Transformed microorganism, and production method of polyhydroxyalkanoic acid

Abstract

Provided is a transformed mesophilic microorganism which has a polyhydroxyalkanoic acid synthase gene and into which a phaA gene derived from a thermophilic microorganism and / or a phaB gene derived from a thermophilic microorganism have been introduced. The transformed mesophilic microorganism may belong to the genus Cupriavidus. A polyhydroxyalkanoic acid can be produced by culturing the transformed mesophilic microorganism in the presence of a carbon source.

Description

Transformed microorganism and method for producing polyhydroxyalkanoic acid

[0001] The present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.

[0002] Against the backdrop of growing awareness of environmental issues, food issues, health and safety, and a growing preference for natural or natural products, the significance and importance of substance production using microorganisms (fermentation production, bioconversion, etc.) is increasing, and substance production using microorganisms is also being applied to the production of protein pharmaceuticals, nucleic acids for gene therapy, etc. For example, the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria is being actively applied industrially.

[0003] One example is the production of polyhydroxyalkanoic acid (hereinafter also referred to as PHA) by microorganisms (see Non-Patent Document 1). PHA is a thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species, and is biodegradable. Currently, with growing environmental awareness, non-petroleum-derived plastic alternatives are attracting attention, and PHA produced and accumulated intracellularly by microorganisms is expected to have a small adverse impact on the ecosystem, as it is incorporated into the natural carbon cycle process. In PHA production using microorganisms, for example, it is known that PHA is produced by feeding sugars, vegetable oils, or fatty acids as carbon sources to Capriavidus bacteria, allowing PHA to accumulate intracellularly (see Non-Patent Documents 2 and 3).

[0004] However, the cost of producing PHA using microorganisms can be higher than the cost of producing general-purpose petroleum-derived plastics. Improving productivity and reducing production costs are key challenges for the widespread use of PHA.

[0005] Anderson AJ. , et al. , Int. J. Biol. Macromol. , 12, 201-105 (1990) Sato S. , et al. , J. Biosci. Bioeng. , 120(3), 246-251 (2015) Insomphun C. , et al. , Metab. Eng. , 27, 38-45 (2015)

[0006] As described above, improving the productivity of polyhydroxyalkanoic acid is an important industrial issue since it will greatly contribute to reducing production costs.

[0007] In view of the above-mentioned current situation, an object of the present invention is to provide a transformed microorganism with improved productivity of polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid by culturing the microorganism.

[0008] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that the productivity of polyhydroxyalkanoic acid can be improved by introducing a gene encoding β-ketothiolase (PhaA) derived from a thermophilic microorganism and / or a gene encoding acetoacetyl-CoA reductase (PhaB) derived from a thermophilic microorganism into a mesophilic microorganism capable of producing polyhydroxyalkanoic acid, and thus completed the present invention.

[0009] Specifically, the present invention relates to a transformed mesophilic microorganism having a polyhydroxyalkanoate synthase gene and into which a phaA gene derived from a thermophilic microorganism and / or a phaB gene derived from a thermophilic microorganism have been introduced. The present invention also relates to a method for producing polyhydroxyalkanoate, which comprises culturing the transformed mesophilic microorganism in the presence of a carbon source.

[0010] According to the present invention, it is possible to provide a transformed microorganism with improved productivity of polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid by culturing the microorganism. According to the present invention, it is possible to improve the efficiency of polyhydroxyalkanoic acid production by a microorganism and reduce the production cost of polyhydroxyalkanoic acid.

[0011] The present disclosure relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid (hereinafter also referred to as PHA), and a method for producing PHA by culturing the transformed microorganism.

[0012] The transformed microorganism according to the present disclosure may be a transformed microorganism whose host is a mesophilic microorganism, which has a PHA synthase gene and into which a gene encoding β-ketothiolase (PhaA) derived from a thermophilic microorganism has been introduced. Alternatively, the transformed microorganism may be a transformed microorganism whose host is a mesophilic microorganism, which has a PHA synthase gene and into which a gene encoding acetoacetyl-CoA reductase (PhaB) derived from a thermophilic microorganism has been introduced. Alternatively, the transformed microorganism may be a transformed microorganism whose host is a mesophilic microorganism, which has a PHA synthase gene and into which a gene encoding PhaA derived from a thermophilic microorganism and a gene encoding PhaB derived from a thermophilic microorganism have been introduced.

[0013] In this disclosure, mesophilic microorganisms refer to microorganisms whose optimum growth temperature is within the range of 25 to 40°C, and thermophilic microorganisms refer to microorganisms that can grow at temperatures of 50°C or higher.

[0014] The host of the transformed microorganism according to the present disclosure may be a wild-type strain inherently containing a PHA synthase gene, a mutant strain obtained by artificially mutating such a wild-type strain, or a transformed strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques. The method for introducing the exogenous gene is not particularly limited, and may include directly inserting or substituting the gene onto the host's chromosome, directly inserting or substituting the gene onto a megaplasmid possessed by the host, or placing the gene onto a vector such as a plasmid, phage, or phagemid for introduction. Two or more of these methods may also be used in combination. Considering the stability of the introduced gene, a method in which the gene is directly inserted or substituting onto the host's chromosome or onto a megaplasmid possessed by the host is preferred, and a method in which the gene is directly inserted or substituting onto the host's chromosome is more preferred.

[0015] The host of the transformed microorganism according to the present disclosure is not particularly limited as long as it is a mesophilic microorganism, and may be a microorganism that originally contains a PHA synthase gene, a gene encoding β-ketothiolase (PhaA), and a gene encoding acetoacetyl-CoA reductase (PhaB).

[0016] Examples of hosts for transformed microorganisms according to the present disclosure include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, etc., which have an optimal growth temperature within the range of 25 to 40°C. From the viewpoints of safety and PHA productivity, preferred are bacteria belonging to the genus Ralstonia, Cupriavidus, or Wautersia, with an optimum growth temperature within a range of 25 to 40°C, more preferred are bacteria belonging to the genus Cupriavidus, with an optimum growth temperature within a range of 25 to 40°C, and particularly preferred is Cupriavidus necator.

[0017] (PHA) The type of PHA produced by the transformed microorganism according to the present disclosure is not particularly limited as long as it is a PHA that can be produced by a microorganism. However, preferred examples include a homopolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, a copolymer of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid (e.g., 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and another hydroxyalkanoic acid.

[0018] Particularly preferred PHAs are homopolymers of 3-hydroxyalkanoic acids having 4 carbon atoms, or copolymers containing 3-hydroxyalkanoic acids having 4 carbon atoms. Examples include P(3HB), which is a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV), a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), P(3HB-co-3HH) (abbreviation: P3HB3HH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), P(3HB-co-4HB), and PHAs containing lactic acid (abbreviation: LA) as a constituent, such as P(LA-co-3HB), from the viewpoint of a wide range of applications as a polymer. However, these are not limited to these.

[0019] The type of PHA to be produced can be appropriately selected depending on the purpose, such as the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic gene involved in the synthesis, and the culture conditions.

[0020] (PHA synthase gene) The PHA synthase (PhaC) gene possessed by the transformed microorganism according to the present disclosure may be one inherent to the host or may be exogenous. Examples of PHA synthase genes include, but are not limited to, PHA synthase genes derived from Aeromonas charae, Aeromonas hydrophila, Pseudomonas SP 61-3, or Capriavidus necator; chimeric PHA synthase genes combining two or more of the above PHA synthase genes; and genes encoding proteins consisting of amino acid sequences showing 90% or more sequence identity to the amino acid sequences of the above-mentioned PHA synthases. The sequence identity is preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more. The number of PHA synthase genes possessed by the transformed microorganism according to the present disclosure may be one or more. Furthermore, when multiple PHA synthase genes are possessed, they may be the same gene or different genes.

[0021] (phaA gene and phaB gene) The phaA gene and phaB gene encode β-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB), respectively. These genes are possessed by many microorganisms that can naturally accumulate PHA, and are involved in the biosynthesis of 3HB-CoA, the most common PHA biosynthetic substrate. Specifically, PhaA is involved in catalyzing the reaction that produces acetoacetyl-CoA by condensing two molecules of acetyl-CoA, and PhaB is involved in catalyzing the reaction that produces 3HB-CoA by reducing acetoacetyl-CoA.

[0022] The transformed microorganism according to the present disclosure has a mesophilic host into which a phaA gene derived from a thermophilic microorganism and / or a phaB gene derived from a thermophilic microorganism have been introduced. The introduction of these genes can increase the productivity of PHA by the transformed microorganism. The transformed microorganism can produce PHA with good productivity even under high stress, for example, at a relatively high culture temperature.

[0023] In the present disclosure, only the phaA gene derived from a thermophilic microorganism may be introduced, only the phaB gene derived from a thermophilic microorganism may be introduced, or both genes may be introduced. However, it is preferable to introduce at least the phaA gene derived from a thermophilic microorganism, and it is particularly preferable to introduce both the phaA gene derived from a thermophilic microorganism and the phaB gene derived from a thermophilic microorganism.

[0024] When the transformed microorganism according to the present disclosure is one into which only the phaA gene derived from a thermophilic microorganism has been introduced, it is preferable that the transformed microorganism also has the phaB gene that the host originally has. In this case, the transformed microorganism may or may not also have the phaA gene that the host originally has.

[0025] When the transformed microorganism according to the present disclosure is one into which only the phaB gene derived from a thermophilic microorganism has been introduced, it is preferable that the transformed microorganism has the phaA gene that the host originally has. In this case, the transformed microorganism may or may not further have the phaB gene that the host originally has.

[0026] When the transformed microorganism according to the present disclosure has both the phaA gene derived from a thermophilic microorganism and the phaB gene derived from a thermophilic microorganism, the transformed microorganism does not need to have the phaA gene and the phaB gene originally possessed by the host. However, since this further improves PHA productivity, it is preferable that the transformed microorganism also has the phaA gene and the phaB gene originally possessed by the host.

[0027] The thermophilic microorganism is not particularly limited as long as it is a microorganism that can grow at 50°C or higher and has the phaA gene and / or the phaB gene. Examples of such microorganisms include the genera Cupriavidus, Caldimonas, Schlegelella, Thermus, Chelatococcus, Bacillus, Aneurinibacillus, Pseudomonas, Synechococcus, and Spirulina. Among these, the genera Cupriavidus, Caldimonas, and Schlegelella are preferred.

[0028] Examples of genes encoding β-ketothiolase (PhaA) derived from thermophilic microorganisms include, but are not limited to, a gene derived from Cupriavidus sp. strain S-6 that encodes PhaA having the amino acid sequence set forth in SEQ ID NO: 1, a gene derived from Caldimonas manganoxidans that encodes PhaA having the amino acid sequence set forth in SEQ ID NO: 2, a gene derived from Schlegelella thermodepolymerans that encodes PhaA having the amino acid sequence set forth in SEQ ID NO: 3, and genes having an amino acid sequence that shows 90% or more sequence identity to these amino acid sequences and having a nucleotide sequence that encodes a protein that exhibits β-ketothiolase activity.

[0029] The sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more. The sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 3 is also preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0030] Examples of genes encoding acetoacetyl-CoA reductase (PhaB) derived from thermophilic microorganisms include, but are not limited to, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 4 derived from Cupriavidus sp. strain S-6, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 5 derived from Caldimonas manganoxidans, a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 6 derived from Schlegelella thermodepolymerans, and genes having an amino acid sequence showing 90% or more sequence identity to these amino acid sequences and having a nucleotide sequence encoding a protein exhibiting acetoacetyl-CoA reductase activity.

[0031] The sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 is preferably 90% 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. The sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or 6 is also preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0032] The method for introducing a target gene into a host is not particularly limited, and may include directly inserting or substituting the target gene onto the chromosome of the host, directly inserting or substituting the target gene onto a megaplasmid carried by the host, or placing the target gene on a vector such as a plasmid, phage, or phagemid and introducing it, or two or more of these methods may be used in combination.

[0033] Considering the stability of the introduced gene, a method of directly inserting or substituting the gene of interest onto the host chromosome or onto a megaplasmid carried by the host is preferred, and a method of directly inserting or substituting the gene of interest onto the host chromosome is even more preferred.

[0034] To ensure the 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" that the host originally has, or so that it is located downstream of an exogenous "gene expression regulatory sequence." In the present disclosure, a "gene expression regulatory sequence" is a DNA sequence that includes a base sequence (e.g., a promoter sequence) that controls the transcription level of the gene and / or a base sequence (e.g., a Shine-Dalgarno sequence) that controls the translation level of messenger RNA transcribed from the gene. As a "gene expression regulatory sequence," any base sequence that exists in nature can be used, or an artificially constructed or modified base sequence can be used.

[0035] Examples of promoter sequences and Shine-Dalgarno sequences contained in the "gene expression regulatory sequence" include, but are not limited to, the base sequences shown in any of SEQ ID NOs: 7 to 14, or base sequences containing parts of these base sequences.

[0036] Substitution, deletion, insertion, and / or addition of at least a portion of genomic DNA can be performed by methods well known to those skilled in the art, such as a method utilizing transposons and the mechanism of homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)) and a method based on the principle of site-specific integration caused by the mechanism of homologous recombination and subsequent loss by a second step of homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)). Alternatively, a method can be used in which the sacB gene from Bacillus subtilis is coexisted, and a microbial strain in which the gene has been lost by second-stage homologous recombination is easily isolated as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195-1204 (1992), Lenz et al., J. Bacteriol., 176:4385-4393 (1994)). As another method, genome editing technology using the CRISPR / Cas9 system to modify target DNA (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732) can also be used. In the CRISPR / Cas9 system, the guide RNA (gRNA) has a sequence that can bind to a portion of the genomic DNA sequence to be modified and serves to transport Cas9 to the target.

[0037] The method for introducing a vector into a cell is not particularly limited, and examples thereof include the calcium chloride method, electroporation, polyethylene glycol method, and spheroplast method.

[0038] (Production of PHA) PHA can be accumulated in the cells by culturing the transformed microorganism according to the present disclosure. The method for culturing the transformed microorganism according to the present disclosure can be a conventional microbial culture method, and the culture can be carried out in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, method of adding the carbon source, culture scale, aeration and agitation conditions, culture temperature, culture time, etc. The carbon source is preferably added to the medium continuously or intermittently.

[0039] The transformed microorganism according to the present disclosure can produce PHA with good productivity even at relatively high culture temperatures, for example, even when cultured at temperatures of 35°C or higher.

[0040] Any carbon source can be used as a carbon source during cultivation as long as it can be assimilated by the transformed microorganism of the present disclosure. Examples include, but are not limited to, sugars such as glucose, fructose, and sucrose; 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 myristic acid, as well as their derivatives, and glycerol. Some or all of the oils and fats may be degraded oils. Degraded oils refer to oils and fats that have been thermally denatured or altered by reaction with oxygen and / or water under heat. The term "degraded oil" is not limited, and includes those commonly referred to as waste oil, discarded oil, waste cooking oil, waste vegetable oil, and used oil. Furthermore, when the transformed microorganism according to the present disclosure is capable of utilizing gases or alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources.

[0041] In the production of PHA according to the present disclosure, it is preferable to culture the microorganism using a medium containing the carbon source, a nitrogen source as a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. 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.

[0042] After culturing for an appropriate time to accumulate PHA in the cells, PHA can be recovered from the cells using a known method. The recovery method is not particularly limited, but for example, after the end of the culture, the cells are separated from the culture solution using a centrifuge or a separation membrane, etc., and dried, and then PHA is extracted from the dried cells using an organic solvent such as chloroform, and cellular components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA, and the supernatant is removed by filtration or centrifugation, and the PHA can be recovered by drying. Alternatively, cellular components other than PHA can be dissolved in water using a surfactant, alkali, enzyme, etc., and then PHA particles can be separated from the aqueous phase by filtration or centrifugation, dried, and recovered.

[0043] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to them. [Item 1] A transformed mesophilic microorganism having a polyhydroxyalkanoic acid synthase gene, into which a phaA gene derived from a thermophilic microorganism and / or a phaB gene derived from a thermophilic microorganism have been introduced. [Item 2] The transformed mesophilic microorganism according to Item 1 or 2, wherein the phaA gene derived from the thermophilic microorganism is a gene encoding an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [Item 3] The transformed mesophilic microorganism according to Item 1 or 2, wherein the phaB gene derived from the thermophilic microorganism is a gene encoding an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. [Item 4] The transformed mesophilic microorganism according to any one of Items 1 to 3, wherein the transformed mesophilic microorganism belongs to the genus Capriavidus. [Item 5] The transformed mesophilic microorganism according to Item 4, which is a transformed microorganism of Capriavidus necator. [Item 6] A method for producing polyhydroxyalkanoic acid, comprising a step of culturing the transformed mesophilic microorganism according to any one of Items 1 to 5 in the presence of a carbon source. [Item 7] A method for producing polyhydroxyalkanoic acid according to Item 6, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid. [Item 8] A method for producing polyhydroxyalkanoic acid according to Item 7, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyric acid as a monomer unit. [Item 9] A method for producing polyhydroxyalkanoic acid according to Item 7 or 8, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

[0044] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. The overall genetic manipulation can be carried out as described in, for example, Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, and the like used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. The enzymes used are not particularly limited as long as they can be used in genetic manipulation.

[0045] The KNK005 trc-phaJ4b / ΔphaZ1,2,6 strain used in this example (hereinafter sometimes referred to as "KNK005dZ / trc-J4b strain") is a strain in which the phaZ1 gene, phaZ2 gene, and phaZ6 gene on the chromosome of the Capriavidus necator H16 strain have been deleted, the PHA synthase gene on the chromosome has been replaced with a modified version of the PHA synthase gene derived from the genus Aeromonas (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 15, i.e., the N149S / D171G mutant (NSDG) gene), and expression of the R-isomer-specific enoyl-CoA hydratase gene on the chromosome (phaJ4b gene) has been enhanced, and can be prepared in accordance with the method described in PCT Publication WO 2015 / 115619.

[0046] (Microbial Strain Preparation Example 1) Preparation of a Strain Introduced with the phaA Gene Derived from a Thermophilic Microorganism First, a plasmid for expressing the phaA gene (phaAsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6 was prepared. This was done as follows. PCR using synthetic oligo DNA was used to obtain a DNA fragment (SEQ ID NO: 16) containing the lacN17 promoter, a modified Escherichia coli lac promoter. This DNA fragment was digested with the restriction enzymes EcoRI and MunI, and the resulting DNA fragment was ligated to a plasmid vector pCUP2 described in WO 2007 / 049716 that had been cleaved with MunI. The DNA fragment ligated in an orientation such that the SpeI restriction enzyme recognition sequence of pCUP2 was located downstream of the lacN17 promoter was selected, yielding pCUP2-lacN17. Next, PCR using synthetic oligo DNA was performed to obtain a DNA fragment (SEQ ID NO: 17) having the nucleotide sequence of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 1. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain the phaAsp expression plasmid pCUP2-lacN17-phaAsp.

[0047] Next, the phaAsp expression plasmid pCUP2-lacN17-phaAsp was introduced into the KNK005dZ / trc-J4b strain, and the resulting strain was designated KNK005dZ / trc-J4b / pCUP2-lacN17-phaAsp (hereinafter, also referred to as the "thermophilic microorganism-derived phaA gene-introduced strain").

[0048] Introduction of the plasmid vector into cells was carried out by electroporation as follows. A Biorad Gene Pulser was used as the gene introduction device, and a Biorad gap 0.2 cm cuvette was used. 400 μl of competent cells and 20 μl of expression vector were injected into the cuvette and set in the pulse device, and an electric pulse was applied under the conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance of 800 Ω. After pulsing, the bacterial solution in the cuvette was cultured with shaking at 30 ° C. for 3 hours in Nutrient Broth medium (DIFCO), and then cultured on a selection plate (Nutrient Agar medium (DIFCO), kanamycin 100 mg / L) at 30 ° C. for 2 days to obtain the grown thermophilic microorganism-derived phaA gene-introduced strain.

[0049] (Microbial Strain Preparation Example 2) Preparation of a Strain Introduced with the phaB Gene Derived from a Thermophilic Microorganism First, a plasmid for expressing the phaB gene (phaBsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6 was prepared. This was done as follows. PCR using synthetic oligo DNA yielded a DNA fragment (SEQ ID NO: 18) having the nucleotide sequence of a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 4. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain the phaBsp expression plasmid pCUP2-lacN17-phaBsp.

[0050] Next, the phaBsp expression plasmid pCUP2-lacN17-phaBsp was introduced into the KNK005dZ / trc-J4b strain by electroporation, and the resulting strain was designated KNK005dZ / trc-J4b / pCUP2-lacN17-phaBsp strain (hereinafter, also referred to as the "thermophilic microorganism-derived phaB gene-introduced strain").

[0051] (Microbial Strain Preparation Example 3) Preparation of a Strain Introduced with the phaAB Genes Derived from a Thermophilic Microorganism First, a plasmid for expressing the phaA gene (phaAsp) and the phaB gene (phaBsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6 was prepared. The preparation was carried out as follows. PCR using synthetic oligo DNA was performed to obtain a DNA fragment (SEQ ID NO: 19) having the nucleotide sequences of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 1 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 4. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated to pCUP2-lacN17 cleaved with MunI and SpeI to obtain a plasmid pCUP2-lacN17-phaABsp for expressing phaAsp and phaBsp.

[0052] Next, the phaAsp and phaBsp expression plasmid pCUP2-lacN17-phaABsp was introduced into the KNK005dZ / trc-J4b strain by electroporation, and the resulting strain was designated KNK005dZ / trc-J4b / pCUP2-lacN17-phaABsp (hereinafter, also referred to as the "thermophilic microorganism-derived phaAB gene-introduced strain").

[0053] (Microbial Strain Preparation Example 4) Preparation of Strain 1 with Chromosomal Substitution of phaAB Genes Derived from a Thermophilic Microorganism First, a plasmid for disrupting the host phaAB genes was prepared. This was done as follows. PCR using synthetic oligo DNA was used to obtain a DNA fragment (SEQ ID NO: 20) containing the base sequence upstream of the phaA structural gene and downstream of the phaB structural gene of the KNK005dZ / trc-J4b strain. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated with the vector pNS2X-sacB described in JP 2007-259708 A, which had also been digested with SwaI, using DNA ligase (Ligation High (Toyobo Co., Ltd.)) to prepare the plasmid vector pNS2X-sacB+phaABUD for disrupting the host phaAB genes.

[0054] Next, a host phaAB gene-disrupted strain was prepared using the host phaAB gene-disruption plasmid vector pNS2X-sacB+phaABUD as follows: Escherichia coli S17-1 strain (ATCC47055) was transformed with the host phaAB gene-disruption plasmid vector pNS2X-sacB+phaABUD, and the resulting transformed microorganism was mixed-cultured with KNK005dZ / trc-J4b strain on Nutrient Agar medium (Difco) for conjugative transfer. The resulting culture medium was inoculated onto Simmons agar medium containing 250 mg / L 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 the strains that grew on the agar medium were selected to obtain a strain in which the plasmid was integrated onto the chromosome of the KNK005dZ / trc-J4b strain. This strain was cultured for two generations in Nutrient Broth medium (manufactured by Difco), and then diluted and spread onto Nutrient Agar medium containing 15% sucrose. The grown strain was obtained as a strain from which the plasmid had been lost. Furthermore, one strain in which the phaAB genes on the chromosome were deleted was isolated by PCR and DNA sequencer analysis. This gene-disrupted strain was designated KNK005dZ / trc-J4b / dphaAB strain.

[0055] Furthermore, a plasmid for introducing the phaA gene (phaAsp) and phaB gene (phaBsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6 was constructed as follows. PCR using synthetic oligo DNA was performed to obtain a DNA fragment (SEQ ID NO: 21) having the nucleotide sequences upstream of the phaA structural gene and downstream of the phaB structural gene of the KNK005dZ / trc-J4b strain, as well as the nucleotide sequences of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 1 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 4. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated to the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)) to prepare the plasmid vector pNS2X-sacB+phaABsp for introducing the phaA gene (phaAsp) and phaB gene (phaBsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6.

[0056] Next, the plasmid vector pNS2X-sacB+phaABsp for introducing the phaA gene (phaAsp) and phaB gene (phaBsp) derived from the thermophilic microorganism Cupriavidus sp. strain S-6 was introduced into the KNK005dZ / trc-J4b / dphaAB strain by the same conjugal transfer method as above. Furthermore, by culturing in the same manner as above and selecting using Nutrient Agar medium containing 15% sucrose, one strain in which phaAsp and phaBsp were introduced into the chromosomal position where the phaA and phaB genes of the original host were present was isolated. The resulting strain was designated KNK005dZ / trc-J4b / phaAB::phaABsp (hereinafter, also referred to as "thermophilic microorganism-derived phaAB gene chromosome substitution strain 1").

[0057] (Microbial Strain Preparation Example 5) Preparation of Strain 2 with Chromosomal Substitution of phaAB Genes Derived from a Thermophilic Microorganism First, a plasmid for introducing the phaA gene (phaAcm) and phaB gene (phaBcm) derived from the thermophilic microorganism Caldimonas manganoxidans was prepared. The preparation was carried out as follows. PCR using synthetic oligo-DNA was performed to obtain a DNA fragment (SEQ ID NO: 22) having the nucleotide sequences upstream of the phaA structural gene and downstream of the phaB structural gene of the KNK005dZ / trc-J4b strain, as well as the nucleotide sequences of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 2 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 5. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated to the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), to prepare a plasmid vector pNS2X-sacB+phaABcm for introducing the phaA gene (phaAcm) and phaB gene (phaBcm) derived from the thermophilic microorganism Caldimonas manganoxidans.

[0058] Next, the plasmid vector pNS2X-sacB+phaABcm for introducing the phaA gene (phaAcm) and phaB gene (phaBcm) derived from the thermophilic microorganism Caldimonas manganoxidans was introduced into the KNK005dZ / trc-J4b / dphaAB strain by the same conjugation transfer method as above. Furthermore, by culturing in the same manner as above and selecting using Nutrient Agar medium containing 15% sucrose, one strain was isolated in which phaAcm and phaBcm were introduced at the position on the chromosome where the original host had the phaA and phaB genes. The resulting strain was named KNK005dZ / trc-J4b / phaAB::phaABcm strain (hereinafter, sometimes referred to as "thermophilic microorganism-derived phaAB gene chromosome replacement strain 2").

[0059] (Microbial Strain Preparation Example 6) Preparation of Strain 3 with Chromosomal Substitution of phaAB Genes Derived from a Thermophilic Microorganism First, a plasmid for introducing the phaA gene (phaAst) and phaB gene (phaBst) derived from the thermophilic microorganism Schlegelella thermodepolymerans was prepared. The preparation was carried out as follows. PCR using synthetic oligo-DNA was performed to obtain a DNA fragment (SEQ ID NO: 23) having the nucleotide sequences upstream of the phaA structural gene and downstream of the phaB structural gene of the KNK005dZ / trc-J4b strain, as well as the nucleotide sequences of a gene encoding PhaA having the amino acid sequence set forth in SEQ ID NO: 3 and a gene encoding PhaB having the amino acid sequence set forth in SEQ ID NO: 6. This DNA fragment was digested with the restriction enzyme SwaI, and the resulting DNA fragment was ligated to the vector pNS2X-sacB described in JP-A-2007-259708, which had also been digested with SwaI, using DNA ligase (Ligation High (manufactured by Toyobo Co., Ltd.)), to prepare a plasmid vector pNS2X-sacB+phaABst for introducing the phaA gene (phaAst) and phaB gene (phaBst) derived from the thermophilic microorganism Schlegelella thermodepolymerans.

[0060] Next, the plasmid vector pNS2X-sacB+phaABst for introducing the phaA gene (phaAst) and phaB gene (phaBst) derived from the thermophilic microorganism Schlegelella thermodepolymerans was introduced into the KNK005dZ / trc-J4b / dphaAB strain by the same conjugal transfer method as above. Furthermore, by culturing in the same manner as above and selecting using Nutrient Agar medium containing 15% sucrose, one strain was isolated in which phaAst and phaBst had been introduced into the chromosomal position where the phaA and phaB genes of the original host were located. The resulting strain was designated KNK005dZ / trc-J4b / phaAB::phaABst (hereinafter, sometimes referred to as "thermophilic microorganism-derived phaAB gene chromosomal replacement strain 3").

[0061] (Comparative Example 1) PHA production by KNK005dZ / trc-J4b strain Cultivation studies were carried out using the KNK005dZ / trc-J4b strain under the following conditions.

[0062] (Culture medium) The composition of the seed culture medium is 1 w / v% Meat extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast extract, 0.9 w / v% Na ２ HPO ４ ・12H ２ O, 0.15w / v% KH ２ P.O. ４ , (pH 6.8).

[0063] The composition of the pre-culture medium was 1.1 w / v% Na ２ HPO ４ ・12H ２ O, 0.19w / v%KH ２ P.O. ４ , 1.29w / v% (NH ４ ) ２ SO ４ , 0.1w / v% MgSO ４ ・7H ２ O, 2.5 w / v% palm olein oil, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid ３ ・6H ２ O, 1w / v% CaCl ２ ・2H ２ O, 0.02w / v% CoCl ２ ・6H ２ O, 0.016w / v% CuSO ４ ・5H ２ O, 0.012w / v% NiCl ２ ・6H ２ O was dissolved).

[0064] The composition of the PHA production medium was 0.385 w / v% Na ２ HPO ４ ・12H ２ O, 0.067w / v% KH ２ P.O. ４ , 0.291w / v% (NH ４ ) ２ SO ４ , 0.1w / v% MgSO４ ・7H ２ 0, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl in 0.1 N hydrochloric acid) ３ ・6H ２ O, 1w / v% CaCl ２ ・2H ２ O, 0.02w / v% CoCl ２ ・6H ２ O, 0.016w / v% CuSO ４ ・5H ２ O, 0.012w / v% NiCl ２ ・6H ２ O was dissolved).

[0065] (Method for measuring PHA production amount) The PHA production amount was measured as follows. The cells were collected from the culture medium by centrifugation, washed with ethanol, and freeze-dried to obtain dried cells. The weight was measured to calculate the dry cell concentration in the culture medium. 100 ml of chloroform was added to 1 g of the obtained dried cells, and the mixture was stirred at room temperature for 24 hours to extract the PHA within the cells. The cell residue was filtered and then concentrated in an evaporator to a total volume of 30 ml. 90 ml of hexane was then gradually added, and the mixture was left to stand for 1 hour with slow stirring. The precipitated PHA was filtered and then vacuum-dried at 50°C for 3 hours. The weight of the dried PHA obtained from 1 g of dried cells was measured, and the PHA production amount per culture medium was calculated.

[0066] (PHA production culture) PHA production culture was carried out as follows. First, a glycerol stock (50 μl) of the KNK005dZ / trc-J4b strain was inoculated into a seed medium (10 ml) and cultured for 24 hours to carry out seed culture. Next, the seed culture solution was inoculated at 1.0 v / v% into a 3 L jar fermenter (MDL-300 model, manufactured by Marubishi Bioengine) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 30°C, an agitation speed of 500 rpm, and an aeration rate of 1.8 L / min, and the culture was continued for 28 hours while controlling the pH between 6.7 and 6.8 to carry out preculture. A 14% aqueous ammonium hydroxide solution was used for pH control.

[0067] Next, the preculture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (MDS-U50 model, manufactured by Marubishi Bioengine) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 36°C, an agitation speed of 420 rpm, and an aeration rate of 2.1 L / min, and the pH was controlled between 6.7 and 6.8. A 25% aqueous solution of ammonium hydroxide was used for pH control. The carbon source was added intermittently. Palm olein oil was used as the carbon source. After 48 hours of culture, the amount of PHA produced per culture solution was measured using the method described above.

[0068] (Examples 1 to 6) Cultivation studies were carried out using each of the strains obtained in Microorganism Production Examples 1 to 6, and the amount of PHA produced was measured under the same conditions as in Comparative Example 1. Table 1 shows the amount of PHA produced by each strain as a relative value to the amount of PHA produced measured in Comparative Example 1, which is set as a reference value.

[0069]

[0070] From Table 1, it was confirmed that the amount of PHA produced by a microorganism was improved by introducing the phaA gene derived from a thermophilic microorganism and / or the phaB gene derived from a thermophilic microorganism.

Claims

1. Possessing the polyhydroxyalkanoate synthase gene, A transformed mesophilic microorganism, which is a transformed microorganism of Capriavidus necator into which a phaA gene derived from a thermophilic microorganism and / or a phaB gene derived from a thermophilic microorganism has been introduced.

2. The transformed mesophilic microorganism according to claim 1, wherein the phaA gene derived from the thermophilic microorganism is a gene encoding an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

3. The transformed mesophilic microorganism according to claim 1 or 2, wherein the phaB gene derived from the thermophilic microorganism is a gene encoding an amino acid sequence showing 90 to 100% sequence identity to the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:

6.

4. A method for producing a polyhydroxyalkanoic acid, comprising culturing the transformed mesophilic microorganism according to claim 1 or 2 in the presence of a carbon source.

5. 5. The method for producing a polyhydroxyalkanoic acid according to claim 4, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids.

6. 6. The method for producing a polyhydroxyalkanoic acid according to claim 5, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxybutyric acid as a monomer unit.

7. 6. The method for producing a polyhydroxyalkanoic acid according to claim 5, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.