sequence encoding the amino acid sequence of PhaB1 shown in SEQ ID NO: 1 was digested with restriction enzymes MunI and SpeI. This DNA fragment was ligated with a pCUP2 vector described in Japanese Patent Application Publication No. 2007-259708, which had been digested with MunI and SpeI, using DNA ligase to prepare the pCUP2-PhaB1 plasmid.

[0076] Next, a DNA fragment (SEQ ID NO: 19) containing the nucleotide sequence of the trc promoter shown in SEQ ID NO: 7 was digested with restriction enzymes MunI and EcoRI. This DNA fragment was ligated with pCUP2-Ptrc-PhaB1 digested with MunI using DNA ligase to create pCUP2-Ptrc-PhaB1, in which the PhaB1 gene sequence was located downstream of the trc promoter. pCUP2-Ptrc-PhaB1 is a plasmid that expresses PhaB1 under the trc promoter.

[0077] (Production Example 3) Preparation of pCUP2-Ptrc-PhaB1-V95L-I202V A DNA fragment (SEQ ID NO: 20) containing the base sequence encoding the amino acid sequence of PhaB1-V95L-I202V was digested with restriction enzymes MunI and SpeI. This DNA fragment was ligated with pCUP2-Ptrc-PhaB1 digested with MunI and SpeI using DNA ligase to create pCUP2-Ptrc-PhaB1-V95L-I202V, in which the PhaB1-V95L-I202V gene sequence is located downstream of the trc promoter. pCUP2-Ptrc-PhaB1-V95L-I202V is a plasmid that expresses PhaB1-V95L-I202V under the trc promoter.

[0078] PhaB1-V95L-I202V is a mutant acetoacetyl CoA reductase in which the amino acid sequence shown in Sequence ID No. 1 has been modified in which the 95th valine from the N-terminus is replaced with leucine, and the 202nd isoleucine is replaced with valine.

[0079] (Production Example 4) Introduction of each plasmid into strain KNK005dZ / dphaJ4a / dphaB1 / dbktB / dA1528 First, strain KNK005dZ / dphaJ4a / dphaB1 / dbktB / dA1528 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 the resulting cells were suspended in 2 mL of distilled water. The cell suspension was mixed with the pCUP2 vector or the plasmid solutions prepared in Production Examples 2-3, injected into a cuvette, and electroporation was performed. Electroporation was performed using a MicroPulser electroporator (Bio-Rad) under the conditions of voltage 1.5 kV, resistance 800 Ω, and current 25 μF. After electroporation, the bacterial cell solution was collected and 5 mL of Nutrient Broth medium was added, and it was incubated at 30°C for 3 hours. The resulting culture 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.

[0080] (Comparative Example 1) PHA production by KNK005dZ / dphaJ4a / dphaB1 / dbktB / dA1528 / pCUP2 strain A strain into which the pCUP2 vector was introduced was cultured under the following conditions. The composition of the parent 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, pH 6.8.

[0081] 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).

[0082] 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 to obtain the culture solution, which was used as the seed stock. Next, 0.5 ml of the seed stock 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 carried out for 72 hours. After the culture was completed, the bacterial cells were collected from the culture solution, washed with ethanol, and vacuum dried. The dry cell weight (g / L) was measured and is shown in Table 1.

[0083] (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) and 3HH (6 carbon atoms), and the ratio of each monomer was calculated. In addition, the weight 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 PHA weight (g / L), PHA content in dried bacterial cells (wt%), and monomer ratio (mol%), are shown in Table 1.

[0084] (Comparative Example 2) A strain into which the PHA-producing plasmid pCUP2-Ptrc-PhaB1 was introduced using the KNK005dZ / dphaJ4a / dphaB1 / dbktB / dA1528 / pCUP2-Ptrc-PhaB1 strain was cultured in the same manner as in Comparative Example 1. The dry cell weight, PHA weight, PHA content in the dry cells, and monomer ratio were calculated in the same manner as in Comparative Example 1, and the results are shown in Table 1.

[0085] (Example 1) A strain into which the PHA-producing plasmid pCUP2-Ptrc-PhaB1-V95L-I202V was introduced was cultured in the same manner as in Comparative Example 1. The dry cell weight, PHA weight, PHA content in the dry cells, and monomer ratio were calculated in the same manner as in Comparative Example 1, and the results are shown in Table 1.

[0086]

[0087] <Discussion> From the results in Table 1, the bacterial strain of Example 1, in which a gene encoding a mutant acetoacetyl CoA reductase having the V95L and I202V mutations was introduced to the wild-type acetoacetyl CoA reductase having the amino acid sequence shown in Sequence ID No. 1, produced PHA at the same rate as the bacterial strain of Comparative Example 2, in which a gene encoding wild-type acetoacetyl CoA reductase without these mutations was introduced, and an increase in the 3HH ratio contained in the PHA was observed.

[0088] These results indicate that mutant acetoacetyl-CoA reductase having a mutation at the 95th or 202nd amino acid from the N-terminus of the amino acid sequence shown in Sequence ID No. 1 is useful for producing PHA copolymers with a high 3HH ratio while maintaining PHA productivity.

Claims

1. A mutant acetoacetyl-CoA reductase 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 both of the following mutations (a) to (b). Mutation (a): A mutation in which the 95th valine from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with an amino acid other than valine. Mutation (b): A mutation in which the 202nd 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 acetoacetyl-CoA reductase according to claim 1, having mutation (a), wherein the amino acid other than valine is leucine.

3. The mutant acetoacetyl-CoA reductase according to claim 1, having mutation (b), wherein the amino acid other than isoleucine is valine.

4. The mutant acetoacetyl-CoA reductase according to claim 1, having both mutation (a) and mutation (b), wherein the amino acid other than valine in mutation (a) is leucine, and the amino acid other than isoleucine in mutation (b) is valine.

5. A gene encoding a mutant acetoacetyl-CoA reductase according to any one of claims 1 to 4.

6. A transformant having the gene described in claim 5.

7. The transformant according to claim 6, wherein the host is a bacterium.

8. The transformant according to claim 7, wherein the eubacterium is a bacterium belonging to the genus Capriavidus.

9. A method for producing polyhydroxyalkanoic acid, comprising the steps of culturing the transformant described in claim 6 in the presence of a carbon source, and recovering polyhydroxyalkanoic acid from the transformant.

10. The method for producing polyhydroxyalkanoic acid according to claim 9, wherein the polyhydroxyalkanoic acid contains 3-hydroxyhexanoic acid as a monomer unit.

11. The method for producing polyhydroxyalkanoic acid according to claim 10, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

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