Transformed microorganism, and method for producing polyhydroxyalkanoic acid

Abstract

Provided is a transformed microorganism having a foreign polyhydroxyalkanoic acid synthase gene, and reduced expression of the glpV gene. The microorganism may further have enhanced expression of the pyridine nucleotide transhydrogenase gene. Also provided is a method for producing a polyhydroxyalkanoic acid, said method including a step of culturing the transformed 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 that produces polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the microorganism.

[0002] In response to growing global interest in sustainability, including the SDGs, and growing concern about environmental issues such as marine microplastics, efforts to convert existing petroleum-derived non-biodegradable plastics to biodegradable materials are being promoted, primarily in industries such as packaging, food service, biomedicine, and agriculture. Examples of biodegradable materials that have seen active industrial production in recent years include polylactic acid (PLA) and polyhydroxyalkanoic acid (hereinafter also referred to as PHA). Among these, PHA has excellent biodegradability in a wide range of environments and is one of the few biodegradable materials that can biodegrade in seawater, and as such, it is gaining increasing attention as a solution to the marine microplastics problem and other environmental issues.

[0003] PHA is a natural thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species. Generally, PHA is industrially produced by culturing PHA-accumulating microorganisms while supplying them with a nutrient source. Increasing PHA productivity is essential for reducing the cost per batch. One known method for improving PHA productivity is to modify microorganisms by genetic recombination (see, for example, Patent Document 1).

[0004] Patent No. 7071283

[0005] There is a demand for further improvement in PHA productivity by microbial culture from the viewpoint of reducing PHA production costs, etc. An object of the present invention is to provide a polyhydroxyalkanoic acid-producing transformed microorganism capable of producing polyhydroxyalkanoic acid with higher productivity, and a production method for producing polyhydroxyalkanoic acid with higher productivity by culturing the transformed microorganism.

[0006] The present inventors discovered that reducing the expression of the glpV gene in a transformed microorganism harboring an exogenous polyhydroxyalkanoate synthase gene improves the productivity of polyhydroxyalkanoate by the transformed microorganism, leading to the present invention. In addition, they discovered that enhancing the expression of the pyridine nucleotide transhydrogenase gene further improves PHA productivity, leading to the present invention.

[0007] Specifically, the present invention relates to a transformed microorganism having an exogenous polyhydroxyalkanoic acid synthase gene and reduced expression of the glpV gene. The present invention also relates to a method for producing polyhydroxyalkanoic acid, which comprises culturing the transformed microorganism in the presence of a carbon source.

[0008] According to the present invention, there are provided a polyhydroxyalkanoic acid-producing transformed microorganism capable of producing polyhydroxyalkanoic acid with higher productivity, and a production method for producing polyhydroxyalkanoic acid with higher productivity by culturing the transformed microorganism. According to the present invention, the amount of polyhydroxyalkanoic acid produced per culture can be improved, thereby enabling efficient production of polyhydroxyalkanoic acid and reducing production costs.

[0009] Embodiments of the present invention are described in detail below. One aspect of the present invention relates to a transformed microorganism having an exogenous polyhydroxyalkanoic acid (hereinafter also referred to as PHA) synthase gene and reduced expression of the glpV gene. Another aspect relates to a transformed microorganism having an exogenous PHA synthase gene, reduced expression of the glpV gene, and enhanced expression of the pyridine nucleotide transhydrogenase gene.

[0010] (Host) The host for the transformed microorganism according to this embodiment is not particularly limited, but is preferably a bacterium that inherently has the glpV gene. Preferred examples of the bacterium include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Burkholderia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, Bacillus, Azotobacter, Nocardia, Sphingomonas, and Comamonas. From the viewpoints of safety and PHA productivity, bacteria belonging to the genus Ralstonia or Cupriavidus are more preferred, bacteria belonging to the genus Cupriavidus are even more preferred, and Cupriavidus necator is particularly preferred.

[0011] The host of the transformed microorganism according to this embodiment 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 strain into which an exogenous PHA synthase gene has been introduced by genetic engineering techniques. The method for introducing the exogenous PHA synthase 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 introducing the gene by placing it on a vector such as a plasmid, phage, or phagemid. 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. Furthermore, the PHA synthase gene originally possessed by the host does not need to be inactivated or deleted; a host that still retains the PHA synthase gene originally possessed may be used, or a host in which the gene has been inactivated or deleted may be used, or a host in which the gene has been inactivated or deleted and then reintroduced may be used.

[0012] (PHA synthase gene) The transformed microorganism according to this embodiment has an exogenous PHA synthase gene. Examples of such PHA synthase genes include, but are not limited to, PHA synthase genes derived from organisms belonging to the genera Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, as well as variants thereof. Examples of such variants include nucleotide sequences encoding PHA synthases in which one or more amino acid residues have been deleted, added, inserted, or substituted. Examples include genes having a nucleotide sequence encoding a polypeptide represented by the amino acid sequence set forth in any of SEQ ID NOS: 3 to 7, and genes having a nucleotide sequence encoding a polypeptide represented by an amino acid sequence having 90% or more sequence identity to the amino acid sequence and having PHA synthase activity. The sequence identity is preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0013] (PHA) The type of PHA produced by the transformed microorganism according to this embodiment is not particularly limited as long as it is a PHA that can be produced by a microorganism, but preferred are homopolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, copolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and other hydroxyalkanoic acids (e.g., 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and copolymers of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms. Examples of PHAs include, but are not limited to, P(3HB), 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: PHBH), a copolymer of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), P(3HB-co-4HB), a copolymer of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), and PHAs containing lactic acid (abbreviation: LA) as a constituent, such as P(LA-co-3HB), etc. Among these, PHBH is preferred from the viewpoint of its wide range of applications as a polymer. The type of PHA produced can be appropriately selected depending on the purpose, the type of PHA synthase gene possessed by the microorganism used or introduced separately, the type of metabolic system gene involved in its synthesis, the culture conditions, etc.

[0014] (glpV Gene) The transformed microorganism according to this embodiment is transformed so that expression of the glpV gene is reduced, thereby improving the PHA productivity of the transformed microorganism.

[0015] The glpV gene is thought to encode a substrate-binding protein of the glycerol transport system. Its expression product is one of several proteins (GlpP, GlpQ, GlpS, GlpT, GlpV) responsible for transporting substances in and out of cells, called ABC transporters, and is thought to be involved in glycerol transport. GlpV is said to bind to glycerol and assist in its transport. Although little is known about the ABC transporter system for glycerol, the system has been extensively studied. Therefore, researchers can easily deduce that the four proteins primarily involved in glycerol transport are GlpP, GlpQ, GlpS, and GlpT, and that GlpV is a cofactor and not essential for transport. There has been no report on the relationship between the expression level of the glpV gene and PHA productivity.

[0016] In this embodiment, the glpV gene is preferably a gene encoding a polypeptide comprising an amino acid sequence encoded by any of the genes described in Entry: K17321 (https: / / www.genome.jp / entry / K17321) of the KEGG ORTHOLOGY DATABASE, or a gene encoding a polypeptide comprising an amino acid sequence having 90% or more sequence identity to the amino acid sequence.

[0017] Preferably, the glpV gene is a gene having a nucleotide sequence encoding a protein containing an amino acid sequence encoded by any of the genes derived from the genera Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium among the genes listed in Entry K17321 of the KEGG ORTHOLOGY DATABASE, or a homologous protein thereof, more preferably a gene having a nucleotide sequence encoding a protein containing an amino acid sequence encoded by any of the genes derived from the genus Capriavidus, or a homologous protein thereof. Even more preferably, the glpV gene is a gene having a nucleotide sequence encoding a protein containing the amino acid sequence of the H16_A2498 protein of Capriavidus necator H16, or a homologous protein thereof, and particularly preferably a gene having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. The sequence identity is preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0018] (Decreased gene expression) In this embodiment, "decreased gene expression" refers to a state in which the transcription level of the target gene or the expression level of the polypeptide encoded by the target gene is reduced compared to a host that has not been transformed to reduce the expression of the target gene. The amount of reduction is not particularly limited, but may be less than 1-fold, preferably 0.8-fold or less, more preferably 0.5-fold or less, even more preferably 0.3-fold or less, and even more preferably 0.2-fold or less, relative to the expression level in a strain in which the expression of the target gene has not been reduced. The transcription level of the target gene or the expression level of the polypeptide encoded by the target gene may be zero. In addition, gene expression can also be considered to be reduced when the polypeptide encoded by the gene does not exhibit its original function, for example, by modifying the nucleotide sequence of the target gene. Furthermore, the expression of the target gene can also be reduced by genetically modifying a microorganism containing a PHA synthase gene so that it produces a metabolite or protein that inhibits the function of the polypeptide.

[0019] In this embodiment, the method for reducing gene expression is not particularly limited, but examples include a method for deleting a portion or the entirety of a target gene, a method for modifying a "gene expression regulatory sequence" involved in the expression of a target gene, a method for eliminating or reducing the original function of a polypeptide encoded by a target gene by modifying the nucleotide sequence of the target gene, and a method for reducing the stability of transcribed messenger RNA by modifying the nucleotide sequence of the target gene and / or its surrounding nucleotide sequence. The method for modifying the nucleotide sequence is not particularly limited, and can be carried out by substituting, deleting, inserting, and / or adding at least a portion of the nucleotide sequence of the target gene and / or its surrounding nucleotide sequence, and can be carried out by methods well known to those skilled in the art. Furthermore, the expression of a target gene may be reduced without modifying the nucleotide sequence of the target gene and / or its surrounding nucleotide sequence by using antisense RNA, RNA interference (RNAi), CRISPR interference (CRISPRi), or the like on a transformed microorganism having a PHA synthase gene.

[0020] (Pyridine nucleotide transhydrogenase gene) The transformed microorganism according to this embodiment may have enhanced expression of the pyridine nucleotide transhydrogenase gene, which can further improve the PHA productivity of the transformed microorganism.

[0021] Pyridine nucleotide transhydrogenase is an enzyme that catalyzes the following reaction: NADH + NADP ＋ = NAD ＋ +NADPH (Formula 1)

[0022] In this embodiment, the pyridine nucleotide transhydrogenase gene is not particularly limited as long as it encodes a protein or protein complex that catalyzes the reaction of Formula 1. Preferably, the gene encodes a pyridine nucleotide transhydrogenase containing the amino acid sequence of an enzyme having the EC number EC 1.6.1.1 or EC 7.1.1.1, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. More preferably, the gene encodes a pyridine nucleotide transhydrogenase containing the amino acid sequence of an enzyme having the EC number EC 1.6.1.1, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. Even more preferably, the gene encodes a pyridine nucleotide transhydrogenase containing the amino acid sequence set forth in SEQ ID NO: 2 (also referred to as UdhA, SthA, Sth, etc.) derived from Escherichia coli, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. The sequence identity is preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0023] (Enhanced gene expression) In this embodiment, enhanced gene expression refers to a state in which the transcription level of the pyridine nucleotide transhydrogenase gene or the expression level of the polypeptide encoded by the gene is increased compared to a host that has not been transformed to enhance expression of the gene. The amount of increase is not particularly limited, but it should be more than 1-fold, preferably 1.1-fold or more, more preferably 1.2-fold or more, even more preferably 1.5-fold or more, and even more preferably 2-fold or more, compared to a strain in which expression of the pyridine nucleotide transhydrogenase gene is not enhanced.

[0024] In this embodiment, the method for enhancing the expression of the pyridine nucleotide transhydrogenase gene is not particularly limited, but a method for introducing a target gene into a host, a method for enhancing the expression level of a target gene originally present in the genomic DNA of the host, or both can be selected.

[0025] 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.

[0026] Considering the stability of the introduced gene, a preferred method is to directly insert or replace the target gene onto the host chromosome or onto a megaplasmid possessed by the host, and a more preferred method is to directly insert or replace the target gene onto the host chromosome. 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" originally possessed by the host, or so that it is located downstream of an exogenous "gene expression regulatory sequence." A "gene expression regulatory sequence" is a DNA sequence containing a nucleotide sequence (e.g., a promoter sequence) that controls the transcription level of the gene and / or a nucleotide sequence (e.g., a Shine-Dalgarno sequence) that controls the translation level of messenger RNA transcribed from the gene. The "gene expression regulatory sequence" may be any naturally occurring nucleotide sequence, or may be an artificially constructed or modified nucleotide sequence.

[0027] Furthermore, methods for enhancing the expression level of a target gene that a host originally has on its genomic DNA are not particularly limited, but include methods of modifying a "gene expression regulatory sequence" located upstream of the target gene, methods of introducing an exogenous "gene expression regulatory sequence" upstream of the target gene, and methods of improving the stability of transcribed messenger RNA by modifying the base sequence of the target gene and / or its surrounding area.

[0028] 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: 8 to 16, or base sequences containing parts of these base sequences.

[0029] 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, including a method utilizing transposons and the mechanism of homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)) and a method based on site-specific integration via the mechanism of homologous recombination followed by loss via a second step of homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)). In addition, a method in which the sacB gene derived 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)) can also be used. 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, guide RNA (gRNA) has a sequence that can bind to a part of the base sequence of the genomic DNA to be modified, and plays a role in transporting Cas9 to the target.

[0030] 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.

[0031] (Culturing) By culturing the transformed microorganism according to this embodiment, PHA can be accumulated in the cells. The culturing can be carried out according to a conventional microbial culturing method, provided that the culture is 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. It is preferable to add the carbon source to the medium continuously or intermittently.

[0032] Any carbon source can be used as a carbon source during cultivation as long as it can be assimilated by the transformed microorganism of this embodiment. Examples include, but are not limited to, sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil, palm kernel oil, or their fractionated oils (e.g., low-melting-point fractions such as palm olein, palm double olein, and palm kernel 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. The oils and fats may be degraded oils. Furthermore, if the transformed microorganism of this embodiment can utilize gases or alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources.

[0033] The transformation according to this embodiment can improve PHA productivity even when degraded oil is used as a carbon source. Degraded oil refers to oils and fats that have been thermally denatured or that have been altered by reacting with oxygen and / or water under heat. The term "degraded oil" is not limited, and includes oils and fats known as waste oil, discarded oil, waste cooking oil, waste vegetable oil, used oil, etc.

[0034] The origin of the degraded oil is not particularly limited, but may be, for example, oil and fat used for cooking fried foods and the like and discharged from ordinary households, restaurants, or food manufacturing companies. It may also be oil and fat discharged from one or more sources and collected by a collection company. Alternatively, it may be oil and fat obtained by removing moisture and impurities from discharged or collected oil and fat.

[0035] The type of fat or oil contained in the degraded oil is not particularly limited, and may be, for example, palm oil, palm kernel oil, or fractionated oils thereof (for example, palm olein, palm double olein, palm kernel oil olein, etc., which are fractionated low-melting point fractions), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, jatropha oil, or other fats or oil fractions thereof, or refined by-products thereof.

[0036] The degree of deterioration of deteriorated oil can be quantified using commonly used indicators, such as acid value, peroxide value, anisidine value, and polymer content (%).

[0037] Generally, fats and oils are oxidized and hydrolyzed when exposed to water, air, or light including ultraviolet light, or when heated to high temperatures. This state is generally referred to as fat and oil deterioration. For example, when fats and oils react with oxygen, peroxides can be produced. These peroxides can be quantified using the peroxide value as an indicator. Furthermore, when peroxides decompose, carbonyl compounds (aldehydes, ketones, etc.) can be produced. These carbonyl compounds can be quantified using the anisidine value as an indicator. Furthermore, peroxides can polymerize to produce polymers. These polymers can be quantified using the polymer (%) as an indicator. There are various theories about the mechanism of fat and oil deterioration, and the mechanism of fat and oil deterioration in this embodiment is not limited.

[0038] Fats and oils can be hydrolyzed by reacting with water under heating to produce free fatty acids, diacylglycerols, and / or monoacylglycerols. These hydrolysis products can be quantified using the acid value as an indicator.

[0039] In this embodiment, degraded oil can be distinguished from unused, undegraded oils and fats using at least one indicator selected from the group consisting of peroxide value, anisidine value, polymer (%), and acid value.

[0040] The deteriorated oil used in this embodiment preferably has an acid value of 1 mg / g or more, more preferably 3 mg / g or more. The peroxide value is preferably 8 meq / kg or more. The anisidine value is preferably 2 or more, more preferably 5 or more, more preferably 10 or more, more preferably 20 or more, even more preferably 40 or more, and particularly preferably 60 or more. The polymer (%) is preferably 1 or more, more preferably 2 or more, and even more preferably 4 or more.

[0041] In this application, the acid value and anisidine value are values ​​analyzed according to the Standard Testing Methods for Analysis of Fats, Oils, and Related Materials established by the Japan Oil Chemists' Society. The peroxide value is a value analyzed according to the potentiometric titration method of the Standard Testing Methods for Analysis of Fats, Oils, and Related Materials. The polymer (%) is a value analyzed according to Provisional Method 16 of the Standard Testing Methods for Analysis of Fats, Oils, and Related Materials.

[0042] In the production of PHA in this embodiment, 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.

[0043] 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 culture is completed, 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.

[0044] By culturing the transformed microorganism according to this embodiment, good PHA productivity can be achieved.

[0045] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to them. [Item 1] A transformed microorganism having an exogenous polyhydroxyalkanoic acid synthase gene and reduced expression of the glpV gene. [Item 2] The transformed microorganism according to Item 1, wherein the glpV gene is a gene encoding GlpV comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. [Item 3] The transformed microorganism according to Item 1 or 2, further comprising enhanced expression of a pyridine nucleotide transhydrogenase gene. [Item 4] The transformed microorganism according to Item 3, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase comprising the amino acid sequence of an enzyme having the EC number EC 1.6.1.1, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. [Item 5] The transformed microorganism according to Item 4, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity to the amino acid sequence. [Item 6] The transformed microorganism according to any one of Items 1 to 5, which is a transformed microorganism of a microorganism belonging to the genus Capriavidus. [Item 7] The transformed microorganism according to Item 6, which is a transformed microorganism of Capriavidus necator. [Item 8] A method for producing polyhydroxyalkanoic acid, comprising a step of culturing the transformed microorganism according to any one of Items 1 to 7 in the presence of a carbon source. [Item 9] The method for producing polyhydroxyalkanoic acid according to Item 8, wherein the carbon source comprises a fat or oil. [Item 10] The method for producing polyhydroxyalkanoic acid according to Item 9, wherein the fat or oil is a degraded oil. [Item 11] The method for producing polyhydroxyalkanoic acid according to any one of Items 8 to 10, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acids. [Item 12] The method for producing a polyhydroxyalkanoic acid according to Item 11, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.[Item 13] The method for producing a polyhydroxyalkanoic acid according to Item 12, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0046] 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 performed, for example, as described in 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 are not particularly limited as long as they can be used in genetic manipulation. Synthetic DNA containing the DNA sequence constituting a gene and plasmid vectors into which the synthetic DNA has been cloned can also be purchased from commercial suppliers and used.

[0047] (Production Example 1) Preparation of a glpV-disrupted strain First, a plasmid for gene deletion / disruption was prepared. This was done as follows. A DNA fragment (SEQ ID NO: 17) containing the nucleotide sequence upstream and downstream of the H16_A2498 structural gene was obtained by PCR using synthetic oligo DNA and the genomic DNA of the Capriavidus necator H16 strain as a template. 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-glpVUD for gene deletion / disruption, which contains the nucleotide sequence upstream and downstream of the H16_A2498 structural gene.

[0048] Next, a glpV-disrupted strain was prepared using the gene deletion / disruption plasmid vector pNS2X-sacB-glpVUD as follows. Escherichia coli strain S17-1 (ATCC 47055) was transformed with the gene deletion / disruption plasmid vector pNS2X-sacB-glpVUD, and the resulting transformed microorganism was mixed-cultured with strain KNK-005 on Nutrient Agar medium (manufactured by Difco) for conjugative transfer. Strain KNK-005 is a transformant in which a PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 5) has been introduced onto the chromosome of Capriavidus necator strain H16, and can be prepared according to the method described in U.S. Pat. No. 7,384,766.

[0049] The resulting culture medium was inoculated onto Simmons agar medium (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) containing 250 mg / L of kanamycin, and strains that grew on the agar medium were selected to obtain a strain in which the plasmid had been integrated into the chromosome of KNK-005 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 identified as a strain from which the plasmid had been lost. Furthermore, one strain in which the region from the start codon to the stop codon of the glpV gene on the chromosome was deleted was isolated by PCR and DNA sequencer analysis. This isolated strain was designated the glpV-disrupted strain.

[0050] (Production Example 2) Preparation of a glpV-disrupted / udhA plasmid-enhanced strain A plasmid vector for expressing the udhA gene, pCUP2-trc-udhA, was prepared. The preparation was carried out as follows. A DNA fragment (SEQ ID NO: 18) having a base sequence containing the trc promoter was obtained by PCR using synthetic oligo DNA. The obtained DNA fragment was digested with the restriction enzymes EcoRI and MunI. This DNA fragment was ligated to a plasmid vector pCUP2 described in WO 2007 / 049716 that had been cleaved with the restriction enzyme MunI. Of the obtained plasmid vectors, a plasmid vector in which the insertion orientation of the DNA fragment was confirmed by PCR to be such that the restriction enzyme SpeI site of the plasmid vector pCUP2 was located downstream of the DNA fragment was obtained as the plasmid vector pCUP2-trc. Next, a DNA fragment (SEQ ID NO: 19) was obtained having a Shine-Dalgarno sequence, which is a ribosome binding site, a terminator sequence, and a nucleotide sequence encoding the amino acid sequence of udhA derived from Escherichia coli K-12 strain (SEQ ID NO: 2). The obtained DNA fragment was digested with restriction enzymes MunI and SpeI. This DNA fragment was ligated to a plasmid vector pCUP2-trc that had been cleaved with restriction enzymes MunI and SpeI to obtain a plasmid vector pCUP2-trc-udhA.

[0051] Next, the plasmid vector pCUP2-trc-udhA was introduced into the glpV-disrupted strain to obtain a glpV-disrupted, udhA plasmid-enhanced strain. The plasmid vector was introduced into the cells by electrotransfer as follows. A Biorad Gene Pulser was used as the gene transfer device, and a 0.2 cm gap cuvette, also manufactured by Biorad, was used. 400 μl of competent cells of the glpV-disrupted strain and 20 μl of the expression vector were poured into the cuvette, which was then placed in the pulser. 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 in Nutrient Broth medium (DIFCO) at 30°C for 3 hours with shaking, and then cultured on a selection plate (Nutrient Agar medium (DIFCO), kanamycin 100 mg / L) at 30°C for 2 days to isolate one grown transformant strain. This isolated strain was named the glpV-disrupted, udhA plasmid-enhanced strain.

[0052] (Production Example 3) Preparation of Strain A with Enhanced glpV Disruption and Enhanced udhA Genome Integration First, a plasmid was prepared for enhancing the udhA gene by integrating it into the genome. The preparation was carried out as follows. A commercial supplier was requested to prepare a plasmid vector for expressing the udhA gene, pNS2X-sacB-dZ1-udhA, in which a DNA fragment (SEQ ID NO: 20) having a base sequence encoding the upstream and downstream nucleotide sequences of the phaZ1 structural gene of Capriavidus necator, the Shine-Dalgarno sequence serving as a ribosome binding site, a terminator sequence, and the amino acid sequence of udhA derived from Escherichia coli K-12 strain (SEQ ID NO: 2) was inserted into the restriction enzyme SwaI site of the vector pNS2X-sacB described in JP 2007-259708 A, and the vector was obtained.

[0053] Next, using the udhA gene expression plasmid vector pNS2X-sacB-dZ1-udhA, a glpV-disrupted / udhA genome integration-enhanced strain A was prepared as follows: Escherichia coli S17-1 strain (ATCC 47055) was transformed with the udhA gene expression plasmid vector pNS2X-sacB-dZ1-udhA, and the resulting transformed microorganism was mixed and cultured with a glpV-disrupted strain on Nutrient Agar medium (manufactured by Difco) for conjugative transfer.

[0054] The resulting culture medium was inoculated onto Simmons agar medium (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) containing 250 mg / L of kanamycin, and strains grown on the agar medium were selected to obtain strains in which the plasmid had been integrated into the chromosome of the glpV-disrupted 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 strains were identified as strains from which the plasmid had been lost. Furthermore, PCR and DNA sequence analysis isolated one strain in which the initiation codon to termination codon of the phaZ1 gene on the chromosome had been replaced with DNA containing a Shine-Dalgarno sequence, a udhA gene sequence, and a terminator sequence. This isolated strain was named glpV-disrupted / udhA genome integration-enhanced strain A. In glpV-disrupted / udhA genome integration-enhanced strain A, the udhA gene is transcribed by the phaZ1 promoter, and udhA gene expression is enhanced compared to the glpV-disrupted strain.

[0055] (Production Example 4) Preparation of Strain B with Enhanced glpV Disruption and Enhanced udhA Genome Integration First, a plasmid was prepared to enhance the udhA gene by integrating it into the genome together with a promoter. The preparation was carried out as follows. A commercial supplier was requested to prepare a plasmid vector for expressing the udhA gene, pNS2X-sacB-dZ1-trp-udhA, in which a DNA fragment (SEQ ID NO: 21) having a base sequence encoding the upstream and downstream nucleotide sequences of the phaZ1 structural gene of Capriavidus necator, a trp promoter sequence serving as a ribosome binding site, a Shine-Dalgarno sequence, a terminator sequence, and the amino acid sequence of udhA derived from Escherichia coli K-12 strain (SEQ ID NO: 2) was inserted into the restriction enzyme SwaI site of the vector pNS2X-sacB described in JP 2007-259708 A, and the vector was obtained.

[0056] Next, the glpV-disrupted / udhA genome integration-enhanced strain B was prepared using the udhA gene expression plasmid vector pNS2X-sacB-dZ1-trp-udhA as follows: Escherichia coli S17-1 strain (ATCC 47055) was transformed with the udhA gene expression plasmid vector pNS2X-sacB-dZ1-trp-udhA, and the resulting transformed microorganism was mixed and cultured with the glpV-disrupted strain on Nutrient Agar medium (Difco) for conjugative transfer.

[0057] The resulting culture medium was inoculated onto Simmons agar medium (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) containing 250 mg / L of kanamycin, and strains grown on the agar medium were selected to obtain strains in which the plasmid had been integrated into the chromosome of the glpV-disrupted 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 strains were identified as strains from which the plasmid had been lost. Furthermore, PCR and DNA sequence analysis isolated one strain in which the phaZ1 gene on the chromosome from its start codon to its stop codon had been replaced with DNA containing the trp promoter sequence, Shine-Dalgarno sequence, udhA gene sequence, and terminator sequence. This isolated strain was named glpV-disrupted / udhA genome integration-enhanced strain B. In glpV-disrupted / udhA genome integration-enhanced strain B, the udhA gene is transcribed by the phaZ1 promoter and trp promoter, and udhA gene expression is enhanced compared to the glpV-disrupted strain and the glpV-disrupted / udhA genome integration-enhanced strain A.

[0058] Comparative Example 1: PHA production by KNK-005 strain using rapeseed oil as a carbon source. A culture study was carried out using the KNK-005 strain under the following conditions. (Culture medium) The composition of the seed culture medium was 1 w / v% meat extract, 1 w / v% bactotryptone, 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).

[0059] 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 , 1.29 w / 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 Palm olein oil was added as a carbon source at a concentration of 10 g / L all at once.

[0060] 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% MgSO ４ ・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).

[0061] (PHA production culture) PHA production culture was carried out as follows. First, a glycerol stock (20 μl) of the KNK-005 strain was inoculated into a seed medium (20 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 33°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.

[0062] Next, the preculture solution was inoculated at 5.0 v / v% into a 5 L jar fermenter (Marubishi Bioengine MDS-U50 model) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 33-34°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 ammonium hydroxide solution was used for pH control. The carbon source was added intermittently. Rapeseed oil was used as the carbon source. The culture was continued for 72 hours from the start of culture.

[0063] The rapeseed oil used in Comparative Example 1 was measured for its acid value, peroxide value, anisidine value, and polymer content (%). The results are shown in Table 4.

[0064] (Purification) After the culture was completed, the culture solution was weighed into a centrifuge tube and the weight of the culture solution was measured. The bacterial cells were collected by centrifugation and suspended in a 3.3 wt / v% aqueous solution of sodium lauryl sulfate (SDS), and the cellular components of the bacterial cells were disrupted using an ultrasonic disrupter to extract PHA. The PHA was collected by centrifugation, washed with water and ethanol in turn, and then vacuum dried at 60 ° C for 3 hours to obtain dried PHA. The weight of the obtained dried PHA was measured and divided by the weight of the culture solution measured initially to calculate the weight of PHA per 1 g of culture solution obtained 72 hours after the start of culture.

[0065] (Method of calculating PHA productivity) PHA productivity (%) was calculated using the following formula as the ratio of the PHA weight (g) per gram of culture solution obtained in the Example 72 hours after the start of culture to the PHA weight (g) per gram of culture solution obtained in the Comparative Example 72 hours after the start of culture: PHA productivity (%) = [PHA weight (g) per gram of culture solution obtained in the Example] / [PHA weight (g) per gram of culture solution obtained in the Comparative Example] × 100

[0066] Example 1 PHA Production by a glpV-Disrupted Strain Using Rapeseed Oil as a Carbon Source A culture study was carried out using a glpV-disrupted strain under the same conditions as in Comparative Example 1. The results of measuring PHA productivity (%) using Comparative Example 1 as a comparison are shown in Table 1. As a result of the culture study, PHA productivity (%) was improved by 9% compared to Comparative Example 1.

[0067]

[0068] (Comparative Example 2) PHA production by strain KNK-005 using degraded oil A as a carbon source Cultivation studies were carried out using strain KNK-005 under the same conditions as in Comparative Example 1, except that the carbon source used in the PHA production culture was changed from rapeseed oil to degraded oil A. As described above, the weight of PHA per 1 g of culture solution obtained 72 hours after the start of culture was calculated.

[0069] The acid value, peroxide value, anisidine value, and polymer (%) of the deteriorated oil A used in Comparative Example 2 were measured, and the results are shown in Table 4.

[0070] Example 2: PHA production by a glpV-disrupted strain using degraded oil A as a carbon source A culture study was carried out using a glpV-disrupted strain under the same conditions as in Comparative Example 2. As described above, the weight of PHA per gram of culture solution obtained 72 hours after the start of culture was calculated, and PHA productivity (%) was calculated in the same manner as above, except that the comparison was changed to Comparative Example 2. The results are shown in Table 2. As a result of the culture study, PHA productivity (%) was improved by 7% compared to Comparative Example 2.

[0071] (Example 3) PHA production by a glpV-disrupted / udhA plasmid-enhanced strain using degraded oil A as a carbon source A culture study was carried out using a glpV-disrupted / udhA plasmid-enhanced strain under the same conditions as in Comparative Example 2. As described above, the weight of PHA per gram of culture solution obtained 72 hours after the start of culture was calculated, and PHA productivity (%) was calculated in the same manner as above, except that the comparison was changed to Comparative Example 2. The results are shown in Table 2. As a result of the culture study, PHA productivity (%) was improved by 12% compared to Comparative Example 2 and by 5% compared to Example 2.

[0072]

[0073] (Comparative Example 3) PHA production by strain KNK-005 using degraded oil B as a carbon source Cultivation studies were carried out using strain KNK-005 under the same conditions as in Comparative Example 1, except that the carbon source used in the PHA production culture was changed from rapeseed oil to degraded oil B. As described above, the weight of PHA per 1 g of culture solution obtained 72 hours after the start of culture was calculated.

[0074] The acid value, peroxide value, anisidine value, and polymer (%) of the deteriorated oil B used in Comparative Example 3 were measured, and the results are shown in Table 4.

[0075] Example 4 PHA Production by a glpV-Disrupted Strain Using Degraded Oil B as a Carbon Source A culture study was carried out using a glpV-disrupted strain under the same conditions as in Comparative Example 3. As described above, the weight of PHA per gram of culture solution obtained 72 hours after the start of culture was calculated, and PHA productivity (%) was calculated in the same manner as above, except that the comparison was changed to Comparative Example 3. The results are shown in Table 3. As a result of the culture study, PHA productivity (%) was improved by 5% compared to Comparative Example 3.

[0076] Example 5 PHA Production by Strain A with Enhanced glpV-Disruption and Enhanced udhA Genome Integration Using Degraded Oil B as a Carbon Source A culture study was carried out using Strain A with enhanced glpV-disruption and enhanced udhA genome integration under the same conditions as in Comparative Example 3. As described above, the weight of PHA per gram of culture solution obtained 72 hours after the start of culture was calculated, and PHA productivity (%) was calculated in the same manner as above, except that the comparison was changed to Comparative Example 3. The results are shown in Table 3. As a result of the culture study, PHA productivity (%) was improved by 8% compared to Comparative Example 3 and by 3% compared to Example 4.

[0077] Example 6 PHA Production by Strain B with Enhanced glpV-Disruption and Enhanced udhA Genome Integration Using Degraded Oil B as a Carbon Source A culture study was carried out using Strain B with enhanced glpV-disruption and enhanced udhA genome integration under the same conditions as in Comparative Example 3. As described above, the weight of PHA per gram of culture solution obtained 72 hours after the start of culture was calculated, and PHA productivity (%) was calculated in the same manner as above, except that the comparison was changed to Comparative Example 3. The results are shown in Table 3. As a result of the culture study, PHA productivity (%) was improved by 8% compared to Comparative Example 3 and by 3% compared to Example 4.

[0078]

[0079] From each table, it can be seen that the transformed strains in which the expression of the glpV gene was reduced had improved PHA productivity compared to the control strain. Furthermore, from Tables 2 and 3, it can be seen that the transformed strains in which the expression of the udhA gene was enhanced in addition to the reduced expression of the glpV gene showed even higher PHA productivity.

[0080]

[0081] From Table 4, it can be seen that the acid value, anisidine value, and polymer (%) of degraded oils A and B were greater than those of virgin rapeseed oil, indicating that degraded oils A and B were oils that had undergone advanced deterioration.

Claims

1. A transformed microorganism having an exogenous polyhydroxyalkanoate synthase gene and reduced expression of the glpV gene.

2. The transformed microorganism according to claim 1, wherein the glpV gene is a gene encoding GlpV comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence.

3. The transformed microorganism of claim 1, further comprising enhanced expression of a pyridine nucleotide transhydrogenase gene.

4. The transformed microorganism according to claim 3, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase comprising the amino acid sequence of an enzyme having the EC number EC 1.6.1.1 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence.

5. The transformed microorganism described in claim 4, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence.

6. The transformed microorganism according to claim 1 or 3, which is a transformed microorganism belonging to the genus Capriavidus.

7. The transformed microorganism according to claim 6, which is a transformed microorganism of Capriavidus necator.

8. A method for producing polyhydroxyalkanoic acid, comprising culturing the transformed microorganism according to claim 1 or 3 in the presence of a carbon source.

9. The method for producing a polyhydroxyalkanoic acid according to claim 8, wherein the carbon source comprises fats or oils.

10. The method for producing polyhydroxyalkanoic acid according to claim 9, wherein the oil or fat is a deteriorated oil.

11. The method for producing a polyhydroxyalkanoic acid according to claim 8, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid.

12. The method for producing a polyhydroxyalkanoic acid according to claim 11, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.

13. The method for producing a polyhydroxyalkanoic acid according to claim 12, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

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