Method for producing polyhydroxyalkanoic acid, and transformed microorganism

Abstract

Provided is a method for producing a polyhydroxyalkanoic acid, including a step of culturing a transformed microorganism having the ability to produce a polyhydroxyalkanoic acid in a medium containing a carbon source. The carbon source contains a degraded oil, and the transformed microorganism has enhanced expression of the pyridine nucleotide transhydrogenase gene. More specifically, provided is a transformed microorganism of a microorganism belonging to the genus Cupriavidus, which has the ability to produce a polyhydroxyalkanoic acid and in which the expression of the pyridine nucleotide transhydrogenase gene is enhanced.

Description

Method for producing polyhydroxyalkanoic acid and transformed microorganism

[0001] The present invention relates to a method for producing polyhydroxyalkanoic acid by culturing a microorganism, and to a transformed microorganism that can be used in the method.

[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 nutrient sources such as carbon, nitrogen, and phosphorus. Carbon sources often used in PHA cultivation include sugars such as glucose and fructose, vegetable oils such as palm oil and rapeseed oil, and free fatty acids and their salts.

[0004] However, the carbon sources mentioned above are problematic due to the large amount of carbon dioxide emitted during the raw material production process, which places a heavy burden on the environment. Therefore, in PHA production, the use of degraded oil (also known as waste oil, discarded oil, waste cooking oil, waste vegetable oil, used oil, etc.) as a carbon source is considered promising.

[0005] Although examples of culturing and producing PHA using degraded oil as a carbon source have already been reported (see, for example, Patent Document 1), PHA productivity has not yet reached a level at which it is possible to recover costs industrially.

[0006] Methods for improving PHA productivity include improving the culture method and modifying microorganisms by genetic recombination. As one example of modifying microorganisms by genetic recombination to improve PHA productivity, Non-Patent Document 1 reports the strong expression of pyridine nucleotide transhydrogenase, an enzyme that reversibly catalyzes the conversion between NADH and NADPH, in Escherichia coli. This document reports an example in which E. coli strains containing a plasmid encoding the phb operon of Capriavidus necator and a plasmid encoding the E. coli pyridine nucleotide transhydrogenase udhA gene were cultured using a carbon source containing glucose, resulting in improved productivity of polyhydroxybutyrate (PHB), a type of PHA, compared to strains not containing the plasmid.

[0007] On the other hand, Non-Patent Document 2 reports that even when a strain of Rhodospirillum rubrum S1, a bacterium that originally produces PHA, in which the udhA gene of Escherichia coli has been enhanced, is cultured using fructose as a carbon source, the productivity of a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid, a type of PHA, does not change compared to the strain before enhancement. Note that neither Non-Patent Document 1 nor Non-Patent Document 2 describes cultivation using degraded oils or fats and oils as a carbon source.

[0008] Japanese Patent Application Laid-Open No. 2004-254668

[0009] Sanchez et al. , Biotechnol Prog. , 420-425 (2006) Heinrich et al. , FEMS Microbiol. Lett. , (2015)

[0010] As shown in Comparative Examples 1 to 3 below, when the present inventors cultured a known PHA-producing strain using degraded oil as a carbon source, the PHA productivity was found to be reduced by about 10 to 20% compared to the culture using unused oils and fats as the carbon source (Reference Example 1). Therefore, in order to industrially produce PHA using degraded oil, it is necessary to improve PHA productivity.

[0011] In view of the above-described current situation, the present invention aims to provide a method for producing polyhydroxyalkanoic acid and a polyhydroxyalkanoic acid-producing microorganism that can improve the productivity of polyhydroxyalkanoic acid in microbial culture using degraded oil as a carbon source.

[0012] The inventors discovered that when PHA is produced by culturing PHA-producing microorganisms using degraded oil as a carbon source, it is possible to improve PHA productivity by using a transformed strain with enhanced expression of the pyridine nucleotide transhydrogenase gene as the PHA-producing microorganism, and thus arrived at the present invention.

[0013] Specifically, the present invention relates to a method for producing polyhydroxyalkanoic acid, comprising the step of culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid in a medium containing a carbon source, wherein the carbon source contains degraded oil, and the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene. The present invention also relates to a transformed microorganism belonging to the genus Capriavidus, which has the ability to produce polyhydroxyalkanoic acid and enhanced expression of a pyridine nucleotide transhydrogenase gene.

[0014] According to the present invention, a method for producing polyhydroxyalkanoic acid can be provided that enables improved polyhydroxyalkanoic acid productivity in microbial culture using degraded oil as a carbon source. Furthermore, a polyhydroxyalkanoic acid-producing microorganism can be provided that enables improved polyhydroxyalkanoic acid productivity through culture using degraded oil as a carbon source. According to the present invention, a decrease in polyhydroxyalkanoic acid productivity due to the use of degraded oils and fats can be suppressed. Therefore, it becomes possible to effectively utilize degraded oil as a carbon source in polyhydroxyalkanoic acid-producing culture.

[0015]

[0013] Embodiments of the present invention are described in detail below. One aspect of the present invention relates to a method for producing polyhydroxyalkanoic acid, comprising culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid in a medium containing a carbon source. At least degraded oil is used as the carbon source, and the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene.

[0016] By culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid (hereinafter also referred to as PHA), PHA can be accumulated in the cells. This culture is referred to as PHA production culture. Prior to PHA production culture, preculture (also referred to as seed culture) for cell growth may be performed one or more times. In this embodiment, the step of culturing the transformed microorganism can be performed according to a conventional microbial culture method, and the culture may be performed in a medium containing an appropriate carbon source. The medium composition, carbon source addition method, culture scale, aeration and agitation conditions, culture temperature, culture time, etc. for the PHA production culture and preculture are not particularly limited. However, it is preferable that the carbon source in the PHA production culture is added to the medium continuously or intermittently.

[0017] (Carbon Source) In this embodiment, the carbon source for the PHA production culture contains at least degraded oil. Studies by the present inventors have revealed that when the known PHA-producing strain KNK-005 is cultured using degraded oil as a carbon source, PHA productivity tends to decrease compared to culture using unused, undegraded oil. However, this embodiment can suppress such a decrease in PHA productivity, and good PHA productivity can be achieved even when degraded oil is used as a carbon source.

[0018] Degraded oil refers to oils and fats that have been thermally denatured or that have changed in quality due to reaction with oxygen and / or water under heat. There are no restrictions on the name, and it also includes oils and fats that are called waste oil, discarded oil, used cooking oil, used vegetable oil, used oil, etc.

[0019] 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.

[0020] 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.

[0021] 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 (%).

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The carbon source used in this embodiment may consist solely of degraded oil, or may further contain undegraded, unused fats and oils in addition to the degraded oil. It may also contain a carbon source other than fats and oils (e.g., sugars, fatty acids, glycerol, etc.). However, from the viewpoint of reducing environmental impact, the proportion of degraded oil contained in the carbon source is preferably 10% by weight or more, more preferably 50% by weight or more, and even more preferably 80% by weight or more. It may even be 90% by weight or more.

[0028] The carbon source used in the pre-culture is not particularly limited, and degraded oil may or may not be used. Examples of usable carbon sources include sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil, palm kernel oil, and fractionated oils thereof (e.g., palm olein, palm double olein, palm kernel olein, and the like, which are fractionated low-melting-point fractions), corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, as well as fractionated oils and refined by-products thereof; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, and myrinsic acid, as well as derivatives thereof, and glycerol.

[0029] 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.

[0030] 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.

[0031] The transformed microorganism used in this embodiment is a microorganism that has the ability to produce polyhydroxyalkanoic acid and in which expression of the pyridine nucleotide transhydrogenase gene is enhanced.

[0032] (Pyridine nucleotide transhydrogenase gene) Pyridine nucleotide transhydrogenase is an enzyme that catalyzes the following reaction: NADH + NADP ＋ = NAD ＋ +NADPH (Formula 1)

[0033] In the production of PHA by microorganisms, the use of degraded oil as a carbon source tends to decrease PHA productivity as described above. However, according to the present embodiment, by enhancing the expression of the pyridine nucleotide transhydrogenase gene in polyhydroxyalkanoic acid-producing microorganisms, it is possible to improve PHA productivity when degraded oil is used as a carbon source.

[0034] 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: 1 (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.

[0035] (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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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: 2 to 10, or base sequences containing parts of these base sequences.

[0041] 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 the microbial strain from 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., 5 (7): 721-732 (2016)) 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.

[0042] 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.

[0043] (Host) Preferred examples of hosts for the transformed microorganism according to this embodiment include bacteria belonging to the genera Ralstonia, Cupriavidus, Wautersia, Burkholderia, Aeromonas, Escherichia, Alcaligenes, Pseudomonas, Bacillus, Azotobacter, Nocardia, Sphingomonas, and Comamonas, but are not limited thereto. 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.

[0044] In particular, a transformed microorganism belonging to the genus Capriavidus in which expression of the pyridine nucleotide transhydrogenase gene has been enhanced has not been reported to date. This transformed microorganism also constitutes one aspect of the present invention. This transformed microorganism makes it possible to improve PHA productivity in PHA-producing cultures using degraded oil as a carbon source.

[0045] 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.

[0046] The host of the transformed microorganism according to this embodiment is preferably a strain capable of assimilating fats and oils or fatty acids. This may be a wild-type strain inherently capable of assimilating fats and oils or fatty acids, a mutant strain obtained by artificially mutating such a wild-type strain, or a strain into which enzymes capable of assimilating fats and oils or fatty acids have been introduced by genetic engineering techniques. The method for introducing exogenous enzymes capable of assimilating fats and oils or fatty acids is not particularly limited, and may include direct insertion or replacement of genes onto the host chromosome, direct insertion or replacement of genes onto a megaplasmid possessed by the host, or placement of genes 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, direct insertion or replacement of genes onto the host chromosome or onto a megaplasmid possessed by the host is preferred, and direct insertion or replacement of genes onto the host chromosome is more preferred.

[0047] (PHA synthase gene) The PHA synthase gene possessed by the transformed microorganism according to this embodiment is not particularly limited, and examples thereof include PHA synthase genes derived from organisms similar to the genera Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium, as well as modified versions thereof. Examples of such modified versions include a nucleotide sequence encoding a PHA synthase in which one or more amino acid residues have been deleted, added, inserted, or substituted. Examples include a gene having a nucleotide sequence encoding a polypeptide represented by the amino acid sequence set forth in any of SEQ ID NOS: 11 to 15, and a gene having a nucleotide sequence encoding a polypeptide having PHA synthase activity and represented by an amino acid sequence having 90% or more sequence identity to the 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.

[0048] (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), 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), 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), a copolymer of 3HB and LA. Among these, P(3HB-co-3HH) 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, and the like.

[0049] According to this embodiment, it is possible to improve PHA productivity in PHA production culture using degraded oil as a carbon source. In addition, it is possible to effectively utilize degraded oil in PHA production culture, thereby reducing the environmental load and suppressing the costs required for PHA production culture.

[0050] The following items list preferred embodiments of the present disclosure, but the present invention is not limited to each of them. [Item 1] A method for producing polyhydroxyalkanoic acid, comprising a step of culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid in a medium containing a carbon source, wherein the carbon source contains degraded oil, and the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene. [Item 2] A method for producing polyhydroxyalkanoic acid according to Item 1, 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 3] A method for producing polyhydroxyalkanoic acid according to Item 2, 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: 1, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. [Item 4] The method for producing polyhydroxyalkanoic acid according to any one of Items 1 to 3, wherein the transformed microorganism is a transformed microorganism belonging to the genus Capriavidus. [Item 5] The method for producing polyhydroxyalkanoic acid according to Item 4, wherein the transformed microorganism is a transformed microorganism of Capriavidus necator. [Item 6] The method for producing polyhydroxyalkanoic acid according to any one of Items 1 to 5, wherein the polyhydroxyalkanoic acid is a copolymer of two or more hydroxyalkanoic acids. [Item 7] The method for producing polyhydroxyalkanoic acid according to Item 6, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit. [Item 8] The method for producing polyhydroxyalkanoic acid according to Item 7, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.[Item 9] A transformed microorganism of a microorganism belonging to the genus Capriavidus, which has the ability to produce polyhydroxyalkanoic acid and in which expression of a pyridine nucleotide transhydrogenase gene is enhanced. [Item 10] The transformed microorganism of Item 9, 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 11] The transformed microorganism of Item 10, 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: 1, or an amino acid sequence having 90% or more sequence identity to said amino acid sequence. [Item 12] The transformed microorganism of any one of Items 9 to 11, which is a transformed microorganism of Capriavidus necator.

[0051] 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.

[0052] The KNK-005 strain used in the following production examples is a known PHA-producing microorganism, and is a transformant of the Capriavidus necator H16 strain, prepared in accordance with the method described in U.S. Pat. No. 7,384,766, in which a PHA synthase gene derived from Aeromonas caviae has been introduced onto the chromosome.

[0053] (Production Example 1) Preparation of a 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: 16) 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: 17) 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: 1). 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.

[0054] Next, the plasmid vector pCUP2-trc-udhA was introduced into the KNK-005 strain to obtain a udhA plasmid-enhanced transformant. 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 KNK-005 strain and 20 μl of the expression vector were poured into the cuvette and 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 udhA plasmid-enhanced strain.

[0055] (Production Example 2) Preparation of a Strain with Enhanced Integration of udhA Genome First, a plasmid was prepared to enhance 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: 18) 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: 1) 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, a strain with enhanced udhA genome integration was prepared using the udhA gene expression plasmid vector pNS2X-sacB-dZ1-udhA 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 the KNK-005 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 that grew on the agar medium were selected to obtain a strain in which the plasmid had been integrated into the chromosome of the 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 strains were identified as strains from which the plasmid had been lost. Furthermore, PCR and DNA sequence analysis led to the isolation of one strain in which the phaZ1 gene on the chromosome from its initiation codon to its termination codon had been replaced with DNA containing a Shine-Dalgarno sequence, a udhA gene sequence, and a terminator sequence. This isolated strain was named the udhA genome integration-enhanced strain. In the udhA genome integration-enhanced strain, the udhA gene is transcribed by the phaZ1 promoter, and udhA gene expression is enhanced compared to the KNK-005 strain.

[0058] (Reference Example 1) PHA production by KNK-005 strain using rapeseed oil as a carbon source Cultivation studies were carried out using 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% 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).

[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 ２O, 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 acid value, peroxide value, anisidine value, and polymer (%) of the rapeseed oil used in Reference Example 1 were measured and the results are shown in Table 2.

[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 each example 72 hours after the start of culture to the PHA weight (g) per gram of culture solution obtained in Reference Example 1 72 hours after the start of culture: PHA productivity (%) = [PHA weight (g) per gram of culture solution obtained in each example] / [PHA weight (g) per gram of culture solution obtained in Reference Example 1] × 100

[0066] (Method for calculating PHA content) The PHA content (%) was calculated as the ratio of the PHA weight (g) per 1 g of culture solution obtained 72 hours after the start of culture to the dry cell weight (g) per 1 g of culture solution obtained 72 hours after the start of culture using the following formula: PHA content (%) = [PHA weight (g) per 1 g of culture solution] / [dry cell weight (g) per 1 g of culture solution] × 100 The "dry cell weight (g) per 1 g of culture solution" is a value obtained by obtaining dry cell cells using the same method as in the above (Purification) section, except for the steps of suspending in an SDS aqueous solution and disrupting cellular components with an ultrasonic disrupter, and then measuring the weight of the obtained dry cell cells.

[0067] The results of measuring the PHA productivity (%) and PHA content (%) in Reference Example 1 are shown in Table 1.

[0068] (Reference Example 2) PHA production by udhA plasmid-enhanced strain using rapeseed oil as a carbon source A culture study was carried out using the udhA plasmid-enhanced strain under the same conditions as in Reference Example 1. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, there was no change in PHA productivity (%) and PHA content (%) compared to Reference Example 1.

[0069] (Comparative Example 1) PHA production by strain KNK-005 using degraded oil A as a carbon source A culture study was carried out using strain KNK-005 under the same conditions as in Reference Example 1, except that the carbon source used in the PHA production culture was changed from rapeseed oil to degraded oil A. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, there was no change in PHA content (%) compared to Reference Example 1, but PHA productivity (%) decreased by 9%.

[0070] The acid value, peroxide value, anisidine value, and polymer content (%) of the degraded oil A used in Comparative Example 1 were measured and the results are shown in Table 2. The degraded oil A was obtained from a degraded oil recovery company.

[0071] Example 1 PHA Production by a udhA Plasmid-Enhanced Strain Using Degraded Oil A as a Carbon Source A culture study was carried out using degraded oil A under the same conditions as in Comparative Example 1, except that the transformed strain used was changed from KNK-005 strain to a udhA plasmid-enhanced strain. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, the PHA content (%) was almost the same as in each Reference Example and Comparative Example 1, but PHA productivity (%) was improved by 5% compared to Comparative Example 1.

[0072] (Comparative Example 2) PHA production by strain KNK-005 using degraded oil B as a carbon source A culture study was carried out using strain KNK-005 under the same conditions as in Reference Example 1, except that the carbon source used in the PHA production culture was changed from rapeseed oil to degraded oil B. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, compared to Reference Example 1, the PHA content (%) was reduced by 5% and the PHA productivity (%) was reduced by 17%.

[0073] The acid value, peroxide value, anisidine value, and polymer content (%) of the degraded oil B used in Comparative Example 2 were measured and the results are shown in Table 2. The degraded oil B was obtained from a degraded oil recovery company.

[0074] Example 2 PHA Production by a udhA Plasmid-Enhanced Strain Using Degraded Oil B as a Carbon Source A culture study was carried out using degraded oil B under the same conditions as in Comparative Example 2, except that the transformed strain used was changed from KNK-005 strain to a udhA plasmid-enhanced strain. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, the PHA content (%) was improved by 3%, and the PHA productivity (%) was improved by 6%, compared to Comparative Example 2.

[0075] (Comparative Example 3) PHA production by strain KNK-005 using degraded oil C as a carbon source A culture study was carried out using strain KNK-005 under the same conditions as in Reference Example 1, except that the carbon source used in the PHA production culture was changed from rapeseed oil to degraded oil C. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, compared to Reference Example 1, the PHA content (%) was reduced by 2% and the PHA productivity (%) was reduced by 11%.

[0076] The acid value, peroxide value, anisidine value, and polymer (%) of the degraded oil C used in Comparative Example 3 were measured and the results are shown in Table 2. The degraded oil C was obtained from a degraded oil recovery company.

[0077] Example 3 PHA Production by a Strain with Enhanced udhA Genome Integration Using Degraded Oil C as a Carbon Source A culture study was carried out using degraded oil C under the same conditions as in Comparative Example 3, except that the transformed strain used was changed from KNK-005 strain to a strain with enhanced udhA genome integration. The measurement results of PHA productivity (%) and PHA content (%) are shown in Table 1. As a result of the culture study, the PHA content (%) was improved by 2%, and the PHA productivity (%) was improved by 4%, compared to Comparative Example 3.

[0078] In addition, the obtained PHA was reacted in a mixed solution of methanol and sulfuric acid under high temperature and pressure, and then subjected to HPLC, and it was confirmed that the obtained PHA was P(3HB-co-3HH) in all Reference Examples, Comparative Examples, and Examples.

[0079]

[0080] From the above, it can be seen that when cultured using degraded oil as a carbon source, transformed strains in which expression of the pyridine nucleotide transhydrogenase gene was enhanced showed higher PHA productivity than non-enhanced strains (Comparison between Example 1 and Comparative Example 1, Comparison between Example 2 and Comparative Example 2, or Comparison between Example 3 and Comparative Example 3). On the other hand, when cultured using virgin rapeseed oil as a carbon source, there was no difference in PHA productivity between the transformed strains in which expression of the pyridine nucleotide transhydrogenase gene was enhanced and the non-enhanced strain (Reference Examples 1 and 2). This shows that the effect of improving PHA productivity by transformed strains in which expression of the pyridine nucleotide transhydrogenase gene was enhanced is not achieved when virgin rapeseed oil is used, and is an effect specific to the use of degraded oil as a carbon source.

[0081]

[0082] From Table 2, it can be seen that the acid value, anisidine value, and polymer (%) of the degraded oils A to C were greater than those of virgin rapeseed oil, and that the degraded oils A to C were oils in which degradation had progressed.

Claims

1. A method for producing polyhydroxyalkanoic acid, comprising the step of culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid in a medium containing a carbon source, wherein the carbon source contains degraded oil, and the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene.

2. The method for producing polyhydroxyalkanoic acid according to claim 1, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase having 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.

3. The method for producing polyhydroxyalkanoic acid described in claim 2, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase having 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.

4. The method for producing polyhydroxyalkanoic acid according to any one of claims 1 to 3, wherein the transformed microorganism is a transformed microorganism belonging to the genus Capriavidus.

5. The method for producing polyhydroxyalkanoic acid according to claim 4, wherein the transformed microorganism is a transformed microorganism of Capriavidus necator.

6. The method for producing a polyhydroxyalkanoic acid according to any one of claims 1 to 3, wherein the polyhydroxyalkanoic acid is a copolymer of two or more types of hydroxyalkanoic acid.

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

8. The method for producing a polyhydroxyalkanoic acid according to claim 7, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

9. A transformed microorganism belonging to the genus Capriavidus, which has the ability to produce polyhydroxyalkanoic acid and in which expression of a pyridine nucleotide transhydrogenase gene is enhanced.

10. The transformed microorganism according to claim 9, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase having 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.

11. The transformed microorganism described in claim 10, 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: 1 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence.

12. The transformed microorganism according to any one of claims 9 to 11, which is a transformed microorganism of Capriavidus necator.

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