Recombinant hydrogen-oxidizing bacteria and method for producing polyhydroxyalkanoic acid

Abstract

To provide a method for producing copolymerized PHA with an increased introduction rate of a second component without using a precursor of the second component.SOLUTION: Provided are a recombinant hydrogen-oxidizing bacterium having at least a 2-keto acid decarboxylase (kivd) gene and a phenylacetaldehyde dehydrogenase (padA) gene as monomer supply enhancing genes and capable of producing copolymerized polyhydroxyalkanoic acid (PHA); and a method for producing polyhydroxyalkanoic acid (PHA), comprising: (i) introducing at least a 2-keto acid decarboxylase (kivd) gene and a phenylacetaldehyde dehydrogenase (padA) gene as monomer supply enhancing genes into a hydrogen-oxidizing bacterium having a PHA polymerizing enzyme to obtain a transformant of the hydrogen-oxidizing bacterium; (ii) culturing the obtained transformant in the presence of a carbon source; and (iii) collecting copolymerized PHA from the culture.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a recombinant hydrogen-oxidizing bacterium having the ability to produce copolymer polyhydroxyalkanoic acid (PHA) with a specific monomer supply-enhancing gene. The present invention also relates to a method for producing PHA using a recombinant hydrogen-oxidizing bacterium having a PHA polymerase into which the monomer supply-enhancing gene has been introduced.

Background Art

[0002] Polyhydroxyalkanoic acid (PHA), which is a homopolymer or copolymer of 3-hydroxyalkanoic acid, is a biodegradable polyester accumulated by microorganisms in cells. In recent years, it has attracted attention not only as a biodegradable plastic material but also as a plastic material derived from biomass.

[0003] The most common PHA, a homopolymer composed of (R)-3-hydroxybutyric acid (3HB) (hereinafter, appropriately referred to as "P(3HB)"), is crystalline, so while the processing time can be shortened, it is hard and brittle and lacks practicality. In addition, problems such as polymer degradation during molding processing, such as the polymer becoming low molecular weight during melting, make it unsuitable for industrial production. As one means of improving these physical properties, various copolymers of 3HB and other monomers (hereinafter, the copolymers of PHA are appropriately referred to as "copolymer PHA") have been developed.

[0004] For example, a copolymer PHA containing 3-hydroxyvaleric acid (3HV) and 3-hydroxy-4-methylvaleric acid (3H4MV) as the second component in addition to 3HB has flexible physical properties. The copolymer PHA is biosynthesized by supplying propionic acid or 4-methylvaleric acid to hydrogen-oxidizing bacteria as a precursor.

[0005] Specifically, the inventors of the present invention used 4-methylvaleric acid as a precursor and the PHA polymerase (PhaC1) derived from Pseudomonas sp. strain 61-3 PsHydrogen-oxidizing bacterium Ralstonia eutropha PHB into which a gene has been introduced - Four strains were cultured to biosynthesize P(3HB-co-3HV-co-3H4MV), which is a copolymerized PHA (Non-Patent Document 1). Further, the present inventors used leucine as a precursor and cultured the four strains of Ralstonia eutropha PHB - to biosynthesize P(3HB-co-3HV-co-3H4MV) (Non-Patent Document 2).

[0006] However, precursors such as 4-methylvaleric acid are generally expensive. Also, generally, they are highly toxic to cells, which inhibits cell growth and reduces PHA accumulation in microorganisms. By using genetically engineered hydrogen-oxidizing bacteria, it is also possible to biosynthesize copolymerized PHA containing 3HV and 3H4MV using sugar or carbon dioxide as a single carbon source without a precursor, but the introduction fraction of the monomer components is as low as 1 to 2 mol% (Non-Patent Documents 3 and 4).

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] Therefore, a method for producing a copolymerized PHA with an enhanced introduction fraction of the second component without using a precursor of the second component has been desired.

Means for Solving the Problems

[0009] As a result of intensive studies in view of such a situation, the present inventors have prepared a hydrogen-oxidizing bacterium having at least two specific monomer supply-enhancing genes related to the PHA biosynthetic pathway, and cultured the recombinant hydrogen-oxidizing bacterium in the presence of a carbon source. As a result, it has been found that a copolymerized PHA with an enhanced introduction fraction of the second component can be produced without using a precursor of the second component, and the present invention has been completed. In particular, the present inventors have found that a hydrogen-oxidizing bacterium having three specific monomer supply-enhancing genes can produce a copolymerized PHA in which the introduction fraction of the second component is enhanced and the productivity of PHA is increased.

[0010] That is, the present invention provides the following. (1) A recombinant hydrogen-oxidizing bacterium having at least a 2-ketocarboxylic acid decarboxylase (kivd) gene and a phenylacetaldehyde dehydrogenase (padA) gene as monomer supply-enhancing genes and having the ability to produce a copolymerized polyhydroxyalkanoate (PHA). (2) The recombinant hydrogen-oxidizing bacterium according to (1), further having a 3-ketothiolase (bktB) gene as a monomer supply-enhancing gene. (3) The recombinant hydrogen-oxidizing bacterium according to (1) or (2), wherein the introduction fraction of the second component in the copolymerized PHA is higher than the introduction fraction of the second component in the copolymerized PHA produced by a hydrogen-oxidizing bacterium having no monomer supply-enhancing gene. (4) The recombinant hydrogen-oxidizing bacterium according to (3), wherein the introduction fraction of the second component is at least 1 mol%. (5) The recombinant hydrogen-oxidizing bacterium according to (3) or (4), wherein the second component is 3-hydroxyvaleric acid (3HV) and / or 3-hydroxy-4-methylvaleric acid (3H4MV). (6) The recombinant hydrogen-oxidizing bacterium according to any one of (1) to (5), wherein the hydrogen-oxidizing bacterium is Ralstonia eutropha. (7) The recombinant hydrogen-oxidizing bacterium according to any one of (1) to (6), wherein the monomer supply enhancing gene is present in the genomic DNA of the hydrogen-oxidizing bacterium. (8) A method for producing polyhydroxyalkanoic acid (PHA), (i) A step of introducing at least a 2-ketocarboxylic acid decarboxylase (kivd) gene and a phenylacetaldehyde dehydrogenase (padA) gene as monomer supply enhancing genes into a hydrogen-oxidizing bacterium having a PHA polymerase to obtain a transformant of the hydrogen-oxidizing bacterium, (ii) A step of culturing the obtained transformant in the presence of a carbon source, and (iii) A step of collecting the copolymerized PHA from the culture The production method comprising. (9) The production method according to (8), wherein in the step (i), a 3-ketothiolase (bktB) gene is further introduced as a monomer supply enhancing gene. (10) The production method according to (8) or (9), wherein the monomer supply enhancing gene is introduced by directly integrating it into the genomic DNA of the hydrogen-oxidizing bacterium or by introducing a plasmid having the gene into the hydrogen-oxidizing bacterium. (11) The production method according to (10), wherein the monomer supply enhancing gene is introduced by directly integrating it into the genomic DNA of the hydrogen-oxidizing bacterium. (12) The production method according to any one of (8) to (11), wherein the hydrogen-oxidizing bacterium having the PHA polymerase is obtained by introducing a gene encoding a PHA polymerase with broad substrate specificity into the hydrogen-oxidizing bacterium. (13) The production method according to any one of (8) to (12), wherein the carbon source is sugar or carbon dioxide. (14) The production method according to (13), wherein the sugar is fructose. (15) The production method according to (13), wherein the carbon dioxide is a mixed gas containing carbon dioxide in the range of 1 to 20% (v / v). (16) The production method according to any one of (8) to (15), wherein in the step (ii), a precursor of the second component is not added. (17) The production method according to any one of (8) to (16), wherein the introduction fraction of the second component in the copolymerized PHA is higher than the introduction fraction of the second component in the copolymerized PHA produced by hydrogen-oxidizing bacteria into which no monomer supply-enhancing gene has been introduced. (18) The production method according to (17), wherein the second component is 3-hydroxyvaleric acid (3HV) and / or 3-hydroxy-4-methylvaleric acid (3H4MV). (19) The production method according to any one of (8) to (18), wherein the hydrogen-oxidizing bacteria is Ralstonia eutropha. (20) The production method according to any one of (8) to (19), wherein the PHA polymerase gene encodes a PHA polymerase mutant (NSDG) in which asparagine at position 149 of the polyhydroxyalkanoic acid polymerase derived from Aeromonas caviae is substituted with serine and aspartic acid at position 171 is substituted with glycine, or a PHA polymerase (Psh) derived from Plesiomonas shigelloides.

Advantages of the Invention

[0011] By using recombinant hydrogen-oxidizing bacteria having the kivd gene and the padA gene, it is possible to produce a copolymerized PHA with an enhanced introduction fraction of the second component without using a precursor of the second component. In particular, by using recombinant hydrogen-oxidizing bacteria having the kivd gene, the padA gene, and the bktB gene, it is possible to produce a copolymerized PHA in which the introduction fraction of the second component is enhanced and the productivity of PHA is increased. Further, by directly introducing these genes into the genomic DNA of hydrogen-oxidizing bacteria, it is possible to achieve an enhancement of the introduction fraction of the second component and an increase in the productivity of PHA.

Brief Description of Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0013] Hereinafter, the present invention will be described in detail according to specific embodiments. However, the present invention is by no means limited to the following embodiments, and appropriate modifications can be made and implemented.

[0014] [Method for Producing PHA] The first embodiment of the present invention is a method for producing PHA, comprising: (i) introducing at least the kivd gene and the padA gene as monomer supply enhancing genes (hereinafter, together with the bktB gene described later, appropriately referred to as "monomer supply enhancing genes") into a hydrogen-oxidizing bacterium having a PHA polymerase to obtain a transformant of the hydrogen-oxidizing bacterium; (ii) culturing the obtained transformant in the presence of a carbon source; and (iii) collecting the copolymerized PHA from the culture. The present invention relates to a production method including these steps. The production method of the present invention is characterized in that it can produce a copolymerized PHA with an enhanced introduction fraction of the second component without using a precursor of the second component. In the step (i), it is preferable to further introduce the bktB gene as a monomer supply enhancing gene. By further introducing the bktB gene, the introduction amount of the second component can be further enhanced and the productivity of PHA can be improved.

[0015] In the present invention, the "introduction fraction of the second component" means the ratio (%) of the monomer of the second component constituting the produced PHA. The "second component" refers to a 3-hydroxyalkanoic acid (3HA) unit other than 3HB among the monomer units constituting PHA. Such 3HA units are, for example, 3HV and / or 3H4MV. In the present invention, when the introduction fraction of the second component in the copolymerized PHA produced by a hydrogen-oxidizing bacterium into which a monomer supply enhancing gene has been introduced is "higher" than the introduction fraction of the second component in the copolymerized PHA produced by a hydrogen-oxidizing bacterium into which no monomer supply enhancing gene has been introduced, although it depends on the type of the second component, for example, it is judged to be "higher" when the introduction fraction of the second component becomes twice or more. In this specification, instead of "higher", it may be expressed as "enhanced", "enhanced" or "enhanced". Note that PHA is appropriately referred to as copolymerized PHA.

[0016] The copolymer PHA of the present invention can be produced by any method as long as it is a method using hydrogen-oxidizing bacteria into which the above monomer supply-enhancing gene has been introduced. For example, the monomer supply-enhancing gene may be introduced into a plasmid vector or an artificial chromosome and then introduced into hydrogen-oxidizing bacteria (for example, Examples 1 to 2 described later). Alternatively, using known gene recombination techniques, the monomer supply-enhancing gene may be directly incorporated into the genomic DNA of hydrogen-oxidizing bacteria (for example, Examples 3 to 5 described later). "Direct incorporation into genomic DNA" in the present invention means introducing the required gene into a targeted genomic site, rather than introducing the target gene into an arbitrary site on the genome. The method of directly incorporating into genomic DNA is preferable because it can suppress fluctuations in the expression level, profile, etc. of the target gene and insert the target gene into a genomic site with accurate and well-characterized features.

[0017] The method of directly incorporating into genomic DNA is not particularly limited, and site-specific recombination is mentioned, for example. In the examples described later, this site-specific recombination is referred to as "genome editing" in the sense of modifying the genome. Site-specific recombination is a phenomenon in which DNA recombination occurs between specific homologous base sequences and is induced by a site-specific recombinase specific to the homologous base sequences. As a system for inducing site-specific recombination by a site-specific recombinase, for example, the Cre / loxP system derived from phage is generally used (for example, Sternberg, N. and Hamilton, D. (1981). “Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites”. J. Mol. Biol. 150 (4): 467-486. doi:10.1016 / 0022-2836(81)90375-2; Lee, G., Saito, I. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216, 55-65 (1998) https: / / doi.org / 10.1016 / S0378-1119(98)00325-4; U.S. Patent No. 4,959,317; U.S. Patent No. 5,658,772). Cre recombinase (sometimes simply referred to as “Cre”) is a DNA recombinase derived from phage P1, recognizes a 34-bp recognition sequence called the loxP sequence, and induces site-specific recombination. In addition to loxP, there are also mutant sequences such as lox511 (Hoess, R., et al. Nucleic Acids Res. 14, 2287-2300 (1986) doi: 10.1093 / nar / 14.5.2287), lox2272 (Lee, G., Saito, I. Gene 216, 55-65 (1998) doi: 10.1016 / S0378-1119(98)00325-4, Missirlis, P., et al., BMC Genom. 7:73 (2006) doi:1 0.1186 / 1471-2164-7-73), lox5171 (identical to lox2272 in the literature), loxFAS, etc. The Cre / loxP system is widely used for modifying gene structures on genomic DNA, such as deletion, substitution, or inversion of the DNA region sandwiched between two loxP sequences, two mutant loxP sequences, or a loxP sequence and a mutant loxP sequence.When two loxP sequences are arranged in opposite directions to each other, the action of Cre causes the direction of the DNA between the loxP to be reversed. Examples of site-specific recombination systems include, in addition to the Cre / loxP system, the Flp / FRT system derived from yeast plasmid 2μ, the Dre / rox system derived from enterobacterial phage D6, the R / RS system derived from soy sauce yeast, and the like.

[0018] Some wild-type hydrogen-oxidizing bacteria originally have the ability to metabolize 2-ketoisovalerate to isobutyryl-CoA, although at a low level, and the ability to metabolize isobutyryl-CoA to 3H4MV-CoA and propionyl-CoA to 3HV-CoA. However, 2-ketoisovalerate, which is a precursor of the monomer component 3H4MV, is used for the biosynthesis of branched-chain amino acids in the cell, and the enzymatic reaction involved in the biosynthesis of 2-ketoisovalerate from pyruvate is subject to feedback inhibition by the produced branched-chain amino acids, resulting in a low introduction fraction of the second component of 3HV and 3H4MV (Non-Patent Documents 3 and 4).

[0019] Figure 1 shows a typical biosynthetic pathway of P(3HB-co-3HV-co-3H4MV), which is an example of the copolymerized PHA obtained by the production method of the present invention. The sugar as a carbon source is converted to pyruvate and then to acetyl-CoA. This acetyl-CoA is dimerized by β-ketothiolase (PhaA) and reduced by acetoacetyl-CoA reductase (PhaB) to be converted to 3HB-CoA (referred to as Pathway (A)). On the other hand, pyruvate is converted to 2-ketoisovalerate, and then 2-ketoisovalerate is converted to isobutyryl-CoA (referred to as Pathway (B)). This isobutyryl-CoA is condensed with acetyl-CoA by BktB and then reduced by PhaB to be converted to 3H4MV-CoA (referred to as Pathway (C)). Isobutyryl-CoA is converted to propionyl-CoA, then condensed with acetyl-CoA by BktB, and then reduced by PhaB to be converted to 3HV-CoA (referred to as Pathway (D)). The monomers supplied through routes (A) to (D) are utilized as substrates for the PHA polymerase, and P(3HB-co-3HV-co-3H4MV) is biosynthesized.

[0020] The main enzymes that catalyze route B are Kivd and PadA, and the main enzyme that catalyzes routes C and D is BktB. When denoted as "kivd", "padA", or "bktB", it means the gene, and when denoted as "Kivd", "PadA", or "BktB", it means the enzyme encoded by the corresponding gene.

[0021] Kivd is not particularly limited as long as it has the action of converting 2-ketoisovaleric acid to isobutyraldehyde. The origin of the kivd is not particularly limited, but those derived from prokaryotes are preferred. As an example, 2-ketoacid decarboxylase (GenBank: ADA65057.1) derived from Lactococcus lactis subsp. lactis KF147 can be mentioned. PadA is not particularly limited as long as it has the action of converting isobutyraldehyde to isobutyric acid. Examples include phenylacetaldehyde dehydrogenase (Gene ID: 945933) derived from Escherichia coli str. K-12, and from other organisms, those derived from Shigella dysenteriae (WP_000138640.1), Enterobacter cloacae complex (WP_028017554.1), Pluralibacter gergoviae (WP_048284376.1), Buttiauxella agrestis (WP_034495388.1), Klebsiella quasipneumoniae (CEL82223.1), etc. As BktB, there is no particular limitation as long as it has the action of converting isobutyryl-CoA into 3-oxo-4-methylvalerate-CoA and converting propionyl-CoA into 3-oxovalerate-CoA. Examples include 3-ketothiolase derived from Ralstonia eutropha H16.

[0022] Kivd, PadA, and BktB may be their variants. Such variants consist of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of each wild-type enzyme, and have corresponding activities. Specifically, the variant of Kivd has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the amino acid sequence of wild-type Kivd, and has 2-ketoacid decarboxylase activity. The variant of PadA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the amino acid sequence of wild-type PadA, and has phenylacetaldehyde dehydrogenase activity. The variant of BktB has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the amino acid sequence of wild-type BktB, and has 3-ketothiolase activity.

[0023] The "identity" of two amino acid sequences refers to the ratio at which the same amino acid residues appear at each corresponding position when the two amino acid sequences are aligned. The "homology" of two amino acid sequences refers to the ratio at which similar amino acid residues appear at each corresponding position when the two amino acid sequences are aligned. The two amino acid sequences to be compared are appropriately aligned, the identical residues existing in each sequence are determined, the number of matching sites is determined, and then the number of matching sites is divided by the total number of residues in the sequence region to be compared, and the resulting value is multiplied by 100. For example, it can be determined using a program such as BLAST (Basic Local Alignment Search Tool) (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

[0024] Each of the genes kivd, padA, and bktB encodes Kivd, PadA, and BktB, respectively. The DNA encoding each enzyme can be easily obtained by well-known methods such as PCR using genomic DNA or cDNA derived from the respective microorganisms as a template. Each of the genes kivd, padA, and bktB may be a variant thereof. Such a variant consists of a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added in the nucleotide sequence of the wild-type gene.

[0025] Specifically, the variant of the kivd gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the nucleotide sequence of the gene. The variant of the padA gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the nucleotide sequence of the gene. The mutant of the bktB gene has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the nucleotide sequence of the said gene.

[0026] The "identity" of two nucleotide sequences refers to the ratio of the same nucleotides appearing at each corresponding position when the two nucleotide sequences are aligned, and the "homology" of two nucleotide sequences refers to the similarity or correlation between the two nucleotide sequences. For example, it can be determined using programs such as the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

[0027] The host microorganism used in the present invention is not particularly limited as long as it is a hydrogen-oxidizing bacterium (also referred to as "hydrogen bacterium"). "Hydrogen-oxidizing bacterium" means a bacterium that oxidizes free hydrogen and uses the energy generated by this reaction to perform carbon assimilation. Examples of hydrogen-oxidizing bacteria include bacteria of the genus Ralstonia such as Ralstonia eutropha, bacteria of the genus Alcaligenes such as Alcaligenes latus, and bacteria of the genus Hydrogenovibrio such as Hydrogenovibrio marinus. Among these, Ralstonia eutropha is preferred in terms of its ability to grow under conditions where gaseous carbon dioxide or carbonate is the sole carbon source, the fact that its whole genome information has been analyzed, and the fact that a gene recombination method has been established (Cramm, R. et al., J. Mol. Microbiol. Biotechnol., 16, 38-52(2009)). Examples include Ralstonia eutropha H16 strain (ATCC17699), Ralstonia eutropha PHB - 4 strain (DSM541).

[0028] In the Ralstonia eutropha H16 strain, usually, the reaction of acetolactate synthase small subunit (IlvH) in the PHA biosynthesis pathway is inhibited by branched-chain amino acids. Therefore, it becomes difficult to overproduce branched-chain amino acids intracellularly, and as a result, the introduction fraction of monomer components decreases. Therefore, when using the Ralstonia eutropha H16 strain as a hydrogen-oxidizing bacterium, it is preferable to introduce a mutation (substituting alanine at the 36th position with tyrosine) into IlvH in order to suppress feedback inhibition by branched-chain amino acids. On the other hand, Ralstonia eutropha 1F2 is known as a mutant strain in which the said feedback inhibition is suppressed.

[0029] In the present invention, the "PHA polymerase" in the "hydrogen-oxidizing bacterium having a PHA polymerase" may be a PHA polymerase originally possessed by the hydrogen-oxidizing bacterium or one externally provided. Some hydrogen-oxidizing bacteria originally have the ability to produce PHA, but that is mainly the ability to produce a homopolymer of HB (P(HB)), and they can hardly synthesize a copolymerized PHA further containing HA units other than HB. Therefore, the main PHA polymerase originally possessed by the hydrogen-oxidizing bacterium is an enzyme that produces P(HB). Therefore, it is preferable to externally provide a PHA polymerase even for a hydrogen-oxidizing bacterium that originally has a PHA polymerase. Such an externally provided PHA polymerase is referred to as a "broad-substrate-specific PHA polymerase" or simply "PHA polymerase". That is, the "broad-substrate-specific PHA polymerase" refers to an enzyme whose polymerization activity for synthesizing PHA is not limited to only (R)-HB-CoA. By introducing a gene encoding a broad-substrate-specific PHA polymerase (hereinafter, appropriately referred to as "PHA polymerase gene" or "phaC") into a host microorganism, a copolymerized PHA further containing HA units other than HB can be synthesized, and the composition of the copolymerized PHA can be controlled. Therefore, in step (i) of the production method of the present invention, it is preferable to further introduce a PHA polymerase gene into the hydrogen-oxidizing bacterium.

[0030] The PHA polymerase gene of the present invention may be introduced into hydrogen-oxidizing bacteria, which are host microorganisms, using a plasmid vector containing the gene, or may be directly introduced onto the genomic DNA possessed by hydrogen-oxidizing bacteria. When using a plasmid vector, it may be introduced into the same plasmid vector as, for example, a plasmid vector into which a monomer supply enhancement gene has been introduced, or it may be introduced into a separate plasmid vector. Specifically, a method can be used in which the PHA polymerase gene of the present invention, a known monomer supply gene, and a monomer supply enhancement gene are inserted into a broad-host-range vector for expressing the target gene in a host microorganism to obtain a plasmid, and the plasmid vector is introduced into the host microorganism. When not using a plasmid vector, known gene recombination techniques, for example, the methods described for the method of introducing a monomer supply enhancement gene, can be used. The PHA polymerase gene of the present invention may be introduced by any of the above methods, but from the viewpoint of PHA productivity, it is preferably directly introduced onto the DNA possessed by hydrogen-oxidizing bacteria.

[0031] As the broad-host-range vector for expressing the target gene in the host, a known vector having a promoter, a ribosome binding site, a gene cloning site, a terminator, etc. can be used.

[0032] Examples of known monomer supply genes include the β-ketothiolase gene (phbA, phaA) derived from Ralstonia eutropha, the acetoacetyl-CoA reductase gene (phbB, phaB) (Peoples, O. P. and Sinskey, A. J., J. Biol. Chem. 264:15293-15297 (1989)), the R-hydratase gene (phaJ) derived from Aeromonas caviae (Fukui, T. and Doi, Y., J. Bacteriol. 179:4821-4830 (1997)), and the like.

[0033] Examples of PHA synthase genes include those derived from microorganisms selected from Pseudomonas sp. 61-3 strain, Pseudomonas stutzeri, Pseudomonas sp. A33, Allochromatium vinosum, Bacillus megaterium, Bacillus cereus, Bacillus sp. INT005, Lamprocystis roseopersicina, Nocardia corallina, Rhodobactor shaeroides, Ralstonia eutropha, Rhodococcus sp. NCIMB 40126, Thiocapsa pfennigii, Aeromonas caviae, and Aeromonas hydrophila. Examples of PHA synthase genes also include those derived from microorganisms selected from Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii (PCT / JP2023 / 037027).

[0034] In addition, as the PHA polymerase gene, a gene encoding a mutant of the above PHA polymerase may also be used. Such a mutant is a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the wild-type PHA polymerase, and having PHA polymerization activity. The mutant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or homology to the amino acid sequence of the wild-type PHA polymerase, and having PHA polymerization activity. The definition of identity or homology is as described above. By using such a mutant, the production amount of the PHA polymer is increased. For example, as the PHA polymerase mutant used in the present invention, mutants including substitution of the 149th asparagine from the N-terminus of the PHA polymerase derived from Aeromonas caviae with serine, and / or substitution of the 171st aspartic acid of PhaC with glycine can be mentioned.

[0035] Furthermore, upstream of the PHA polymerase gene, a gene encoding a protein called phasin (hereinafter, appropriately referred to as "PhaP") is introduced (see, for example, JP-A-2013-42697). Phasin is known to co-localize with PHA granules in bacterial cells and is considered to be involved in the formation and stabilization of PHA granules. The microorganism from which PhaP is derived is, for example, a microorganism of the genus Aeromonas, and specifically, Aeromonas caviae can be mentioned.

[0036] As PhaP, mutants having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid region from the N-terminus to the 20th amino acid are also preferably used. For example, a mutant in which the 4th asparagine from the N-terminus of PhaP is substituted with asparagine can be mentioned (see, for example, JP-A-2013-42697). The mutant of phasin similarly improves the productivity of PHA.

[0037] Variants that include all of the above-mentioned substitution of the 149th amino acid of PhaC, substitution of the 171st amino acid of the same enzyme, and substitution of the 4th amino acid of PhaP are also preferred. Hereinafter, a variant in which the 4th aspartic acid of PhaP is substituted with asparagine will be referred to as "PhaP Ac D4N", and a double mutant in which the 149th asparagine of PhaC is substituted with serine and the 171st aspartic acid of PhaC is substituted with glycine will be referred to as "PhaC Ac NSDG" or simply "NSDG". PhaP Ac The method for producing D4N is disclosed, for example, in JP-A-2013-42697, and the method for producing PhaC Ac The method for producing NSDG is described in detail, for example, in Tsuge T, et al., FEMS Microbial Lett 277(2007)217-222.

[0038] As the carbon source, for example, sugars, carboxylic acids, oils and fats, carbon dioxide, etc. can be used. Among them, sugars are preferred because they have a weak inhibitory effect on cell growth. Also, carbon dioxide is preferred because of its low environmental impact. Examples of sugars include glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, starch, starch hydrolysates, etc. Among them, fructose is preferred because certain hydrogen-oxidizing bacteria selectively assimilate fructose. Examples of carboxylic acids include acetic acid, lactic acid, etc. As oils and fats, vegetable oils are preferred, such as soybean oil, corn oil, cottonseed oil, peanut oil, coconut oil, palm oil, palm kernel oil, or their fractionated oils, such as palm W olein (low-boiling fraction obtained by solvent-free fractionation of palm oil twice), palm kernel olein (low-boiling fraction obtained by solvent-free fractionation of palm kernel oil once), or synthetic oils obtained by chemically or biochemically treating these oils and their fractions, or their mixed oils.

[0039] When using carbon dioxide, examples include air enriched with carbon dioxide by bubbling. In the present invention, since hydrogen-oxidizing bacteria are used as host microorganisms, a mixed gas containing hydrogen (H2), oxygen (O2), and carbon dioxide (CO2) can be used. Further, the mixed gas may contain other components such as ammonia, nitrogen, hydrocarbons, carbon monoxide, formaldehyde, water vapor, etc. The proportion of carbon dioxide in the mixed gas is, for example, 1 to 20% (v / v), preferably 1 to 15% (v / v), more preferably 3 to 15% (v / v).

[0040] The culture temperature is a temperature at which the bacteria can grow, preferably 15 to 40°C, particularly preferably 20 to 40°C, and even more preferably 28 to 34°C. The culture time is not particularly limited, but for example, in batch culture, 1 to 7 days is preferable, and continuous culture is also possible. The culture medium is not particularly limited as long as it can be utilized by the host of the present invention. A medium containing a nitrogen source, inorganic salts, other organic nutrient sources, etc. in addition to a carbon source can be used. Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, diammonium hydrogen phosphate, etc., peptone, meat extract, yeast extract, etc. Examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium hydrogen phosphate, magnesium sulfate, sodium chloride, etc. Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, proline, etc.; vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, vitamin C, etc.

[0041] The recovery of the copolymerized PHA of the present invention from the bacterial cells can be carried out, for example, by the following method. After the completion of the culture, the bacterial cells are separated from the culture broth using a centrifuge or the like, and the bacterial cells are washed with distilled water, methanol or the like and dried. Then, from the dried bacterial cells, the copolymer is extracted using an organic solvent such as chloroform. Next, the bacterial cell components are removed from the organic solvent solution containing the copolymer by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the copolymer. The supernatant can be removed from the precipitated copolymer by filtration or centrifugation and dried to recover the copolymer. As another means, the recovery of the copolymerized PHA from the bacterial cells can also be carried out by the following method. The bacterial cells containing the copolymerized PHA are dispersed in an aqueous solvent containing a surfactant or the like, and the bacterial cells are disrupted using an ultrasonic crusher or a high-pressure homogenizer. Then, the insoluble components are recovered by filtration or centrifugation. The recovered insoluble components are washed with an appropriate solvent such as water and dried to recover the copolymerized PHA. The analysis of the obtained copolymer can be carried out, for example, by gas chromatography, nuclear magnetic resonance method or the like.

[0042] In the copolymerized PHA of the present invention, the introduction fraction of the second component in the copolymerized PHA is at least twice as high as the introduction fraction of the second component in the copolymerized PHA biosynthesized using a hydrogen-oxidizing bacterium having no monomer supply enhancing gene, and depending on the type of the second component, it is enhanced several times to about 10 times. The introduction fraction of the second component is at least 1 mol% in the copolymerized PHA composition, preferably 2 mol% or more, more preferably 4 mol% or more, still more preferably 6 mol% or more, and particularly preferably 10 mol% or more. In particular, the introduction fraction of 3HV as the second component is significantly higher compared to a hydrogen-oxidizing bacterium having no monomer supply enhancing gene at all, and is at least 4 mol% in the copolymerized PHA composition. Therefore, high flexibility can be expected. This indicates that a copolymerized PHA having high flexibility can be biosynthesized even in the absence of the precursor of the second component. Flexible physical properties are a great advantage during processing as a plastic material. Therefore, the copolymerized PHA of the present invention can be used for applications such as films, sheets, containers, bottles, packaging materials and the like.

[0043] [Recombinant hydrogen-oxidizing bacteria] The second embodiment of the present invention relates to a recombinant hydrogen-oxidizing bacterium having at least the kivd gene and the padA gene as monomer supply-enhancing genes and having the ability to produce copolymerized PHA. It is preferable that the recombinant hydrogen-oxidizing bacterium further has the bktB gene as a monomer supply-enhancing gene. It is preferable that the recombinant hydrogen-oxidizing bacterium of the present invention has the monomer supply-enhancing gene in the genomic DNA of the hydrogen-oxidizing bacterium (particularly, a specific site of the genomic DNA). A recombinant hydrogen-oxidizing bacterium having such a monomer supply-enhancing gene in the genomic DNA of the hydrogen-oxidizing bacterium can be produced by the above-mentioned "direct integration into genomic DNA". In the present invention, "having the ability to produce copolymerized PHA" means that the recombinant hydrogen-oxidizing bacterium only needs to have the ability to produce copolymerized PHA, and the ability to produce copolymerized PHA may be inherent in the hydrogen-oxidizing bacterium itself or may be imparted to the hydrogen-oxidizing bacterium. Even if the hydrogen-oxidizing bacterium itself has the ability to produce copolymerized PHA, when the biosynthesis of the copolymer cannot be carried out or the ability to control the composition of the copolymer is low, the ability to produce copolymerized PHA may be imparted from the outside. Specific examples of hydrogen-oxidizing bacteria having the ability to produce PHA include those belonging to the genus Ralstonia, the genus Cupriavidus, and the like.

[0044] In the recombinant hydrogen-oxidizing bacterium, the introduction fraction of the second component in the copolymerized PHA is higher than that of the copolymerized PHA produced by a hydrogen-oxidizing bacterium having no monomer supply-enhancing gene at all. The introduction fraction of the second component is at least 1 mol% in the copolymerized PHA composition, preferably 2 mol% or more, more preferably 4 mol% or more, still more preferably 6 mol% or more, and particularly preferably 10 mol% or more. In particular, the introduction fraction of 3HV as the second component is significantly higher compared to a hydrogen-oxidizing bacterium having no three monomer supply-enhancing genes and is at least 4 mol% in the copolymerized PHA composition.

[0045] Note that the definition of the "second component" is as described in the first embodiment of the present invention. Also, the kivd gene, the padA gene, and the bktB gene, as well as the method for producing the recombinant hydrogen-oxidizing bacterium, are as described in the first embodiment.

[0046] The third embodiment of the present invention is a recombinant hydrogen-oxidizing bacterium having any one of the monomer supply-enhancing genes of the kivd gene, the padA gene, and the bktB gene and having the ability to produce copolymerized PHA, and a method for producing the same. Such a recombinant hydrogen-oxidizing bacterium also has a higher introduction fraction of the second component in the copolymerized PHA than the introduction fraction of the second component in the copolymerized PHA produced by a hydrogen-oxidizing bacterium having no monomer supply-enhancing gene at all.

[0047] Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples at all.

Examples

[0048] Example 1 Production of PHA Using Fructose as a Carbon Source (1) Construction of Plasmid MCS-3_P tac -bktb-kivd-padA First, for the kivd gene, the kivd gene derived from Lactobacillus lactis KF147 (GenBank: ADA65057.1) was used, and for the padA gene, the padA gene derived from Escherichia coli K-12 strain (Gene ID: 945933) was used. In both cases, the codons were optimized for Escherichia coli and chemically synthesized products were obtained from Eurofins Genomics Co., Ltd. The nucleotide sequences are shown.

[0049] Nucleotide Sequence of kivd Gene (SEQ ID NO: 1)

[0050] Base sequence of padA gene (SEQ ID NO: 2)

[0051] The bktB gene was obtained from Ralstonia eutropha H16 (Kyoto Encyclopedia of Genes and Genomes: KEGG) (https: / / www.genome.jp / kegg / catalog / org_list.html).

[0052] The nucleotide sequence of the bktB gene (SEQ ID NO: 3)

[0053] Next, using the kivd and padA genes as DNA templates, PCR amplification was performed using forward primer 1, reverse primer 2, forward primer 3, and reverse primer 4. Forward primer 1: 5’-TCTGAGGTTAGCCTTGGTACCgaaggagatataca-3’ (SEQ ID NO: 4) Reverse primer 2: 5’-tgtatatctccttcctcgagtcaggatttgttct-3’ (SEQ ID NO: 5) Forward primer 3: 5’-agaacaaatcctgactcgaggaaggagatataca-3’ (SEQ ID NO: 6) Reverse primer 4: 5’-AAAGGGAACAAAAGCTGGGTACCtcaatagcgtac-3’ (SEQ ID NO: 7) Next, the obtained PCR product was used as a DNA template, and overlap PCR was performed using forward primer 1 and reverse primer 4 to obtain a kivd-padA DNA fragment (3.2 kb). Subsequently, the kivd-padA DNA fragment was digested with the restriction enzyme KpnI, and PBBR1”C1 Ps AB Re _P tac The plasmid pBBR1”C1 inserted into the KpnI site of bktB (Miyahara Y, Yamamoto M, Thorbecke R, Mizuno S, Tsuge T (2020), Biotechnol. Lett., 42, 1655-1662) Ps AB Re _P tac bktB-kivd-padA (14.5 kb) (Figure 2) was constructed.

[0054] Next, pBBR1”C1 Ps AB Re _P tac Using bktB-kivd-padA as a DNA template, PCR amplification was performed using forward primer 5, reverse primer 6, forward primer 7, and reverse primer 8. Forward primer 5: 5’-aaagggaacaaaagctgggtacctcaatagcgtac-3’ (SEQ ID NO: 8) Reverse primer 6: 5’-ttcgagcgtatctgaggttagccttgaaggagata-3’ (SEQ ID NO: 9) Forward primer 7: 5’-atgtatatctccttcaaggctaacctcagatacgc-3’ (SEQ ID NO: 10) Reverse primer 8: 5’-ggccgctctagaactagtggatcccccgggctg-3’ (SEQ ID NO: 11)

[0055] Thereby, gene fragments of the tac promoter (P tac ), bktB, kivd, and padA were obtained. Next, the broad-host-range vector pBBR1MCS-3 (Kovach ME, Elzer PH, Hill DS et al., Gene 1995; 166: 175-6. DOI: 10.1016 / 0378-1119(95)00584-1) (5.2 kbp) was digested with the restriction enzymes KpnI and SpeI. Using the Takara In-Fusion (registered trademark) HD Cloning Kit (manufactured by Takara Bio Inc.), the gene fragments of P tac , bktB, kivd, and padA were inserted into pBBR1MCS-3. Thereby, plasmid MCS-3_P tac -bktb-kivd-padA (9.8 kbp) (Figure 3) was obtained.

[0056] (2) Construction of plasmid MCS-3_P tac -bktb (1) The plasmid MCS-3_P tac -bktb-kivd-padA obtained in (1) was digested with the restriction enzyme KpnI, blunt-ended using T4 DNA polymerase, and then self-ligated to obtain plasmid MCS-3_P tac -bktb.

[0057] (3) Plasmid pJRD215_PAc Construction of NSDG pBBREE”P Ac The NSDG vector (7.5 kb) (Watanabe Y, Ichinomiya Y, Shimada D, Saika A, Abe H, Taguchi S, Tsuge T, J. Biosci. Bioeng., 2012, 113:286-292, DOI: 10.1016 / j.jbiosc.2011.10.015) and the pJRD215 vector (Davison J, Heusterspreute M, Chevalier N, Ha-Thi V, Brunel F, Gene, 1987, 51, 275-280) were digested with the restriction enzymes EcoRI and BamHI, and the pha promoter of Aeromonas caviae FA440 strain and the phaCNSDG gene of Aeromonas caviae FA440 strain were inserted into the EcoRI and BamHI sites of the pJRD215 vector. Thereby, pJRD215_P, a plasmid for PHA polymerization, was obtained. Ac NSDG (12.1 kbp) was obtained. Note that phaCNSDG is a double mutant of PHA polymerase in which the 149th asparagine of PhaC is replaced by serine and the 171st aspartic acid of PhaC is replaced by glycine, and the method for producing the same is described in detail in, for example, Tsuge T, et al., FEMS Microbial Lett 277(2007)217-222.

[0058] (4) Transformation of hydrogen-oxidizing bacteria The enzyme involved in the biosynthesis of 2-ketoisovaleric acid (acetolactate synthase small subunit: IlvH) has its enzymatic reaction inhibited by branched amino acids, making it difficult to overproduce branched amino acids intracellularly. As a result, the introduction fraction of monomer components decreases. In the copolymerization PHA biosynthesis, Ralstonia eutropha 1F2, a mutant strain in which a mutation (A36T) was introduced into the ilvH gene and feedback inhibition by branched amino acids was suppressed, was used as the host (Journal of Biotechnology, vol. 197, No. 8, 2015; doi:10.1128 / JB.02474-14). The plasmid pJRD215_P prepared in (3) was introduced into Ralstonia eutropha 1F2 using the electroporation method (applied voltage 1.5 kV). Ac -NSDG and MCS-3_P constructed in (1) tac -bktB-kivd-padA were introduced to produce recombinant strains. Also, for comparison, pJRD215_P Ac -NSDG and the plasmid MCS-3_P prepared in (2) tac -bktB were introduced to produce recombinant strains.

[0059] (5) Biosynthesis and analysis of PHA The prepared recombinant strains were inoculated into 2 mL of NR medium (2.0 g / L yeast extract, 10 g / L Bacto-tryptone, 10 g / L dried bonito extract) and cultured with shaking overnight at 30 °C to obtain preculture solutions. Subsequently, 100 mL of MS medium (9.0 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO 4、1 mL of the preculture was inoculated into (0.5 g / L NH4Cl, 0.2 g / L MgSO4·7H2O, 1 mL trace elements solution), and shake culture was carried out in a Sakaguchi flask at 30 °C for 72 hours. The "trace elements solution" contains 0.218 g / L CoCl2·6H2O, 20.5 g / L FeCl3·6H2O, 7.8 g / L CaCl2, 0.118 g / L NiCl2·6H2O, 0.105 g / L CrCl3·6H2O, and 0.156 g / L CuSO4·5H2O in 0.1 N HCl. In addition, in all cultures, 100 μg / mL kanamycin and 10 μg / mL tetracycline were added to maintain the plasmid intracellularly.

[0060] After the culture was completed, the culture broth was transferred to a centrifuge tube and centrifuged at 5000×g for 10 minutes to recover the cells. Then, the cell pellet was suspended in 50 mL of pure water and centrifuged again. This operation was performed twice to remove the remaining medium components and metabolites contained in the culture supernatant. The obtained cell pellet was dried using a freeze dryer to obtain dry cell powder. The obtained dry cells were suspended in 15 mL of a 4% aqueous sodium dodecyl sulfate solution, and ultrasonic disruption was performed at 12 W for 10 minutes to extract the PHA accumulated intracellularly. Approximately 10 - 15 mg of the extracted PHA sample was dissolved in 1 mL of deuterated chloroform (CDCl3), 1 and subjected to 1H NMR (BioSpin Avance III 400A (Bruker) and BioSpin Avance III HD 500) analysis to determine the monomer composition. The monomer composition of PHA was determined by calculating the ratio between the peak areas in the methine region of the NMR spectrum. The results are shown in Table 1.

[0061]

Table 1

[0062] The results in Table 1 indicate that the introduction fractions of 3HV and 3H4MV monomers were enhanced by introducing three genes, bktB, kivd, and padA, into hydrogen-oxidizing bacteria (Run 3).

[0063] Example 2 Production of PHA Using Carbon Dioxide as a Carbon Source The strains used and the preculture method were the same as in Example 1. 1 mL of the preculture solution was inoculated into a 250 mL jar fermenter (ABLE Corporation) containing 100 mL of MS medium. Subsequently, a mixed gas (H2:O2:CO2:N2 = 3.8:7.3:13.0:75.9% (v / v)) was supplied at a flow rate of 5 mL / min or 10 mL / min, and culturing was performed at a stirring speed of 1200 rpm and a culture temperature of 30 °C for 162 hours. To prevent foaming of the culture solution, 0.01 wt% of Antifoam 204 (manufactured by Sigma) was added to the medium. In all cultures, 100 μg / mL of kanamycin and 10 μg / mL of tetracycline were added for culturing to maintain the plasmid intracellularly.

[0064] In the same manner as in Example 1, cell collection and preparation of dried cells were carried out. The PHA content and PHA monomer composition were measured by gas chromatography (GC) using a Shimadzu GC-2014s apparatus (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. The preparation of the GC sample was carried out as follows. 20 mg of dried cells were weighed into a screw-cap test tube, 2 mL of sulfuric acid methanol (15 vol% sulfuric acid) and 2 mL of chloroform were added, and a methyl esterification reaction was carried out at 100 °C for 140 minutes. After completion of the reaction, 1 mL of ultrapure water was added and stirred vigorously, and the chloroform layer was recovered. An equal volume of chloroform solution containing 0.1% (w / v) methyl-n-octane as an internal standard was added to the recovered solution to prepare a final sample for GC analysis. The sample was injected through a GC capillary column InertCap 1 (30 m × 0.25 mm, GL Science). The column temperature was initially held at 90 °C for 2 minutes, then raised to 110 °C at a rate of 5 °C / min, and then further raised to 280 °C at a rate of 20 °C / min. From the obtained signal peak areas, the total PHA content and the monomer composition of PHA were calculated. The results are shown in Table 2.

[0065]

Table 2

[0066] The results in Table 2 show that the introduction fractions of 3HV and 3H4MV monomers were enhanced by introducing the three genes of bktB, kivd, and padA into hydrogen-oxidizing bacteria.

[0067] Example 3 Production of PHA Using Fructose as a Carbon Source (1) Preparation of Ralstonia eutropha 1F2ΔphaC1::phaCNSDG (a) Construction of Plasmid pk18-phaCNSDG To insert the phaCNSDG gene represented by SEQ ID NO: 12 into the Ralstonia eutropha 1F2ΔphaC1 strain (PHA polymerization ability-deficient strain), plasmid pk18-phaCNSDG was constructed. The plasmid pJRD215_P prepared in (3) of Example 1 Ac Using NSDG as a template, PCR amplification was performed using forward primer 1 (SEQ ID NO: 13) and reverse primer 2 (SEQ ID NO: 14) to amplify a phaCNSDG gene fragment (1.8 kb). Next, using the genomic DNA of Ralstonia eutropha H16 as a template, DNA fragments of 0.8 - 1 kb upstream and downstream of the phaC1 gene on the genome were obtained using forward primer 3, reverse primer 4, forward primer 5, and reverse primer 6 (SEQ ID NOs: 15 - 18 in order). Next, overlap PCR was performed using the obtained DNA fragments as templates. As a result, a fragment (3.6 kb) (SEQ ID NO: 19) was obtained by connecting the gene fragments upstream and downstream of the phaC1 gene of Ralstonia eutropha H16 and the phaCNSDG gene fragment. The obtained DNA fragment was inserted into the SphI and EcoRI sites of pK18mobsacB (DOI: 10.1016 / 0378 - 1119(94)90324 - 7) to construct the plasmid pk18 - phaCNSDG.

[0068] The nucleotide sequence of the phaCNSDG gene (SEQ ID NO: 12)

[0069] Forward primer 1: 5’-AGAGACAATCAAATCATGAGCCAACCATCTTATGG-3’ (SEQ ID NO: 13) Reverse primer 2: 5’-GCACTCATGCAAGCGTCATGCGGCGTCCTCCTCTG-3’ (SEQ ID NO: 14) Forward primer 3: 5’-aaaGAATTCCGGGCAAGTACCTTGCCGACAT-3’ (SEQ ID NO: 15) Reverse primer 4: 5’-GTTGGCTCATGATTTGATTGTCTCTCTGCCGTCAC-3’ (SEQ ID NO: 16) Forward primer 5: 5’-CGCCGCATGACGCTTGCATGAGTGCCGGCGTGCGT-3’ (SEQ ID NO: 17) Reverse primer 6: 5’-aaaGCATGCACTCGGCGCGCGACAGGGCGCGCTTG-3’ (SEQ ID NO: 18)

[0070] Nucleotide sequence of the 3.6 kb DNA fragment obtained by overlap PCR (SEQ ID NO: 19)

[0071] (b) Construction of Ralstonia eutropha 1F2ΔphaC1::phaCNSDG strain In order to replace the phaC1 gene of Ralstonia eutropha 1F2 strain with the phaCNSDG gene represented by SEQ ID NO: 12, homologous recombination was performed using the plasmid pk18-phaCNSDG constructed in (a). First, the plasmid pk18-phaCNSDG was introduced into Escherichia coli S17-1 strain by the heat shock method to transform the Escherichia coli S17-1 strain. This transformant was inoculated into an LB medium (5.0 g / L yeast extract, 10 g / L bacto-tryptone, 10 g / L NaCl) containing 50 μg / mL kanamycin and cultured with shaking at 37°C overnight. On the other hand, the Ralstonia eutropha 1F2 strain was inoculated into an NR medium (2.0 g / L yeast extract, 10 g / L bacto-tryptone, 10 g / L skipjack extract) and cultured with shaking at 30°C overnight. Next, 2 mL of the culture solution of the transformant of Escherichia coli S17-1 strain and the culture solution of Ralstonia eutropha 1F2 strain were each collected, centrifuged at 10000×g for 2 minutes to prepare cell pellets. The cell pellets were washed 3 times with 1 mL of NR medium to remove the antibiotics contained in the medium. The washed cell pellets were suspended in 50 μL of NR medium to prepare a cell suspension in which the two types of cells were mixed. 100 μL of the cell suspension was dropped onto an NR agar medium and incubated at 30°C for 24 hours to perform conjugation transfer.

[0072] Subsequently, the cultured cells were suspended in physiological saline and inoculated onto Simmons citrate agar medium (2 g / L trisodium citrate dihydrate, 5.0 g / L NaCl, 1.0 g / L KH2PO4, 1.0 g / L NH4H2PO4, 0.2 g / L MgSO4·7H2O) containing 200 μg / mL kanamycin and 10 μg / mL gentamicin, and cultured at 30 °C for 2 days. The formed colonies were inoculated into NR medium (2 mL) containing 200 μg / mL kanamycin and cultured at 30 °C overnight. The culture solution was suspended in physiological saline to prepare a dilution solution, which was inoculated onto MSY medium (MS medium (refer to Miyahara Y, Wang Chih-Ting, Ishii-Hyakutake M, Tsuge T, Bioengineering 2022, 9(10), 586. DOI: https: / / doi.org / 10.3390 / bioengineering9100586) with 1.0 g / L yeast extract added) containing 150 g / L sucrose, and cultured at 30 °C for 2 days. The formed colonies were collected, and colony PCR was performed using Primer 3 and Primer 6 to confirm the construction of the Ralstonia eutropha 1F2ΔphaC1::phaCNSDG strain.

[0073] (2) Preparation of recombinant strains MCS-3_Ptac-bktB-kivd-padA prepared in Example 1 was introduced into Ralstonia eutropha 1F2ΔphaC1::phaCNSDG prepared in (1) using the electroporation method (applied voltage 1.5 kV) to prepare a recombinant strain. Also, for comparison, plasmid MCS-3 was introduced into Ralstonia eutropha 1F2ΔphaC1::phaCNSDG to prepare a recombinant strain.

[0074] (3) Biosynthesis and analysis of PHA The prepared recombinant strain was inoculated into 2 mL of NR medium (2.0 g / L yeast extract, 10 g / L Bacto-tryptone, 10 g / L skipjack extract), and cultured with shaking overnight at 30 °C to obtain a preculture solution. Next, 1 mL of the preculture solution was inoculated into 100 mL of MS medium (9.0 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4, 0.5 g / L NH4Cl, 0.2 g / L MgSO4·7H2O, 1 mL trace elements solution) containing 10 g / L fructose, and cultured with shaking at 30 °C for 72 hours. The "trace elements solution" contains 0.218 g / L CoCl2·6H2O, 20.5 g / L FeCl3·6H2O, 7.8 g / L CaCl2, 0.118 g / L NiCl2·6H2O, 0.105 g / L CrCl3·6H2O, 0.156 g / L CuSO4·5H2O in 0.1 N HCl. In addition, in all cultures, 10 μg / mL of tetracycline was added to maintain the plasmid intracellularly.

[0075] After the culture was completed, the culture solution was transferred to a centrifuge tube and centrifuged at 9000×g for 10 minutes to collect the cells. Next, the cell pellet was suspended in 40 mL of pure water and centrifuged again. This operation was performed twice to remove the remaining medium components and metabolites contained in the culture supernatant. The obtained cell pellet was dried using a freeze dryer to obtain a dried cell powder.

[0076] The PHA content and PHA monomer composition were measured by gas chromatography (GC) using a Shimadzu GC-2014s apparatus (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. The preparation of the GC sample was carried out as follows. 20 mg of dry cells were weighed into a screw-cap test tube, 2 mL of methanol sulfate (15 vol% sulfuric acid) and 2 mL of chloroform were added, and a methyl esterification reaction was carried out at 100 °C for 140 minutes. After the reaction was completed, 1 mL of ultrapure water was added and stirred vigorously, and the chloroform layer was recovered. An equal volume of chloroform solution containing 0.1% (w / v) methyl-n-octane as an internal standard was added to the recovered solution to prepare a final sample for GC analysis. The sample was injected through a GC capillary column InertCap 1 (30 m × 0.25 mm, GL Science). The column temperature was first held at 90 °C for 2 minutes, then raised to 110 °C at a rate of 5 °C / min, and then further raised to 280 °C at a rate of 20 °C / min. From the obtained signal peak areas, the total PHA content and 3HA monomer composition were calculated (Table 3).

[0077]

Table 3

[0078] Example 4 Production of PHA Using Carbon Dioxide as a Carbon Source The strain used and the preculture method were the same as in Example 3. 1 mL of the preculture solution was inoculated into a 250 mL jar fermenter (ABLE Corporation) containing 100 mL of MS medium. Then, a mixed gas (H2:O2:CO2:N2 = 3.8:7.3:13.0:75.9% (v / v)) was supplied at a flow rate of 50 mL / min, and culturing was carried out at a stirring speed of 1200 rpm and a culture temperature of 30 °C for 120 hours. To prevent foaming of the culture solution, 0.01 wt% of Antifoam 204 (manufactured by Sigma) was added to the medium. In addition, in all cultures, 10 μg / mL of tetracycline was added to maintain the plasmid intracellularly during culturing. In the same manner as in Example 3, bacteria collection and preparation of dried bacterial cells were carried out. Using the obtained dried bacterial cells, the PHA content and PHA monomer composition were calculated in the same manner as in Example 3 (Table 4).

[0079]

Table 4

[0080] The results in Tables 3 and 4 indicate that the introduction fractions of 3HV and 3H4MV monomers were enhanced by introducing the three genes of bktB, kivd, and padA into hydrogen-oxidizing bacteria.

[0081] Example 5 1. Preparation of plasmid for introducing polymerase gene into genomic DNA of Ralstonia eutropha H16 strain (1) Preparation of plasmid pK18-d-phaC1 pK18-d-phaC1 was prepared as follows to delete the PHA polymerase gene (phaC1) of Ralstonia eutropha H16 strain. Genome editing regarding the phaC gene and ilvH gene was carried out using homologous recombination with pK18mobsacB. The primers used in PCR amplification are shown in Table 1. Using a sample of genomic DNA extracted from Ralstonia eutropha H16 strain as a template, PCR reactions were performed using primers D-phaC1-ufw2, D-PhaC1-urv, D-PhaC1-dfw, and D-PhaC1-drv, and two PCR fragments of approximately 1 kb were obtained respectively. Overlap PCR was carried out using the obtained fragments and primers D-phaC1-ufw2 and D-PhaC1-drv to obtain a 1.8-kb fragment. The obtained fragment was digested with restriction enzymes EcoRI and SphI and inserted into the pK18mobsacB vector (A Schaefer et al., 1994, DOI: 10.1016 / 0378-1119(94)90324-7) to prepare plasmid pK18-d-phaC1.

[0082] (2) Plasmid pK18-d-phaC NSDG Preparation To replace the PHA polymerase gene (phaC1) of Ralstonia eutropha H16 strain with the polymerase variant phaC NSDG gene derived from Aeromonas caviae, K18-d-phaC NSDG was prepared as follows. Using a sample of genomic DNA extracted from Ralstonia eutropha H16 strain as a template, PCR reactions were performed using primers D-phaC1-ufw2 and D-PhaC1-urv-NSDG, D-PhaC1-drv and D-PhaC1-dfw-NSDG, and two PCR fragments of approximately 1 kb were obtained each. pBBR1-phaC NSDG AB Re J Ac (R.M Sivashankari et al., 2023, DOI 10.3389 / fbioe.2023.1114946) was used as a template, and PCR was performed using primers up-NSDG-fw and down-NSDG-rv to obtain a 1.8-kb fragment. Using the above three fragments as templates, overlap PCR was performed using primers D-phaC1-ufw2 and D-PhaC1-drv to obtain a 3.6-kb fragment. The obtained 3.6-kb fragment was digested with restriction enzymes EcoRI and SphI and inserted into the pK18mobsacB vector to generate pK18-d-phaC NSDG was prepared.

[0083] (3) Preparation of plasmid pK18-d-phaC Psh Preparation To replace the PHA polymerase gene (phaC1) of Ralstonia eutropha H16 strain with the polymerase phaC Psh gene derived from Plesiomonas shigelloides, pK18-d-phaC Psh was prepared as follows. Using a sample of genomic DNA extracted from Ralstonia eutropha H16 strain as a template, PCR reactions were performed using primers D-phaC1-ufw2 and D-PhaC1-urv-psl, D-PhaC1-drv and D-PhaC1-dfw-psl, and two PCR fragments of approximately 1 kb were obtained respectively. pBBR1-phaC Psh AB Re J Ac (R.M Sivashankari et al., 2023, DOI 10.3389 / fbioe.2023.1114946) was used as a template, and PCR was performed using primers up-psl-fw and down-psl-rv to obtain a 1.9-kb fragment. Using the above three fragments as templates, overlap PCR was performed using primers D-phaC1-ufw2 and D-PhaC1-drv to obtain a 3.7-kb fragment. The obtained 3.7-kb fragment was digested with restriction enzymes EcoRI and SphI and inserted into the pK18mobsacB vector to generate pK18-d-phaC Psh was generated.

[0084] (4) Preparation of plasmid pk18mobsacB_ddd-ilvH To delete the acetolactate synthase small subunit gene (ilvH) of Ralstonia eutropha H16 strain, pk18mobsacB_ddd-ilvH was prepared as follows. Using a sample of Ralstonia eutropha H16 strain genomic DNA as a template, PCR reactions were performed using primers ddd-A36T_F and dd-ilvH_R1, dd-ilvH_F2 and ddd-A36T_R, and two PCR fragments of approximately 1.8 kb and 1.3 kb were obtained respectively. Then, overlap PCR was performed using the two obtained fragments and primers ddd-A36T_F and ddd-A36T_R to obtain a 3.1-kb DNA fragment. The obtained 3.1-kb fragment was digested with restriction enzymes EcoRI and SphI and inserted into the pK18mobsacB vector to generate pk18mobsacB_ddd-ilvH.

[0085] (5) Preparation of plasmid pk18mobsacB_ddd-A36T Except for using a sample of genomic DNA extracted from Ralstonia eutropha strain 1F2 as a template, pk18mobsacB_ddd-A36T for introducing the A36T mutation into the acetolactate synthase small subunit gene (ilvH) was prepared in the same manner as (4).

[0086]

Table 5

[0087] 2. Preparation of plasmids for introducing monomer supply enhancement genes into the genomic DNA of Ralstonia eutropha strain H16 (1) Preparation of pK18-A0006::Cre To use the Cre-loxP system in the following examples, the Cre (Cre recombinase) gene was introduced into the A0006 gene locus of Ralstonia eutropha strain H16 to prepare pK18-A0006::Cre. The Cre gene was obtained from GenScript Japan Corporation, which was chemically synthesized by optimizing the codons of the Cre gene derived from Bacteriophage P1 (Genbank ID: X03453.1, Abremski et al., 1983) for Ralstonia eutropha strain H16. The nucleotide sequence is shown below.

[0088] Nucleotide sequence of the chemically synthesized Cre gene (SEQ ID NO: 36)

[0089] The primers used for the preparation of pK18 - A0006::Cre are shown in Table 6, and the synthesized DNA is shown in Table 7. The homologous sequences around the A0006 gene locus were amplified by PCR using primers F17 and R18, the araC - araBAD promoter was amplified using primers F18 and R19, the Cre gene was amplified using primers F19 and R20, the homologous sequences around the A0006 gene locus were amplified using primers F20 and R21, and the pK18mobsacB sequence was amplified using primers F21 and R21, respectively, to synthesize DNA fragments 23 - 27. Subsequently, a circular vector was constructed from the obtained DNA fragments using NEBuilder from New England Biolabs and transformed into Escherichia coli. Plasmids were extracted from the formed colonies to obtain pK18 - A0006::Cre.

[0090]

Table 6 - 1

Table 6 - 2

[0091] Note that the primer F20 contains the lox5171 sequence described in the following table, and the primer R18 contains the lox2272 sequence described in the following table.

[0092]

Table 7

[0093] (2) Preparation of the landing pad Using pK18 - A0006::Cre prepared in (1), a region (hereinafter, appropriately referred to as the "landing pad") where a foreign gene (monomer supply - enhancing gene) lands by specifically recognizing the Cre gene was prepared by homologous recombination (Figure 4).

[0094] (3) Plasmid pK18 - P j5c2 - kivd_PphaC - padA, and plasmid pK18 - P j5c2 - kivd_P g25 - Preparation of padA (3 - 1) Promoter Using the Cre - loxP system, monomer supply - enhancing genes (bktB, kivd, padA) were introduced into the genomic DNA of Ralstonia eutropha H16 strain (Figure 5). For the expression of monomer supply - enhancing genes, three different promoters with different expression intensities available in the Ralstonia eutropha H16 strain: j5[c2] promoter (P j5c2 ), g25 promoter (P g25 ), and pha promoter (P pha ) derived from the pha operon of R. eutropha (for all, Johnson, A., Gonzalez - Villanueva, M., Tee, K., Wong, T. An Engineered Constitutive Promoter Set with Broad Activity Range for Cupriavidus necator H16. ACS Synth. Biol.7,1918 - 1928(2018) https: / / doi.org / 10.1021 / acssynbio.8b00136) were used to control the expression level of the introduced genes. P j5c2 and P g25 were chemically synthesized, and P pha was synthesized by PCR from the genomic DNA of Ralstonia eutropha H16 strain. The strength of the expression level of such introduced genes is in the order of P j5c2 , P g25 , P pha . The nucleotide sequences of each promoter are shown below.

[0095] Nucleotide sequence of phaC1 promoter (SEQ ID NO: 79): caatggccacgatgtacatcaaaaattcatccttctcgcctatgctctggggcctcggcagatgcgagcgctgcataccgtccggtaggtcgggaagcgtgcagtgccgaggcggattcccgcattgacagcgcgtgcgttgcaaggcaacaatggactCaaatgtctcggaatcgctgacgattcccaggtttctccggcaagcatagcgcatggcgtctccatgcgagaatgtcgcgcttgccggataaaaggggagccgctatcggaatggacgcaagccacggccgcagcaggtgcggtcgagggcttccagccagttccagggcagatgtgccggcagaccctcccgctttgggggaggcgcaagccgggtccattcggatagcatctccccatgcaaagtgccggccagggcaatgcccggagccggttcgaatagtgacggcagagagacaatcaaatc Base sequence of the g25 promoter (SEQ ID NO: 80): tatggaaaaataaaaatttcttgataaaattttccaatactattataatattgttAttaaagaggagaaattaac Base sequence of the j5c2 promoter (SEQ ID NO: 81): agcggatataaaaaccgttattgacacaggtggaaatttagaatatactgttagtAaacctaatggatcgaccttagatcttttaagaaggagatatacat

[0096] (3-2) pK18-P j5c2 -kivd_P phaC Production of -padA P at the A0006 gene locus of the landing pad of the "Ralstonia eutropha H16 strain containing the landing pad" in the following 4.(1) j5c2 -kivd_P phaC To insert the -padA gene, pK18-P j5c2 -kivd_P phaC-padA was prepared as follows. The primers used for plasmid construction are shown in Table 6, and the synthesized DNA is shown in Table 8. rrnB T1+T7 terminator 1 was amplified by PCR using primers F01 and R01, rrnB T1+T7 terminator 2 was amplified by PCR using primers F02 and R02, the kivd gene was amplified by PCR using primers F03 and R03, the j5[c2] promoter 1 (P j5c2 ) was amplified by PCR using primers F04 and R04, P pha was amplified by PCR using primers F05 and R05, the j5[c2] promoter 2 (P j5c2 ) was amplified by PCR using primers F04 and R06, the g25 promoter was amplified by PCR using primers F04 and R07, the padA gene was amplified by PCR using primers F07 and R08, terminator 3 was amplified by PCR using primers F08 and R09, the chloramphenicol resistance gene was amplified by PCR using primers F09 and R10, the terminator sequence: loxP sequence was amplified by PCR using primers F10 and R11, and the pK18mobsacB backbone sequence: lox2272 sequence was amplified by PCR using primers F11 and R12, respectively, and DNA fragments 1 to 12 were synthesized. Subsequently, overlap PCR was performed using DNA fragments 2, 3, 4 and primers F02 and R04 to synthesize fragment 13. Similarly, fragment 14 was synthesized using DNA fragments 2, 3, 6 and primers F02 and R06, fragment 15 was synthesized using DNA fragments 5, 8, 9 and primers F05 and R09, fragment 16 was synthesized using DNA fragments 7, 8, 9 and primers F06 and R09, and fragment 17 was synthesized using DNA fragments 10, 11 and primers F09 and R11, respectively. Subsequently, the obtained DNA fragments (fragments 13, 15, 17, 12) were used with NEBuilder from New England Biolabs to construct a circular vector and transform Escherichia coli. Plasmids were extracted from the formed colonies to obtain pK18-P j5c2 -kivd_P phaC -padA.

[0097] (3-3)pK18-P j5c2 -kivd_Pg25 Preparation of -padA For the "Ralstonia eutropha H16 strain containing a landing pad" in the following 4.(1), at the A0006 gene locus of the landing pad, P j5c2 -kivd_P g25 To insert the -padA gene, pK18-P j5c2 -kivd_P g25 -padA was prepared as follows. Similar to (3-2), a circular vector was constructed from the obtained DNA fragments (fragment 14, 16, 17, 12) using NEBuilder and transformed into Escherichia coli. Plasmids were extracted from the formed colonies, and pK18-P j5c2 -kivd_P g25 -padA was obtained.

[0098]

Table 8

[0099] (4) Plasmid pK18-P j5c2 Preparation of -bktB-kivd-padA For the "Ralstonia eutropha H16 strain containing a landing pad" in the following 4.(1), at the A0006 gene locus of the landing pad, P j5c2 To insert the -bktB-kivd-padA gene, plasmid pK18-P j5c2 -bktB-kivd-padA was prepared as follows. pK18-P j5c2The primers used for the preparation of -bktB-kivd-padA are shown in Table 6, and the synthesized DNA is shown in Table 9. The j5[c2] promoter 3 was amplified using primers F12 and R13, the terminator 4 was amplified using primers F13 and R14, the pK18mobsacB backbone sequence-chloramphenicol resistance gene sequence was amplified using primers F14 and R15, the bktB-kivd-padA sequence was amplified using primers F15 and R16, the bktB-kivd-padA sequence was amplified using primers F16 and R17, and the backbone sequence of the pGEM-T easy vector (Promega) was PCR-amplified to synthesize DNA fragments 18 to 22, respectively. Next, from the obtained DNA fragments (fragments 18, 19, 20), plasmid pK18-P was synthesized using NEBuilder. j5c2 Similarly, from the obtained DNA fragments (fragments 21, 22), pGEM-bktB-kivd-padA was synthesized using NEBuilder. Plasmid pK18-P j5c2 was digested with restriction enzymes AgeI and BamHI, and plasmid pGEM-bktB-kivd-padA was digested with restriction enzymes XmaI and BamHI, followed by a ligation reaction. Subsequently, the DNA solution after the ligation reaction was transformed into Escherichia coli, and plasmids were extracted from the formed colonies to obtain pK18-P j5c2 -bktB-kivd-padA.

[0100]

Table 9

[0101] 3. Introduction of the polymerase gene into the genomic DNA of Ralstonia eutropha H16 strain The pK18 plasmid (i.e., pK18-d-phaC1, pK18-d-phaC NSDG 、pK18-d-phaC Psh, gene deletion and gene insertion of Ralstonia eutropha H16 strain were carried out by homologous recombination using (pk18mobsacB_ddd-ilvH, pk18mobsacB_ddd-A36T), and a genome-edited strain (A) was prepared. That is, first, in order to delete the PHA polymerase gene (phaC1) of Ralstonia eutropha H16 strain, Escherichia coli S17-1 was transformed with pK18-d-phaC1 using the heat shock method, electroporation method, etc., and a recombinant strain was obtained on an LB agar medium containing 50 mg / L kanamycin. Next, the obtained recombinant Escherichia coli and Ralstonia eutropha H16 strain were cultured overnight in 2 mL of LB medium or NR medium. The recombinant Escherichia coli was cultured in an LB medium containing 50 mg / L kanamycin. The culture solution of the above recombinant Escherichia coli and the culture solution of the host strain (Ralstonia eutropha H16 strain) for genome editing were transferred to a 15 mL tube, centrifuged at 10,000×g for 2 minutes to recover the cell pellet. To remove kanamycin, the cell pellet was washed 3 times with 1 mL of NR medium. The washed pellet (mixed pellet of recombinant Escherichia coli and host strain) was suspended in 50 μL of NR medium and dropped onto an NR agar medium. The agar medium onto which the suspended bacterial solution was dropped was incubated at 30°C for 24 hours. Subsequently, the cells on the agar medium were scraped off, suspended in 0.8% sterilized physiological saline, and diluted 10 - 1000 times. Then, 100 μL aliquots of the diluted solution were spread onto Simmons citrate agar medium containing 10 mg / L gentamicin and 200 mg / L kanamycin, and cultured at 30°C for 2 - 3 days. After culturing, the formed colonies were collected, inoculated into NR medium (2 mL) containing 200 mg / L kanamycin, and cultured overnight at 30°C. After culturing, the cells were suspended in 0.8% sterilized physiological saline to prepare a 10 - 1000-fold dilution. Next, 100 μL of the diluted solution was spread onto MS minimal medium containing 1 g / L yeast extract, 150 g / L sucrose, and 10 mg / L gentamicin, and cultured at 30°C for 2 - 3 days. Finally, the formed colonies were collected to obtain a genome-edited strain (A) in which the polymerase gene was inserted into the phaC1 gene locus of Ralstonia eutropha H16 strain.

[0102] Note that to replace the PHA polymerase gene (phaC1) of Ralstonia eutropha H16 strain with the polymerase mutant phaC NSDG gene derived from Aeromonas caviae, pK18-d-phaC NSDG was used. To replace phaC1 with the phaC Psh gene derived from Plesiomonas shigelloides, pK18-d-phaC Psh was used respectively. Also, for the preparation of a deletion strain of the acetolactate synthase small subunit gene (ilvH) of Ralstonia eutropha H16 strain and an IlvH mutant (IlvH A36T ) expression strain in which the 36th alanine of IlvH was mutated to tyrosine, pK18mobsacB_ddd-ilvH and pK18mobsacB_ddd-A36T were used respectively.

[0103] 4. Introduction of the monomer supply enhancement gene into the genomic DNA of the genome-edited strain (A) using the Cre-loxP system (1) Insertion of the Cre-loxP landing pad To construct a Cre-loxP expression system on the genomic DNA of the genome-edited strain (A), a landing pad (Figure 4) was inserted into the A0006 locus of the genome-edited strain (A) using pK18-A0006::Cre. The Ralstonia eutropha H16 strain into which this landing pad was inserted is sometimes referred to as the "Ralstonia eutropha H16 strain containing the landing pad". The insertion of the landing pad was introduced into the genome by homologous recombination using the plasmid pK18-A0006::Cre. Specifically, pK18-A0006::Cre was transformed into Escherichia coli NEB (registered trademark) 10-beta Electrocompetent E.coli / pUB307, respectively. The obtained transformants were inoculated into LB medium (2 mL) containing 50 μg / mL of kanamycin and 10 μg / mL of tetracycline, and cultured with shaking overnight at 37 °C to obtain recombinant Escherichia coli. On the other hand, the Ralstonia eutropha H16 strain was inoculated into NR medium (2 mL) and cultured with shaking overnight at 30 °C. Next, 500 μL of the culture solution of the above recombinant Escherichia coli and the culture solution of the Ralstonia eutropha H16 strain were each collected, centrifuged at 5000×g for 1 minute, and each cell pellet was prepared. These pellets were combined and suspended in LB medium (1 mL), and cultured with shaking overnight at 30 °C for conjugation transfer. After culturing, selection was performed on LB agar medium containing 200 μg / mL of kanamycin and 10 μg / mL of gentamicin, and incubated at 30 °C for 24 hours. Subsequently, the cells on the agar medium were scraped off, spread on LB agar medium containing 10 mg / L of gentamicin and 10% of sucrose, and cultured at 30 °C for 2 to 3 days. Finally, the formed colonies were collected to obtain the "Ralstonia eutropha H16 strain containing the landing pad" in which the polymerization enzyme gene was inserted into the A0006 gene locus of the Ralstonia eutropha H16 strain. (2) Introduction of the monomer supply enhancement gene using the Cre-loxP system pK18-P j5c2 -kivd_P phaC -padA, pK18-P j5c2 -kivd_P g25- padA, pK18 - P j5c2 Using any of the plasmids of - bktB - kivd - padA, these monomer supply - enhancing genes were introduced into the above - mentioned landing pad by the Cre - loxP site - specific homologous recombination method (Figure 5). Specifically, pK18 - P j5c2 - kivd_P phaC - padA, pK18 - P j5c2 - kivd_P g25 - padA, pK18 - P j5c2 - bktB - kivd - padA were each transformed into Escherichia coli NEB® 10 - beta Electrocompetent E.coli / pUB307. The obtained transformants were inoculated into LB medium (2 mL) containing 50 μg / mL of chloramphenicol and 10 μg / mL of tetracycline, and cultured with shaking overnight at 37°C to obtain recombinant Escherichia coli. On the other hand, the "Ralstonia eutropha H16 strain containing the landing pad" was inoculated into NR medium (2 mL) and cultured with shaking overnight at 30°C. Next, 500 μL of the culture solution of the above - mentioned recombinant Escherichia coli and the culture solution of Ralstonia eutropha H16 strain were each collected, centrifuged at 5000×g for 1 minute to prepare each cell pellet. These pellets were combined and suspended in LB medium (1 mL) containing 0.2% (w / v) arabinose, and cultured with shaking overnight at 30°C for conjugative transfer. After the culture, selection was carried out on LB agar medium containing 50 μg / mL of chloramphenicol and 10 μg / mL of gentamicin to obtain the final genome - edited strain (B) in which the monomer supply - enhancing gene was inserted into the A0006 gene locus.

[0104] 5. PHA Biosynthesis and Analysis (1) Heterotrophic culture using fructose (Sakaguchi flask) 4. The genome-edited strain (B) prepared in step 4 was inoculated into 2 mL of NR medium (2.0 g / L yeast extract, 10 g / L Bacto-tryptone, 10 g / L skipjack extract), and cultured with shaking overnight at 30 °C to obtain a preculture solution. Next, 1% of the preculture solution was inoculated into 100 mL of MS medium (9.0 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO 4、 0.5 g / L NH4Cl, 0.2 g / L MgSO4·7H2O, 1 mL trace elements solution) containing 10 g / L fructose, and cultured with shaking at 30 °C for 72 hours. "trace elements solution" contains 0.218 g / L CoCl2·6H2O, 20.5 g / L FeCl3·6H2O, 7.8 g / L CaCl2, 0.118 g / L NiCl2·6H2O, 0.105 g / L CrCl3·6H2O, 0.156 g / L CuSO4·5H2O in 0.1 N HCl. After the cultivation, the culture solution was transferred to a centrifuge tube and centrifuged at 5000×g for 10 minutes to collect the cells. Next, the cell pellet was suspended in 50 mL of pure water and centrifuged again. This operation was performed twice to remove the remaining culture medium components and metabolites contained in the culture supernatant. The obtained cell pellet was dried using a freeze dryer to obtain a dried cell powder.

[0105] (2) Heterotrophic culture using fructose (L-shaped test tube) Culture and dried cell powder were prepared in the same manner as in (1) above, except that the amount of the medium was 10 mL, and cells were collected using a 1.5 mL tube (12,000×g, 5 minutes) and washed three times with 1 mL of pure water.

[0106] (3) Autotrophic culture using carbon dioxide (jar fermenter) 1 mL of the preculture was inoculated into a 250 mL jar fermenter (ABLE Corporation) containing 100 mL of MS medium. Subsequently, a mixed gas (H2:O2:CO2:N2 = 3.4:10.0:13.6:73.0% (v / v)) was supplied at a flow rate of 50 mL / min, and cultivation was carried out at a stirring speed of 1200 rpm and a cultivation temperature of 30 °C for 120 hours. To prevent foaming of the culture broth, 0.01 wt% of Antifoam 204 (manufactured by Sigma) was added to the medium.

[0107] The PHA content and PHA monomer composition were measured by the same method as in Example 3. The results are shown below. In Tables 10 and 11, "strain Pi-jg" means a strain having a PHA polymerase (Psh) gene derived from Pseudomonas syringae and the ilvH A36T encoding ilvH A36T gene, and expressing the monomer supply enhancement gene kivd by P j5c2 and expressing the monomer supply enhancement gene padA by P g25 . "Strain Ni-jg" means a strain having the NSDG gene and the above ilvH A36T gene, and expressing the monomer supply enhancement gene kivd by P j5c2 and expressing the monomer supply enhancement gene padA by P g25 . "Strain Pi-j" means a strain having the Psh gene and the above ilvH A36T gene, and expressing the monomer supply enhancement gene group bktB - kivd - padA by P j5c2 . "Strain Ni-j" means a strain having the NSDG gene and the above ilvH A36T gene, and expressing the monomer supply enhancement gene group bktB - kivd - padA by P j5c2 .

[0108]

Table 10

[0109]

Table 11

[0110] The results in Table 10 also show that the introduction fractions of 3HV and 3H4MV monomers were enhanced even by introducing the two genes of kivd and padA into hydrogen-oxidizing bacteria. Further, the results in Table 11 show that the productivity of PHA was improved by directly introducing the monomer supply-enhancing gene into the genomic DNA of the host bacteria.

Industrial Applicability

[0111] The copolymerized PHA having flexible physical properties produced by the production method of the present invention can be used for films and the like.

Claims

1. A recombinant hydrogen-oxidizing bacterium having at least a 2-keto acid decarboxylase (kivd) gene and a phenylacetaldehyde dehydrogenase (padA) gene as monomer supply enhancing genes and having the ability to produce copolymerized polyhydroxyalkanoic acid (PHA).

2. The recombinant hydrogen-oxidizing bacterium according to claim 1, further comprising a 3-ketothiolase (bktB) gene as a monomer supply enhancing gene.

3. The recombinant hydrogen oxidizing bacterium according to claim 1, wherein the introduction rate of the second component in the copolymerized PHA is higher than the introduction rate of the second component in a copolymerized PHA produced by a hydrogen oxidizing bacterium not having any monomer supply enhancing genes.

4. The recombinant hydrogen-oxidizing bacterium according to claim 3 , wherein the introduction ratio of the second component is at least 1 mol %.

5. The recombinant hydrogen-oxidizing bacterium according to claim 3 , wherein the second component is 3-hydroxyvaleric acid (3HV) and / or 3-hydroxy-4-methylvaleric acid (3H4MV).

6. 2. The recombinant hydrogen-oxidizing bacterium of claim 1, wherein the hydrogen-oxidizing bacterium is Ralstonia eutropha.

7. The recombinant hydrogen-oxidizing bacterium according to any one of claims 1 to 6, wherein the recombinant hydrogen-oxidizing bacterium has the monomer supply enhancing gene in its genomic DNA.

8. A method for producing polyhydroxyalkanoic acid (PHA), comprising the steps of: (i) introducing at least a 2-keto acid decarboxylase (kivd) gene and a phenylacetaldehyde dehydrogenase (padA) gene as monomer supply enhancing genes into a hydrogen oxidizing bacterium having a PHA synthase to obtain a transformant of the hydrogen oxidizing bacterium; (ii) culturing the obtained transformant in the presence of a carbon source; and (iii) A step of collecting the copolymerized PHA from the culture A manufacturing method comprising:

9. The method according to claim 8, wherein in the step (i), a 3-ketothiolase (bktB) gene is further introduced as a monomer supply enhancing gene.

10. The method according to claim 8 , wherein the monomer supply enhancing gene is introduced by direct integration into genomic DNA of the hydrogen oxidizing bacterium, or by introducing a plasmid carrying the gene into the hydrogen oxidizing bacterium.

11. The method according to claim 10, wherein the monomer supply enhancing gene is introduced by direct integration into the genomic DNA of the hydrogen-oxidizing bacterium.

12. The method according to claim 7, wherein the hydrogen-oxidizing bacterium having the PHA synthase is obtained by introducing a gene encoding a PHA synthase having broad substrate specificity into the hydrogen-oxidizing bacterium.

13. The method according to claim 8 , wherein the carbon source is sugar or carbon dioxide.

14. The method of claim 13, wherein the sugar is fructose.

15. The method according to claim 13, wherein the carbon dioxide is a mixed gas containing carbon dioxide in the range of 1 to 20% (v / v).

16. The method according to claim 8 , wherein in step (ii), no precursor of the second component is added.

17. The production method according to claim 8, wherein the introduction rate of the second component in the copolymerized PHA is higher than the introduction rate of the second component in a copolymerized PHA produced by a hydrogen-oxidizing bacterium to which no monomer supply enhancing gene has been introduced.

18. The method according to claim 17, wherein the second component is 3-hydroxyvaleric acid (3HV) and / or 3-hydroxy-4-methylvaleric acid (3H4MV).

19. The method according to claim 8, wherein the hydrogen-oxidizing bacteria is Ralstonia eutropha.

20. The method according to claim 8, wherein the PHA synthase gene encodes a PHA synthase mutant (NSDG) in which the 149th asparagine of polyhydroxyalkanoate synthase derived from Aeromonas caviae is replaced with serine and the 171st aspartic acid is replaced with glycine, or a PHA synthase (Psh) derived from Plesiomonas shigelloides.

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