WO2025173696A1 - Method for producing polyhydroxyalkanoic acid, and transformed microorganism - Google Patents

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

The present invention relates to a method for producing polyhydroxyalkanoic acid by microbial culture, and to a transformed microorganism that can be used in this method.

Amid growing global interest in sustainability, including the SDGs, and growing concern over environmental issues such as marine microplastics, there has been a push to convert existing petroleum-derived non-biodegradable plastics to biodegradable materials, 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 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, raising expectations that it could be a solution to the marine microplastics problem and other environmental issues.

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 produced industrially by culturing PHA-accumulating microorganisms while supplying them with nutrient sources such as carbon, nitrogen, and phosphorus. Carbon sources often used in PHA cultivation and production include sugars such as glucose and fructose, vegetable oils such as palm oil and rapeseed oil, and free fatty acids and their salts.

However, the aforementioned carbon sources 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, used cooking oil, waste vegetable oil, used oil, etc.) as a carbon source is seen as promising.

There have been reports of culturing and producing PHA using degraded oil as a carbon source (see, for example, Patent Document 1), but PHA productivity has not yet reached a level where it is industrially possible to recover costs.

Methods for improving PHA productivity include improving cultivation methods and modifying microorganisms through genetic recombination. As one example of improving PHA productivity by modifying microorganisms through genetic recombination, 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 productivity of polyhydroxybutyrate (PHB), a type of PHA, was improved compared to a non-transfected strain by culturing E. coli strains that had been introduced with a plasmid encoding the phb operon of Capriavidus necator and a strain that had been introduced with a plasmid encoding the E. coli pyridine nucleotide transhydrogenase udhA gene using a carbon source containing glucose.

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, which is a type of PHA, does not change compared to the strain before enhancement.
Incidentally, neither Non-Patent Document 1 nor Non-Patent Document 2 describes cultivation using degraded oil or fats and oils as a carbon source.

Japanese Patent Application Laid-Open No. 2004-254668

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

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

In light of the above-mentioned 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.

The inventors discovered that when culturing PHA-producing microorganisms using degraded oil as a carbon source to produce PHA, 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, leading to the present invention.

That is, the present invention provides a method for producing polyhydroxyalkanoic acid, which comprises a step of culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid in a medium containing a carbon source,
the carbon source comprises depleted oil;
The present invention relates to a method for producing polyhydroxyalkanoic acid, wherein 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 in which expression of a pyridine nucleotide transhydrogenase gene is enhanced.

According to the present invention, it is possible to provide a method for producing polyhydroxyalkanoic acid, which can improve the productivity of polyhydroxyalkanoic acid in microbial culture using degraded oil as a carbon source.
Furthermore, it is possible to provide a polyhydroxyalkanoic acid-producing microorganism that can improve the productivity of polyhydroxyalkanoic acid by culturing it using degraded oil as a carbon source.
According to the present invention, it is possible to suppress a decrease in productivity of polyhydroxyalkanoic acid due to the use of degraded oils and fats, and therefore it is possible to effectively utilize degraded oils as a carbon source in polyhydroxyalkanoic acid-producing culture.

Hereinafter, embodiments of the present invention will be described in detail.
One aspect of the present invention relates to a method for producing polyhydroxyalkanoic acid, which comprises culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid in a medium containing a carbon source, wherein at least degraded oil is used as the carbon source, and the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene.

By culturing a transformed microorganism capable of producing polyhydroxyalkanoic acid (hereinafter also referred to as PHA), PHA can be accumulated within 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 conventional microbial culture methods, and the culture may be performed in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, carbon source addition method, culture scale, aeration and agitation conditions, culture temperature, culture time, etc. for PHA production culture and preculture. However, it is preferable to add the carbon source to the medium continuously or intermittently in PHA production culture.

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

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

The origin of degraded oil is not particularly limited, but it may be, for example, oil and fat discharged from ordinary households, restaurants, or food manufacturing companies after cooking fried foods. 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 from which moisture and impurities have been removed from discharged or collected oil and fat.

The original 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 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 their refined by-products.

The degree of deterioration of degraded oil can be quantified using commonly used indicators. Examples of such indicators include acid value, peroxide value, anisidine value, and polymer content (%).

Generally, fats and oils are oxidized and hydrolyzed when exposed to water, air, light including ultraviolet rays, or when heated to high temperatures. This condition is generally called deterioration of fats and oils.
For example, when fats and oils react with oxygen, peroxides can be produced, which can be quantified using the peroxide value as an indicator.
Furthermore, when peroxides decompose, carbonyl compounds (aldehydes, ketones, etc.) can be produced. The carbonyl compounds can be quantified using the anisidine value as an index.
Moreover, peroxides can polymerize to form polymers, which can be quantified using the percentage of polymer as an indicator.
There are various theories about the mechanism of deterioration of fats and oils, and the mechanism of deterioration of fats and oils is not limited in this embodiment.

Oils and fats react with water under heat and can be hydrolyzed to produce free fatty acids, diacylglycerols, and/or monoacylglycerols. These hydrolysis products can be quantified using the acid value as an indicator.

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.

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.
It is also preferable that the peroxide value is 8 meq/kg or more.
The anisidine value is preferably 2 or more, more preferably 5 or more, more preferably 10 or more, even 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.

In the present application, the acid value and the anisidine value are values analyzed in accordance with the Standard 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 Method for Analysis of Fats, Oils and Related Materials.
The polymer (%) is a value obtained by analysis in accordance with Provisional Method 16 of the Standard Methods for the Analysis of Fats, Oils, and Related Materials.

The carbon source used in this embodiment may consist solely of degraded oil, but may also contain undegraded, unused fats and oils in addition to the degraded oil. It may also contain carbon sources other than fats and oils (for example, sugars, fatty acids, glycerol, etc.). However, from the perspective 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.

The carbon source used in the pre-culture described above is not particularly limited, and degraded oil may or may not be used. Usable carbon sources include, for example, sugars such as glucose, fructose, and sucrose; oils and fats such as palm oil, palm kernel oil, or their fractionated oils (for example, palm olein, palm double olein, palm kernel olein, etc., which are fractionated low-melting point fractions), 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.

In the production of PHA in this embodiment, it is preferable to culture the microorganisms using a medium containing the above-mentioned carbon source, a nitrogen source (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.

After culturing for an appropriate period of time to allow PHA to accumulate within the cells, PHA can be recovered from the cells using well-known methods. There are no particular limitations on the recovery method. For example, after culturing is complete, the cells can be separated from the culture medium using a centrifuge or separation membrane, and then dried. PHA can then be extracted from the dried cells using an organic solvent such as chloroform. Cell components can then be removed from the organic solvent solution containing PHA by filtration or other methods. A poor solvent such as methanol or hexane can be added to the filtrate to precipitate the PHA, and the supernatant can be removed by filtration or centrifugation. The PHA can then be recovered by drying. Alternatively, cell components other than PHA can be dissolved in water using surfactants, alkali, enzymes, etc., and the PHA particles can then be separated from the aqueous phase by filtration or centrifugation, dried, and recovered.

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.

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

When producing PHA using microorganisms, using degraded oil as a carbon source tends to result in reduced PHA productivity, as described above. However, according to this 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.

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 above. 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.

(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 the 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 the expression of the pyridine nucleotide transhydrogenase gene is not enhanced.

In this embodiment, the method for enhancing the expression of the pyridine nucleotide transhydrogenase gene is not particularly limited, but it is possible to select 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.

The method for introducing a target gene into a host is not particularly limited, but may include directly inserting or substituting the target gene onto the host's chromosome, 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 then introducing it, and two or more of these methods may be used in combination.

In consideration of the stability of the introduced gene, a method in which the target gene is directly inserted or replaced onto the host chromosome or onto a megaplasmid possessed by the host is preferred, and a method in which the target gene is directly inserted or replaced onto the host chromosome is more preferred. To ensure expression of the introduced gene, it is preferable to introduce the target gene so that it is located downstream of a "gene expression regulatory sequence" that the host originally possesses, or so that it is located downstream of an exogenous "gene expression regulatory sequence." A "gene expression regulatory sequence" is a DNA sequence that includes a base sequence that controls the transcription level of the gene (e.g., a promoter sequence) and/or a base sequence that controls the translation level of messenger RNA transcribed from the gene (e.g., a Shine-Dalgarno sequence). As a "gene expression regulatory sequence," any naturally occurring base sequence can be used, or an artificially constructed or modified base sequence can also be used.

In addition, methods for increasing 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.

Promoter sequences and Shine-Dalgarno sequences included in "gene expression regulatory sequences" 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.

Replacement, 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. Representative methods include a method that utilizes transposons and the mechanism of homologous recombination (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)), and a method based on the principle of site-specific integration caused by the mechanism of homologous recombination and loss by a second stage of homologous recombination (Noti et al., Methods Enzymol., 154:197-217 (1987)). Alternatively, a method can be used in which the sacB gene from Bacillus subtilis is coexisted, and a microbial strain from which the gene has been lost by second-stage homologous recombination can be easily isolated as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195-1204 (1992); Lenz et al., J. Bacteriol., 176:4385-4393 (1994)). Another method is genome editing using the CRISPR/Cas9 system to modify target DNA (Y. Wang et al., ACS Synth Biol., 5(7):721-732 (2016)). In the CRISPR/Cas9 system, guide RNA (gRNA) has a sequence that can bind to part of the base sequence of the genomic DNA to be modified, and serves to transport Cas9 to the target.

The method for introducing vectors into cells is not particularly limited, but examples include the calcium chloride method, electroporation, polyethylene glycol method, and spheroplast method.

(host)
Preferred examples of hosts for the transformed microorganisms 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 to these. 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.

In particular, no transformed microorganisms belonging to the genus Capriavidus have been reported to date in which expression of the pyridine nucleotide transhydrogenase gene has been enhanced. This transformed microorganism also constitutes one aspect of the present invention. This transformed microorganism makes it possible to improve PHA productivity in PHA production cultures using degraded oil as a carbon source.

The host of the transformed microorganism of this embodiment may be a wild-type strain that inherently contains 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 replacing the gene onto the host's chromosome, directly inserting or replacing the gene onto a megaplasmid possessed by the host, or introducing the gene by placing it onto 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 replaced 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 replaced onto the host's chromosome is more preferred.

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

(PHA synthase gene)
The PHA synthase gene possessed by the transformed microorganism of 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 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: 11 to 15, 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.

(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.

According to this embodiment, it is possible to improve PHA productivity in PHA production culture using degraded oil as a carbon source. It also makes it possible to effectively utilize degraded oil in PHA production culture, reducing the environmental burden and suppressing the costs required for PHA production culture.

The following items list preferred aspects of the present disclosure, but the present invention is not limited to the following items.
[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,
the carbon source comprises depleted oil;
A method for producing polyhydroxyalkanoic acid, wherein the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene.
[Item 2]
2. The method for producing a polyhydroxyalkanoic acid according to Item 1, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase comprising an 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 the amino acid sequence.
[Item 3]
3. The method for producing a 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 the amino acid sequence.
[Item 4]
4. The method for producing a 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]
Item 5. The method for producing a polyhydroxyalkanoic acid according to Item 4, wherein the transformed microorganism is a transformed microorganism of Capriavidus necator.
[Item 6]
6. The method for producing a polyhydroxyalkanoic acid according to any one of items 1 to 5, wherein the polyhydroxyalkanoic acid is a copolymer of two or more kinds of hydroxyalkanoic acids.
[Item 7]
Item 7. The method for producing a polyhydroxyalkanoic acid according to Item 6, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit.
[Item 8]
8. The method for producing a 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 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]
10. The transformed microorganism according to Item 9, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding a pyridine nucleotide transhydrogenase comprising an 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]
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]
12. The transformed microorganism according to any one of items 9 to 11, which is a transformed microorganism of Capriavidus necator.

The present invention will be explained in more detail below using examples. However, the present invention is not limited to these examples. The overall genetic manipulation can be performed as described in, for example, Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Enzymes, cloning hosts, etc. used in genetic manipulation can be purchased from commercial suppliers and used according to their instructions. There are no particular restrictions on enzymes, as long as they can be used for genetic manipulation. Synthetic DNA containing the DNA sequence that constitutes a gene, and plasmid vectors into which this synthetic DNA has been cloned, can also be purchased from commercial suppliers and used.

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

(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.

Next, the plasmid vector pCUP2-trc-udhA was introduced into the KNK-005 strain to obtain a transformant udhA plasmid-enhanced strain. The plasmid vector was introduced into the cells by electrical transfer 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, which was then placed in the pulse device. An electrical pulse was applied under conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance of 800 Ω. After pulsing, the bacterial solution in the cuvette was cultured with shaking at 30°C for 3 hours in Nutrient Broth medium (DIFCO), and then cultured on a selection plate (Nutrient Agar medium (DIFCO), kanamycin 100 mg/L) at 30°C for 2 days, and one grown transformant strain was isolated. This isolated strain was named the udhA plasmid-enhanced strain.

(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.

Next, a strain with enhanced udhA genome integration was prepared as follows using the udhA gene expression plasmid vector pNS2X-sacB-dZ1-udhA.
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 KNK-005 strain on Nutrient Agar medium (manufactured by Difco) to carry out conjugative transfer.

The resulting culture medium was plated on Simmons agar medium (sodium citrate 2g/L, sodium chloride 5g/L, magnesium sulfate heptahydrate 0.2g/L, ammonium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, agar 15g/L, pH 6.8) containing 250mg/L 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 (Difco), then diluted and spread on Nutrient Agar medium containing 15% sucrose, and the grown strains were identified as strains that had lost the plasmid. 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 the Shine-Dalgarno sequence, the 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.

(Reference Example 1) PHA production by strain KNK-005 using rapeseed oil as a carbon source Cultivation studies were carried out using strain KNK-005 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, 0.15 w/v% KH ₂ PO ₄ (pH 6.8).

The preculture medium consisted of _1.1 w/v% _{Na2HPO4.12H2O} , 0.19 _w /v% _KH2PO4 , _1.29 w/v% ( _NH4 ) _2SO4 , 0.1 w/v% _MgSO4.7H2O , _2.5 w/v% palm olein oil, and _0.5 v/v% trace metal salt solution ( _1.6 w/v% FeCl3.6H2O, ₁ w/v% _CaCl2.2H2O , 0.02 w _/ v% _CoCl2.6H2O , 0.016 _w /v% _CuSO4.5H2O , and 0.012 _w /v% _NiCl2.6H2O dissolved in _0.1 N hydrochloric acid). Palm olein oil was added as a carbon source at a concentration of 10 g/L all at once.

The composition of the PHA production medium was 0.385 w/v% _{Na2HPO4.12H2O} , _{0.067 w} / _v % _KH2PO4 , 0.291 w/v% ( _NH4 ) _2SO4 , _0.1 w/v% _MgSO4.7H2O , and 0.5 v/v% trace metal salt solution ₍ 1.6 w/v% _FeCl3.6H2O , 1 w/v _% CaCl2.2H2O, 0.02 _w /v% _CoCl2.6H2O , 0.016 _w / _v % _CuSO4.5H2O , and 0.012 _w / _v % _NiCl2.6H2O dissolved in _0.1 N hydrochloric acid).

(PHA production culture)
The 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 was inoculated at 1.0 v/v% into a 3 L jar fermenter (Marubishi Bioengine MDL-300 model) containing 1.8 L of pre-culture 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 pre-culture was carried out for 28 hours while controlling the pH between 6.7 and 6.8. A 14% aqueous ammonium hydroxide solution was used for pH control.

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, with the pH controlled between 6.7 and 6.8. A 25% aqueous solution of ammonium hydroxide was used for pH control. The carbon source was added intermittently. Rapeseed oil was used as the carbon source. The culture was continued for 72 hours from the start of culture.

The measurement results for the acid value, peroxide value, anisidine value, and polymer (%) of the rapeseed oil used in Reference Example 1 are shown in Table 2.

(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 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 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 PHA weight per 1 g of culture solution obtained 72 hours after the start of culture.

(Method of calculating PHA productivity)
The PHA productivity (%) was calculated using the following formula as the ratio of the PHA weight (g) per gram of culture medium obtained 72 hours after the start of culture in each example to the PHA weight (g) per gram of culture medium obtained 72 hours after the start of culture in Reference Example 1.
PHA productivity (%)=[PHA weight (g) per 1 g of culture medium obtained in each example]/[PHA weight (g) per 1 g of culture medium obtained in Reference Example 1]×100

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

The measurement results for PHA productivity (%) and PHA content (%) in Reference Example 1 are shown in Table 1.

(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.

(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%.

Table 2 shows the measurement results for the acid value, peroxide value, anisidine value, and polymer content (%) of the degraded oil A used in Comparative Example 1. Note that degraded oil A was obtained from a degraded oil recovery company.

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.

(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%.

Table 2 shows the measurement results for the acid value, peroxide value, anisidine value, and polymer content (%) of degraded oil B used in Comparative Example 2. Degraded oil B was obtained from a degraded oil recovery company.

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.

(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%.

Table 2 shows the measurement results for the acid value, peroxide value, anisidine value, and polymer content (%) of degraded oil C used in Comparative Example 3. Note that degraded oil C was obtained from a degraded oil recovery company.

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.

Furthermore, the resulting PHA was reacted in a mixture of methanol and sulfuric acid at high temperature and pressure, and then subjected to HPLC, confirming that the resulting PHA in all reference examples, comparative examples, and examples was P(3HB-co-3HH).

 

From the above, it can be seen that when cultured using degraded oil as a carbon source, transformed strains with enhanced expression of the pyridine nucleotide transhydrogenase gene exhibit higher PHA productivity compared to 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 virgin rapeseed oil was used as the carbon source, there was no difference in PHA productivity between the transformed strain in which expression of the pyridine nucleotide transhydrogenase gene was enhanced and the non-enhanced strain (Reference Examples 1 and 2). This indicates that the effect of improving PHA productivity by the transformed strain in which expression of the pyridine nucleotide transhydrogenase gene was enhanced is not achieved when virgin rapeseed oil is used, but is an effect specific to the use of degraded oil as the carbon source.

 

Table 2 shows that the acid value, anisidine value, and polymer (%) of degraded oils A to C are higher than those of virgin rapeseed oil, indicating that degraded oils A to C are oils that have undergone advanced degradation.

Claims (12)

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,
the carbon source comprises depleted oil;
A method for producing polyhydroxyalkanoic acid, wherein the transformed microorganism has enhanced expression of a pyridine nucleotide transhydrogenase gene. The method for producing polyhydroxyalkanoic acid according to claim 1, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding 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. The method for producing polyhydroxyalkanoic acid according to claim 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. 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. The method for producing polyhydroxyalkanoic acid according to claim 4, wherein the transformed microorganism is a transformed microorganism of Capriavidus necator. The method for producing 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. The method for producing polyhydroxyalkanoic acid according to claim 6, wherein the polyhydroxyalkanoic acid is a copolymer containing 3-hydroxyhexanoic acid as a monomer unit. The method for producing polyhydroxyalkanoic acid according to claim 7, wherein the polyhydroxyalkanoic acid is a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. A transformed microorganism belonging to the genus Capriavidus that has the ability to produce polyhydroxyalkanoic acid and has enhanced expression of the pyridine nucleotide transhydrogenase gene. The transformed microorganism according to claim 9, wherein the pyridine nucleotide transhydrogenase gene is a gene encoding 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. The transformed microorganism according to 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. The transformed microorganism described in any one of claims 9 to 11, which is a transformed microorganism of Capriavidus necator.