JP2001275675A - Polyhydroxybutyric acid synthase group and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a polyhydroxybutyric acid(PHB) synthase group useful for PHB synthesis, to provide the corresponding genes respectively encoding the synthases, to provide recombinant vectors respectively integrated with the above genes, to provide transformants afforded by respectively transferring the above recombinant vectors into hosts, to provide a method for producing the PHA synthase group using the above transformants, and to provide a method for producing the corresponding PHBs respectively using the above transformants. SOLUTION: The enzymes belonging to a PHA synthase group or the corresponding PHBs are produced by transferring the respective genes of enzymes belonging to the PHA synthase group obtained from Ralstonia eutropha TB64 strain into respective host organisms to afford the corresponding transformants which are then cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a group of enzymes for the synthesis of poly-3-hydroxybutyric acid (hereinafter referred to as PHB), a gene encoding the group of enzymes, a recombinant vector containing the gene,
The present invention relates to a transformant transformed by the vector, a method for producing a group of PHB synthase enzymes using the transformant, and a method for producing PHB using the transformant.

[0002]

2. Description of the Related Art It has been reported that many microorganisms produce poly-3-hydroxybutyric acid (PHB) or other PHA and accumulate in cells ("Biodegradable Plastic Handbook", Decomposable Plastics Study Group, published by NTT, P178-197, 1
995). These polymers can be used for production of various products by melt processing or the like, similarly to conventional plastics. Furthermore, because it is biodegradable, it has the advantage of being completely degraded by microorganisms in nature,
It does not remain in the natural environment and cause pollution unlike many conventional synthetic polymer compounds. It is also excellent in biocompatibility and is expected to be applied as a soft medical member.

[0003] Such PHA produced by microorganisms depends on the type of microorganism used for the production, the composition of the medium, the culture conditions, and the like.
It is known that various compositions and structures can be obtained, and studies on such composition and structure control have been made mainly from the viewpoint of improving the physical properties of PHA. Particularly, PHB has been studied energetically, but in the case of homopolymer, since it has a high crystallinity and brittle properties, there is a problem in physical properties that the impact resistance is inferior. Was not enough. As a method of overcoming these drawbacks, screening for strains producing the copolymer was performed.

[0004] For example, Alcaligenes eutropus H16 (ATCC No. 1769)
9) and its mutants are capable of producing 3-hydroxybutyric acid (3HB) and 3-hydroxybutyric acid (3HB) by changing the carbon source during the culture.
It has been reported that a copolymer with hydroxyvaleric acid (3HV) is produced at various composition ratios (Japanese Patent Application Laid-Open Nos. 6-15604, 7-14352, 8-19227). No.
etc).

[0005] JP-A-5-74492 discloses a genus of Methylobacterium sp., A genus Paracoccus, and a genus Alcaligenes.
sp.) and a method of producing a copolymer of 3HB and 3HV by contacting a microorganism of the genus Pseudomonas sp. with a primary alcohol having 3 to 7 carbon atoms.

JP-A-5-93049 and JP-A-7-265
No. 065 discloses an Aeromonas cavier.
aviae) using oleic acid or olive oil as a carbon source to produce a two-component copolymer of 3HB and 3-hydroxyhexanoic acid (3HHx).

[0007] In Japanese Patent Application Laid-Open No. Hei 9-91893, Comamonas
Acidovorans IFO13852 strain (Comamonas acid
ovorans IFO13852) discloses producing a polyester having 3HB and 4-hydroxybutyric acid as monomer units by culturing using gluconic acid and 1,4-butanediol as carbon sources.

However, such a method of adding a copolymer component has problems that the polymer productivity is low and the carbon source yield is low, and these methods are difficult to use when using inexpensive natural products or the like as the carbon source. Also lead to an increase in manufacturing costs.

[0009] In addition, although attempts have been reported to produce a high-strength film by subjecting a PHB homopolymer to a stretching treatment, it is necessary to set complicated conditions and perform a multi-step process. Could lead to a rise in economics, which could lead to a rise (Holmes, PA,
In: Developments in Crystalline Polymers-2, Basset
t, DC (ed.) p1-65, Elsevier (1988)).

In this regard, Japanese Patent Application Laid-Open No. H10-176070 discloses a solution using a specific PHB homopolymer having a certain molecular weight or more, but does not mention polymer productivity or carbon source yield at all. It has not been.

[0011]

As described above, in improving the physical properties and productivity of microorganism-produced PHB, several types of compositions and culture conditions can be obtained by changing the type and culture conditions of the microorganism used for the production. Although a structure is obtained,
In a microorganism or a group of PHB synthesizing enzymes, the polymer productivity of PHB with improved physical properties has not yet reached the desired level, and a known microorganism or PHB synthesizing enzyme group alone can produce PHB with improved physical properties in a high yield. It was difficult to synthesize.

Therefore, there has been a demand for the development of a method for producing PHB with improved physical properties simply and with good reproducibility in high yield.

That is, obtaining a gene encoding a group of PHB synthase enzymes from a microorganism capable of accumulating PHB with improved physical properties in a cell at a high yield, a recombinant vector containing the gene system, and transformation with the vector Transformed transformants,
The development of a method for producing a group of PHB synthase enzymes using the transformant and a method for producing PHB using the transformant were considered to be extremely useful and important.

[0014] The present invention relates to the production of PB in such PHB production.
The present invention has been made in view of the usefulness of a group of enzymes involved in HB synthesis, and an object thereof is to provide a novel group of PHB synthesis enzymes. Another object of the present invention is to provide genes encoding these PHB synthesis enzymes. It is still another object of the present invention to provide a recombinant vector incorporating a PHB synthase enzyme group gene and a transformant transformed with the recombinant vector in order to enhance PHB synthase activity. Also,
A method for producing a group of PHB synthase enzymes using the transformant,
It is intended to provide a method for producing high-yield PHB using the transformant.

[0015]

Accordingly, the present inventors aimed at high-yield production of PHB with improved physical properties,
Search for microorganisms having the ability to produce and accumulate B in cells, obtain genes encoding PHB synthase enzymes from such microorganisms, recombinant vectors containing the genes, transformed with the vectors Intensive research on the development of a transformant, a method for producing a PHB synthase group using the transformant, and a method for producing PHB using the transformant, thereby producing a high-yield PHB homopolymer. Ralstonia Eutropha
nia eutropha) TB64 strain, and completed the present invention. The TB64 strain was deposited under the deposit number "FERM BP-
No. 6933 (transferred from P-16980) "to the Ministry of International Trade and Industry, the National Institute of Advanced Industrial Science and Technology, the Institute of Biotechnology and Industrial Technology, and the Patent Microorganisms Depositary Center based on the Budapest Treaty.

The present inventors have conducted intensive studies based on the above problems, and as a result, succeeded in cloning genes of a group of PHB synthase enzymes from the TB64 strain, and completed the present invention.

That is, the PHB synthase according to the present invention has an amino acid sequence represented by SEQ ID NO: 1. In addition, with respect to the amino acid sequence represented by SEQ ID NO: 1, as long as the PHB synthase having the amino acid sequence does not impair the PHB synthesizing activity,
The present invention also includes a PHB synthase having a mutated amino acid sequence in which at least one kind of amino acid deletion, substitution, and addition has been mutated. Furthermore, the present invention includes a PHB synthase gene containing DNAs encoding these amino acid sequences.
And those having a base sequence represented by

The β-ketothiolase according to the present invention has an amino acid sequence represented by SEQ ID NO: 3. Further, the β-ketothiolase having the amino acid sequence represented by SEQ ID NO: 3 has β
-A β-ketothiolase having a mutated amino acid sequence in which at least one kind of mutation of amino acid deletion, substitution and addition is produced within a range not to impair ketothiolase activity is also included in the present invention. Further, the β-ketothiolase gene according to the present invention is a β-ketothiolase gene including a DNA encoding the amino acid sequence of SEQ ID NO: 3 or a mutant sequence thereof. Examples of the DNA include SEQ ID NO: 4
And those having a base sequence represented by

Further, according to the present invention, the acetoacetyl-Co
A reductase has an amino acid sequence represented by SEQ ID NO: 5, and amino acid deletion, substitution and addition of amino acid to the amino acid sequence of SEQ ID NO: 5 within a range that does not impair acetoacetyl-CoA reductase activity. An acetoacetyl-CoA reductase having a mutated amino acid sequence having at least one mutation is also included in the present invention. Further, the present invention relates to an acetoacetyl-CoA reductase gene containing a DNA encoding the amino acid sequence of SEQ ID NO: 5 or a mutant sequence thereof. Examples of the DNA include those having the base sequence of SEQ ID NO: 6. Can be

The present invention further includes a recombinant vector containing the PHB synthase gene or at least one of the β-ketothiolase gene and the acetoacetyl-CoA reductase gene. The body is also included in the present invention.

In the present invention, the above transformant is cultured, and PHB synthase or PHB is synthesized from the resulting culture.
A method for producing a PHB synthase or a group of PHB synthases, which comprises obtaining a group of B synthases,
The method includes a step of culturing the transformant and obtaining PHB from the obtained culture, and includes a method for producing PHB at a high yield.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail.

The PHB synthase enzyme group gene of the present invention has been isolated from the cells of Ralstonia eutropha TB64 strain.

First, chromosomal DNA is obtained from a strain having a PHB synthase enzyme group gene. Known methods can be used for the method of separating chromosomal DNA. For example, TB
After culturing the 64 strains in an LB medium or an M9 medium to which an appropriate carbon source has been added, for example, the method of Marmer et al. (Journal of Molecular Biology, vol. 3, p. 208, 1961)
) To prepare chromosomal DNA.

The chromosomal DNA obtained by the above method is digested with an appropriate restriction enzyme (eg, Sau3AI), and a digest having an appropriate fragment length can be ligated with a restriction enzyme (eg, BamHI). Then, the gene is ligated to the vector cleaved in step 1 to prepare a gene library. Here, the appropriate fragment length means 4000 to 2 when a normal vector is used.
When the cosmid or phage vector is used, it is about 15,000 to 30,000 base pairs. Methods for collecting DNA fragments of an appropriate length include a method using a sucrose density gradient and a method using an agarose gel (Molecular Cloning, Cold Spring Harbor Labo
A known method such as Ratory Publishing (1982) may be used.

As the vector, a phage vector or a plasmid vector capable of autonomously growing in a host microorganism is used. Phage vectors or cosmid vectors include, for example, pWE15, M13, λEMBL3, λEMBL4,
λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon
4A, Charon21A, etc., and plasmid vectors include, for example, pBR, pUC, pBluescriptII, pGEM
System, pTZ system, pET system and the like. In addition, various shuttle vectors such as vectors capable of autonomous propagation in a plurality of host microorganisms such as Escherichia coli and Pseudomonas can also be used. A desired fragment can be obtained from such a vector by digestion with an appropriate restriction enzyme in the same manner as described above.

Ligation of the chromosomal DNA fragment and the vector fragment may be carried out by using DNA ligase, for example, by using a ligation kit (Takara Shuzo Co., Ltd.). Thus, the chromosomal DNA fragment and the vector fragment are ligated to prepare a mixture of recombinant plasmids containing various fragments (hereinafter, referred to as a gene library). Here, in preparing a gene library, in addition to a method using a chromosomal DNA fragment of an appropriate length, mRNA is extracted and purified from the TB64 strain, and cDN is extracted using reverse transcriptase.
Method for synthesizing fragment A (Molecular Cloning, Cold Spr
ing Harbor Laboratory Press, 1982). It is also possible to once transform or transduce a gene library into Escherichia coli and then amplify the gene library in large quantities (Molecular Clonin
g, Cold Spring Harbor Laboratory, 1982).

Introduction of the recombinant vector into the host microorganism
This is performed by a known method. For example, when the host microorganism is Escherichia coli, the calcium chloride method (Journal of Molecular Biolo
gy, 53, 159, 1970), the rubidium chloride method (Metho
ds in Enzymology, 68, 253, 1979) and electroporation (Current Protocols in Molecular Bi
ology, 1, 184 pages, 1994). For transduction using cosmid vectors or phage vectors, the in vitro packaging method (Curr
ent Protocols in Molecular Biology, 1, 571, 199
4 years) can be used. In addition, a method by joint transmission can be used.

Next, a probe for obtaining a DNA fragment containing the gene of the PHB synthesis enzyme group of the TB64 strain is prepared. Regarding the base sequence of the PHB synthesis enzyme group gene,
Several sequences have already been reported (Steinbuchel A.
and Schlegel HG, Mol.Microbiol., 5: 535-542 (199
1)). From these nucleotide sequences, a region having a high degree of conservation is selected, and an oligonucleotide is designed. Oligonucleotides can be synthesized using, for example, a custom synthesis service of Amersham Pharmacia Biotech.

Then, using the designed oligonucleotide as a primer, a polymerase chain reaction (hereinafter referred to as PCR) was carried out using the chromosomal DNA of the TB64 strain as a template to obtain PHB.
Partially amplify the genes of the synthetic enzymes. The PCR-amplified fragment thus obtained is a fragment having nearly 100% homology to the PHB synthase enzyme group gene of the TB64 strain, and is expected to have a high S / N ratio as a probe when performing colony hybridization. And the stringency of hybridization can be easily controlled. The PCR amplified fragment is labeled with an appropriate reagent, and the chromosomal DNA is
Colony hybridization is performed on the library to select PHB synthase group genes (Curren
t Protocols in Molecular Biology, 1, 603, 1994
Year). Here, the label of the PCR amplified fragment is AlkPhosDirect
(Amersham Pharmacia Biotech) and other commercially available kits can be used.

The selection of a gene fragment containing a PHB synthase enzyme group gene can also be based on a method using a phenotype for directly assessing the presence or absence of PHB synthesis, in addition to the above selection method using a genotype. It is. As a method of examining the presence or absence of PHB synthesis, for example, a method of staining with Sudan Black B (Archives of Biotechnology, Vol. 71, 2
83, 1970), and a method of examining the accumulation of PHB by a phase contrast microscope can be used.

An alkaline method (Current Protocols in Molecular Bio
, Vol. 1, pp. 161, 1994) to obtain a DNA fragment containing a PHB synthase enzyme group gene. The nucleotide sequence of the DNA fragment is determined, for example, by the Sanger method (Molecular Clonin).
g, Vol. 2, p. 133, 1989), etc., and can be carried out by a die primer method or a die terminator method using an automatic base sequence analyzer such as a DNA sequencer 377A (Perkin-Elmer). it can.

After the entire base sequence has been determined by the above method, a chemical synthesis method and PCR using chromosomal DNA as a template are performed.
The gene of the present invention can be obtained by hybridization using a probe or a DNA fragment prepared by an arbitrary method such as degradation of a DNA fragment having the base sequence with a restriction enzyme.

[0034] SEQ ID NO: 2 shows the nucleotide sequence of the PHB synthase gene of the present invention, and SEQ ID NO: 1 shows the amino acid sequence encoded by the gene. As long as it has PHB synthesizing activity, some, for example, one to several amino acids may have mutations such as deletion, substitution, and addition. In addition, the gene of the present invention includes, in addition to those having the nucleotide sequence encoding the amino acid represented by SEQ ID NO: 1, degenerate isomers encoding the same polypeptide that differs only in degenerate codons.

In addition, SEQ ID NO: 4 shows the base sequence of the β-ketothiolase gene of the present invention, and SEQ ID NO: 3 shows the amino acid sequence encoded by the gene. As long as the peptide has β-ketothiolase activity, some amino acids may have mutations such as deletion, substitution, and addition. Further, the gene of the present invention includes, in addition to those having the nucleotide sequence encoding the amino acid represented by SEQ ID NO: 3, degenerate isomers encoding the same polypeptide that differs only in degenerate codons.

SEQ ID NO: 6 shows the nucleotide sequence of the acetoacetyl-CoA reductase gene of the present invention, and SEQ ID NO: 5 shows the amino acid sequence encoded by the gene.
As described above, some amino acids may have mutations such as deletion, substitution, and addition as long as the polypeptide having the amino acid sequence has acetoacetyl-CoA reductase activity. Further, the gene of the present invention includes, in addition to those having the nucleotide sequence encoding the amino acid represented by SEQ ID NO: 5, degenerate isomers encoding the same polypeptide that differs only in degenerate codons.

Here, the mutations such as the deletion, substitution, addition and the like described above are performed by a site mutation introduction method (Current Protocols in Mole
cular Biology 1, 811, 1994).

The transformant of the present invention is obtained by introducing a recombinant vector having an enzyme gene of the PHB synthesis enzyme group into a host suitable for the type of expression vector of the recombinant vector, and transforming the transformed vector. Can be obtained. This host may be Escherichia
Genus, Pseudomonas, Ralstonia, Alcaligene
s), Comamonas, Burkholderia, Agrobacterium
ium) genus, Flavobacterium genus,
Vibrio, Enterobact
er), Rhizobium, Gluconobacter, Acinetoba
cter), Moraxella, Nitrosomonas, Aeromonas,
Paracoccus, Bacillus
Genus, Clostridium, Lactobacillus, Corynebacterium
ebacterium), Arthrobacter
Genus, Achromobacter, Micrococcus, Mycob
acterium), Streptococcus
Genus, Streptomyces, Actinomyces, Nocardia
And various bacteria such as the genus Methylobacterium. Also, Saccharomyces (Saccha
romyces), yeasts of the genus Candida, various molds and the like.

Further, in these hosts, P
Selecting a host that does not have HB-degrading enzymes is particularly preferred for producing very high molecular weight PHB.

When a microorganism belonging to the genus Ralstonia or a bacterium such as Escherichia coli is used as a host, the recombinant vector is capable of autonomous replication in the host and has a promoter,
It is preferable that the composition includes a PHB synthesis enzyme group gene, a transcription termination sequence, and the like. As an expression vector, p having an RK2 origin of replication replicated and maintained in a wide range of hosts is used.
PJRD21 with LA2917 (ATCC 37355) or RSF1010 replication origin
5 (ATCC 37533) and the like, and any one having an origin of replication that can be replicated and maintained in a wide range of hosts can be used.

Any promoter can be used as long as it can be expressed in a host. For example, Escherichia coli such as trp promoter, trc promoter, tac promoter, lac promoter, PL promoter, PR promoter, T7 promoter, T3 promoter and the like can be used. And phage-derived promoters. As a method for introducing the recombinant DNA into bacteria, the calcium chloride method, the electroporation method, and the like described above can be used.

When yeast is used as a host, for example, YEp13, YCp50, pRS-based, pYEX-based vectors and the like can be used as expression vectors. As the promoter, for example, a GAL promoter, an AOD promoter and the like can be used. As a method for introducing recombinant DNA into yeast, for example, electroporation (Methods Enzyme)
mol., 194, 182-187 (1990)), spheroplast method (Proc.
Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), lithium acetate method (J. Bacteriol., 153, 163-168 (1983)) and the like can be used.

Further, the recombinant vector includes expression control fragments having various functions for suppressing, amplifying, or inducing expression, markers for selecting transformants, resistance genes to antibiotics, Alternatively, it may further have a gene encoding a signal intended for secretion outside the cells.

The PHB synthase enzyme group of the present invention is obtained by culturing the transformant of the present invention, and culturing it (cultured cells or culture supernatant).
It can be obtained by producing and accumulating the PHB synthase group of the present invention therein, and obtaining the PHB synthase group from the culture. As a method for culturing the transformant of the present invention, a usual method used for culturing a host may be used. As a culturing method, any method used for culturing ordinary microorganisms, such as a batch type, a flow batch type, a continuous culture, a reactor type, and the like can be used.

As a medium for culturing a transformant obtained by using a bacterium such as Escherichia coli as a host, a complete medium or a synthetic medium, for example, LB medium, M9 medium and the like can be mentioned. The culture temperature is aerobically 8 to 72 in the range of 25 to 37 ° C.
By culturing for a period of time, a group of PHB synthesis enzymes is accumulated in the cells and collected. Carbon source is necessary for the growth of microorganisms, for example, glucose, fructose, sucrose, maltose, galactose, sugars such as starch, ethanol, propanol, lower alcohols such as butanol,
Polyhydric alcohols such as glycerin, acetic acid, citric acid, succinic acid, tartaric acid, lactic acid, organic acids such as gluconic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid , Undecanoic acid,
Fatty acids such as dodecanoic acid can be used.

Examples of the nitrogen source include those derived from natural products such as peptone, meat extract, yeast extract, malt extract, casein decomposition product and corn steep liquor, in addition to ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate. Is mentioned. Further, examples of the inorganic substance include monobasic potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. In the culture solution, kanamycin, ampicillin, tetracycline, chloramphenicol,
An antibiotic such as streptomycin may be added.

When culturing a microorganism transformed with an expression vector whose promoter is inducible, an inducer suitable for the type of promoter may be added to the medium. For example, isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indoleacrylic acid (IAA) and the like can be mentioned as the inducer.

For purification of the PHB synthase group, cells or supernatant are centrifuged and collected from the obtained culture, and the cells are disrupted.
Extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration and the like can be performed alone or in an appropriate combination. Confirmation that the obtained purified substance is the target enzyme can be performed by a usual method, for example, SDS polyacrylamide gel electrophoresis, western blotting, or the like.

Cultivation of a transformant using a microorganism as a host, production and accumulation of a PHB synthase enzyme group by the transformant, and recovery of the PHB synthesis enzyme group from the microbial cell are as follows. It is not limited to the above method.

Further, when culturing a transformant using a microorganism as a host for the production of PHA, a medium having a composition corresponding to the host used, the composition of the recombinant vector introduced into the host, etc. Culturing the transformant using culture conditions,
A method for obtaining PHA from a culture can be used. As the medium and culture conditions, for example, those exemplified in the above-mentioned production of PHA synthase can be similarly used.

For the recovery of PHB from the cells, the usual extraction with an organic solvent such as chloroform is the simplest, but in an environment where the organic solvent is difficult to use,
Use a method of removing PHB by removing bacterial components other than PHB by treatment with a surfactant such as SDS, treatment with an enzyme such as lysozyme, or treatment with a chemical such as EDTA, sodium hypochlorite, or ammonia. Can also.

The culture of the transformant using a microorganism as a host, the production and accumulation of PHA by the transformant in the cells, and the recovery of PHA from the cells are limited to those described above. is not.

[0053]

The present invention will be described more specifically with reference to the following examples. However, the present invention does not limit the technical scope to these examples. Example 1 Cloning of Genes of PHB Synthetic Enzyme Group of TB64 Strain TB64 strain was added to 100 ml of LB medium (1%
After culturing overnight at 30 ° C. with polypeptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.4), chromosomal DNA was separated and recovered by the method of Marmer et al. The obtained chromosomal DNA was partially digested with a restriction enzyme Sau3AI. The vector uses pUC18, is cut with a restriction enzyme BamHI, and is dephosphorylated (Molecular Cloning, 1: 572, 1989; Co.
ld Spring Harbor Laboratory), and then using DNA ligation kit Ver.II (Takara Shuzo) for chromosome DN.
The fragment was ligated to the partially degraded Sau3AI fragment of A. Next, this connection D
Using the NA fragment, Escheichia coli HB101 was transformed to prepare a chromosomal DNA library of TB64.

Next, phenotypic screening was carried out to obtain a DNA fragment containing the gene of the PHB synthesis enzyme group of the TB64 strain. The selection medium contains L containing 2% glucose.
Using the B medium, Sudan Black B solution was sprayed at the time when the colonies on the agar plate medium had grown to an appropriate size, and colonies that emitted fluorescence by UV light irradiation were obtained. Plasmids are recovered from the obtained colonies by the alkaline method to obtain DNBs containing PHB synthase enzymes.
A fragment could be obtained.

The gene fragment obtained here was transformed into a vector pBBR122 (Mo BiTe) containing a broad host range replication region not belonging to any of the incompatibility groups IncP, IncQ or IncW.
When the recombinant plasmid was transformed into Ralstonia eutropha TB64m1 strain (a strain deficient in PHB synthesizing ability) by electroporation, the TBBm1 strain recovered the PHB synthesizing ability and showed complementarity.

The nucleotide sequence of the fragment containing the PHB synthesis enzyme gene was determined by the Sanger method. as a result,
It was confirmed that a PHB synthesis enzyme group gene having the nucleotide sequence shown in SEQ ID NO: 2, 4, or 6 was present in the fragment. The amino acid sequences encoded by the nucleotide sequences of SEQ ID NOs: 2, 4, and 6 are shown in SEQ ID NOs: 1, 3, and 5, respectively.

(Example 2: Recombination of TBB strain strain PHB synthase enzyme group gene into expression vector) An oligonucleotide having a base sequence near the start codon of the PHB synthase gene represented by SEQ ID NO: 2 (SEQ ID NO: 2) : 7) and an oligonucleotide (SEQ ID NO: 8) having a base sequence near the stop codon of the acetoacetyl-CoA reductase gene represented by SEQ ID NO: 6 was designed and synthesized (Amersham Pharmacia Biotech), and this oligonucleotide was used as a primer. PCR was performed to amplify a fragment containing the PHB synthase gene, β-ketothiolase gene, and acetoacetyl-CoA reductase gene (LA-PCR kit; Takara Shuzo).

Next, the PCR-amplified fragment obtained above was completely digested with the restriction enzyme BamHI, cut with the restriction enzyme BamHI of the expression vector pTrc99A, and dephosphorylated (Molecular Cloning, vol. 1, 5.7.2, 198
9 years; published by Cold Spring Harbor Laboratory) using DNA ligation kit Ver.II (Takara Shuzo).

Escherichia coli (Esch)
E. coli HB101) was transformed by the calcium chloride method (Takara Shuzo), and the recombinant plasmid recovered from the obtained recombinant was designated as pTB64-phb. Example 3 PHB Production by Genetically Recombinant Escherichia coli of PHB Synthetic Enzyme Group Escherichia coli HB101 was transformed with the recombinant plasmid pTB64-phb obtained in Example 2 by the calcium chloride method. Recombinant plasmid-derived recombinant E. coli was obtained.

The recombinant pTB64-phb was transformed into 200 ml of LB medium.
And cultured with shaking at 37 ° C. and 125 strokes / min. After culturing for 12 hours, 200 ml of the culture solution was inoculated into 200 ml of LB medium containing 2% of glucose (total of 400 ml).
ml), and cultured with shaking at 37 ° C. and 125 strokes / min for 12 hours. The cells thus obtained were collected by centrifugation, washed once with cold methanol and freeze-dried.

The lyophilized pellet was suspended in 100 ml of chloroform and stirred at 60 ° C. for 20 hours to extract PHB. After the extract was filtered through a membrane filter having a pore size of 0.45 μm, the extract was concentrated with a rotary evaporator, the concentrate was reprecipitated in hexane, and only the precipitate was recovered and dried under vacuum to obtain PHB. PHB obtained
Is a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP
-5050, EI method) to identify the methyl esterified product of the PHB monomer unit. Table 1 shows the results.

[0062]

[Table 1]

Further, the molecular weight of the obtained PHB homopolymer was measured by GPC (Tosoh HLC-8020, column: Polymer Laboratory PLgel MIXED-C (5 μm).
m), solvent: chloroform, converted to polystyrene), Mn = 1,720,000, Mw = 3,
It was 560,000.

[0064]

According to the present invention, a group of PHB synthase enzymes,
A gene encoding the PHB synthesis enzyme group, a recombinant vector containing the gene, and a transformant transformed with the recombinant vector can be provided. The gene of the present invention is useful for synthesizing PHB with high yield.

[0065]

[Sequence List] SEQUENCE LISTING <110> Canon INC. <120> Polyhydroxybutyrate synthase and gene encoding the same <121> <130> 405103 <160> 8 <210> 1 <211> 589 <212> PRT <213> Ralstonia eutropha TB64 <220> <223> Polyhydroxybutyrate synthase <400> 1 Met Ala Thr Gly Lys Gly Ala Ala Ala Ser Thr Gln Glu Gly Lys Ser 1 5 10 15 Gln Pro Phe Lys Phe Thr Pro Gly Pro Phe Asp Pro Ala Thr Trp Leu 20 25 30 Glu Trp Ser Arg Gln Trp Gln Gly Thr Glu Gly Asn Gly His Ala Ala 35 40 45 Ala Ser Gly Ile Pro Gly Leu Asp Ala Leu Ala Gly Val Lys Ile Ala 50 55 60 Pro Ala Gln Leu Gly Asp Ile Gln Gln Arg Tyr Met Lys Asp Phe Ser 65 70 75 80 Ala Leu Trp Gln Ala Met Ala Glu Gly Lys Ala Glu Ala Thr Gly Pro 85 90 95 Leu His Asp Arg Arg Phe Ala Gly Asp Ala Trp Arg Thr Asn Leu Pro 100 105 110 Tyr Arg Phe Ala Ala Ala Phe Tyr Leu Leu Asn Ala Arg Ala Leu Thr 115 120 125 Glu Leu Ala Asp Ala Val Glu Ala Asp Ala Lys Thr Arg Gln Arg Ile 130 135 140 Arg Phe Ala Ile Ser Gln Trp Val Asp Ala Met Ser Pro Ala Asn Phe 145 150 155 160 Leu Ala Thr As n Pro Glu Ala Gln Arg Leu Leu Ile Glu Ser Gly Gly 165 170 175 Glu Ser Leu Arg Ala Gly Val Arg Asn Met Met Glu Asp Leu Thr Arg 180 185 190 Gly Lys Ile Ser Gln Thr Asp Glu Ser Ala Phe Glu Val Gly Arg Asn 195 200 205 Val Ala Val Thr Glu Gly Ala Val Val Phe Glu Asn Glu Tyr Phe Gln 210 215 220 Leu Leu Gln Tyr Lys Pro Leu Thr Asp Lys Val His Ala Arg Pro Leu 225 230 235 240 Leu Met Val Pro Pro Cys Ile Asn Lys Tyr Tyr Ile Leu Asp Leu Gln 245 250 255 Pro Glu Ser Ser Leu Val Arg His Val Val Glu Gln Gly His Thr Val 260 265 270 Phe Leu Val Ser Trp Arg Asn Pro Asp Ala Ser Met Ala Gly Ser Thr 275 280 285 Trp Asp Asp Tyr Ile Glu His Ala Ala Ile Arg Ala Ile Glu Val Ala 290 295 300 300 Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val 305 310 315 320 Gly Gly Thr Ile Val Ser Thr Ala Leu Ala Val Leu Ala Ala Arg Gly 325 330 335 Glu His Pro Ala Ala Ser Val Thr Leu Leu Thr Thr Leu Leu Asp Phe 340 345 350 Ala Asp Thr Gly Ile Leu Asp Val Phe Val Asp Glu Gly His Val Gln 355 360 365 365 Leu Arg Glu Ala Th r Leu Gly Gly Gly Ala Gly Ala Pro Cys Ala Leu 370 375 380 Leu Arg Gly Leu Glu Leu Ala Asn Thr Phe Ser Phe Leu Arg Pro Asn 385 390 395 400 Asp Leu Val Trp Asn Tyr Val Val Asp Asn Tyr Leu Lys Gly Asn Thr 405 410 415 Pro Val Pro Phe Asp Leu Leu Phe Trp Asn Gly Asp Ala Thr Asn Leu 420 425 430 Pro Gly Pro Trp Tyr Cys Trp Tyr Leu Arg His Thr Tyr Leu Gln Asn 435 440 445 Glu Leu Lys Val Pro Gly Lys Leu Thr Val Cys Gly Val Pro Val Asp 450 455 460 Leu Ala Ser Ile Asp Val Pro Thr Tyr Ile Tyr Gly Ser Arg Glu Asp 465 470 475 480 His Ile Val Pro Trp Thr Ala Ala Tyr Ala Ser Thr Ala Leu Leu Ala 485 490 495 495 Asn Lys Leu Arg Phe Val Leu Gly Ala Ser Gly His Ile Ala Gly Val 500 505 510 Ile Asn Pro Pro Ala Lys Asn Lys Arg Ser His Trp Thr Asn Asp Ala 515 520 525 Leu Pro Glu Ser Pro Gln Gln Trp Leu Ala Gly Ala Ile Glu His His 530 535 540 Gly Ser Trp Trp Pro Asp Trp Thr Ala Trp Leu Ala Gly Gln Ala Gly 545 550 555 560 Ala Lys Arg Ala Ala Pro Ala Asn Tyr Gly Asn Ala Arg Tyr Arg Ala 565 570 575 Ile Glu Pro Ala Pr o Gly Arg Tyr Val Lys Ala Lys Ala 580 585 <210> 2 <211> 1770 <212> DNA <213> Ralstonia eutropha TB64 <220> <221> CDS <222> (1) ... (1770) Polyhydroxybutyrate synthase encoding sequence <400> 2 atggcgaccg gcaaaggcgc ggcagcttcc acgcaggaag gcaagtccca accattcaag 60 ttcacgccgg ggccattcga tccagccaca tggctggaat ggtcccgcca gtggcagggc 120 actgaaggca acggccacgc tgccgcgtcc ggcattccgg gcctggatgc gctggcaggc 180 gtcaagatcg cgccggcgca gctgggtgat atccagcagc gctacatgaa ggacttctca 240 gcgctgtggc aggccatggc cgagggcaag gccgaggcca ccggtccgct gcacgaccgg 300 cgcttcgccg gcgacgcatg gcgcaccaac ctcccatatc gcttcgctgc cgcgttctac 360 ctgctcaatg cgcgcgcctt gaccgagctg gccgatgccg tcgaggccga tgccaagacc 420 cgccagcgca tccgcttcgc gatctcgcaa tgggtcgatg cgatgtcgcc cgccaacttc 480 cttgccacca atcccgaggc gcagcgcctg ctgatcgagt cgggcggcga atcgctgcgt 540 gccggcgtgc gcaacatgat ggaagacctg acacgcggca agatctcgca gaccgacgag 600 agcgcgtttg aggtcggccg caatgtcgcg gtgaccgaag gcgccgtggt cttcgagaac 660 gagtacttcc agctgttgca gtacaagccg ctgaccgaca agg tgcacgc gcgcccgctg 720 ctgatggtgc cgccgtgcat caacaagtac tacatcctgg acctgcagcc ggagagctcg 780 ctggtgcgcc atgtggtgga gcagggacat acggtgtttc tggtgtcgtg gcgcaatccg 840 gacgccagca tggccggcag cacctgggac gactacatcg agcacgcggc catccgcgcc 900 atcgaagtcg cgcgcgacat cagcggccag gacaagatca acgtgctcgg cttctgcgtg 960 ggcggcacca ttgtctcgac cgcgctggcg gtgctggccg cgcgcggcga gcacccggcc 1020 gccagcgtca cgctgctgac cacgctgctg gactttgccg acaccggcat cctcgacgtc 1080 tttgtcgacg agggccatgt gcagttgcgc gaggccacgc tgggcggcgg cgccggcgcg 1140 ccgtgcgcgc tgctgcgcgg ccttgagctg gccaatacct tctcgttcct gcgcccgaac 1200 gacctggtgt ggaactacgt ggtcgacaac tacctgaagg gcaacacgcc ggtgccgttc 1260 gacctgctgt tctggaacgg cgacgccacc aacctgccgg ggccgtggta ctgctggtat 1320 ctgcgccaca cgtacctgca gaacgagctc aaggtaccgg gcaagctgac cgtgtgcggc 1380 gtgccggtgg acctggccag catcgacgtg ccgacctata tctacggctc gcgcgaagac 1440 catatcgtgc cgtggaccgc ggcctatgcc tcgaccgcgc tgctggcgaa caagctgcgc 1500 ttcgtgctgg gtgcgtcggg ccatatcgcc ggtgtgatca acccgccggc caa gaacaag 1560 cgcagccact ggactaacga tgcgctgccg gagtcgccgc agcaatggct ggccggcgcc 1620 atcgagcatc acggcagctg gtggccggac tggaccgcat ggctggccgg gcaggccggc 1680 gcgaaacgcg ccgcgcccgc caactatggc aatgcgcgct atcgcgcaat cgaacccgcg 1740 cctgggcgat acgtcaaagc caaggcatga 1770 <210> 3 <211> 393 <212> PRT <213> Ralstonia eutropha TB64 <220> <223 > Beta-ketothiolase <400> 3 Met Thr Asp Val Val Ile Val Ser Ala Ala Arg Thr Ala Val Gly Lys 1 5 10 15 Phe Gly Gly Ser Leu Ala Lys Ile Pro Ala Pro Glu Leu Gly Ala Val 20 25 30 Val Ile Lys Ala Ala Leu Glu Arg Ala Gly Val Lys Pro Glu Gln Val 35 40 45 Ser Glu Val Ile Met Gly Gln Val Leu Thr Ala Gly Ser Gly Gln Asn 50 55 60 Pro Ala Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro Ala Met Val Pro 65 70 75 80 Ala Met Thr Ile Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Val Met 85 90 95 Leu Ala Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile Val Val Ala 100 105 110 Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His Val Leu Pro Gly Ser 115 120 125 Arg Asp Gly Phe Arg Met Gly Asp Ala Lys Leu Val Asp Thr Met Ile 130 135 140 Val Asp Gly Leu Trp Asp Val Tyr Asn Gln Tyr His Met Gly Ile Thr 145 150 155 160 Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Ala Gln Asp 165 170 175 Glu Phe Ala Val Gly Ser Gln Asn Lys Ala Glu Ala Ala Gln Lys Ala 180 185 190 Gly Lys Phe Asp Glu Glu Ile Val Pro Val Leu Ile Pro Gln Arg Lys 195 200 205 Gly Asp Pro Val Ala Phe Lys Thr Asp Glu Phe Val Arg Gln Gly Ala 210 215 220 Thr Leu Asp Ser Met Ser Gly Leu Lys Pro Ala Phe Asp Lys Ala Gly 225 230 235 240 Thr Val Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly Ala Ala Ala 245 250 255 Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly Leu Thr Pro 260 265 270 Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly Val Asp Pro Lys Val 275 280 285 285 Met Gly Met Gly Pro Val Pro Ala Ser Lys Arg Ala Leu Ser Arg Ala 290 295 300 Glu Trp Thr Pro Gln Asp Leu Asp Leu Met Glu Ile Asn Glu Ala Phe 305 310 315 320 Ala Ala Gln Ala Leu Ala Val His Gln Gln Met Gly Trp Asp Thr Ser 325 330 335 Lys Val Asn Val Asn Gly Gly Ala Ile Ala Ile Gly HisPro Ile Gly 340 345 350 Ala Ser Gly Cys Arg Ile Leu Val Thr Leu Leu His Glu Met Lys Arg 355 360 365 Arg Asp Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly Gly Gly Met 370 375 380 Gly Val Ala Leu Ala Val Glu Arg Lys 385 390 <210> 4 <211> 1182 <212> DNA <213> Ralstonia eutropha TB64 <220> <221> CDS <222> (1) ... (1182) Beta-ketothiolase encoding sequence <400> 4 atgactgacg ttgtcatcgt atccgccgcc cgcaccgcgg tcggcaagtt tggcggctcg 60 ctggccaaga tcccggcacc ggaactgggt gccgtggtca tcaaggccgc gctggagcgc 120 gccggcgtca agccggagca ggtgagcgaa gtcatcatgg gccaggtgct gaccgccggt 180 tcgggccaga accccgcacg ccaggccgcg atcaaggccg gcctgccggc gatggtgccg 240 gccatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct ggccgccaac 300 gcgatcatgg cgggcgacgc cgagatcgtg gtggccggcg gccaggaaaa catgagcgcc 360 gccccgcacg tgctgccggg ctcgcgcgat ggtttccgca tgggcgatgc caagctggtc 420 gacaccatga tcgtcgacgg cctgtgggac gtgtacaacc agtaccacat gggcatcacc 480 gccgagaacg tggccaagga atacggcatc acacgcgagg cgcaggatga gttcgccgtc 540 ggctcgcaga acaaggccg a agccgcgcag aaggccggca agtttgacga agagatcgtc 600 ccggtgctga tcccgcagcg caagggcgac ccggtggcct tcaagaccga cgagttcgtg 660 cgccagggcg ccacgctgga cagcatgtcc ggcctcaagc ccgccttcga caaggccggc 720 acggtgaccg cggccaacgc ctcgggcctg aacgacggcg ccgccgcggt ggtggtgatg 780 tcggcggcca aggccaagga actgggcctg accccgctgg ccacgatcaa gagctatgcc 840 aacgccggtg tcgatcccaa ggtgatgggc atgggcccgg tgccggcctc caagcgcgcc 900 ctgtcgcgcg ccgagtggac cccgcaggac ctggacctga tggagatcaa cgaggccttc 960 gccgcgcagg cgctggcggt gcaccagcag atgggctggg acacctccaa ggtcaatgtg 1020 aacggcggcg ccatcgccat cggccacccg atcggcgcgt cgggctgccg tatcctggtg 1080 acgctgctgc acgagatgaa gcgccgtgac gcgaagaagg gcctggcctc gctgtgcatc 1140 ggcggcggca tgggcgtggc gctggcagtc gagcgcaaat aa 1182 <210> 5 <211> 246 <212> PRT <213> Ralstonia eutropha TB64 <220> <223> acetoacetyl CoA reductase <400> 5 Met Thr Gln Arg Ile Ala Tyr Val Thr Gly Gly Met Gly Gly Ile Gly 1 5 10 15 Thr Ala Ile Cys Gln Arg Leu Ala Lys Asp Gly Phe Arg Val Val Ala 20 25 30 Gly Cys Gly Pro AsnSer Pro Arg Arg Glu Lys Trp Leu Glu Gln Gln 35 40 45 Lys Ala Leu Gly Phe Asp Phe Ile Ala Ser Glu Gly Asn Val Ala Asp 50 55 60 Trp Asp Ser Thr Lys Thr Ala Phe Asp Lys Val Lys Ser Glu Val Gly 65 70 75 80 Glu Val Asp Val Leu Ile Asn Asn Ala Gly Ile Thr Arg Asp Val Val 85 90 95 Phe Arg Lys Met Thr Arg Ala Asp Trp Asp Ala Val Ile Asp Thr Asn 100 105 110 Leu Thr Ser Leu Phe Asn Val Thr Lys Gln Val Ile Asp Gly Met Ala 115 120 125 Asp Arg Gly Trp Gly Arg Ile Val Asn Ile Ser Ser Val Asn Gly Gln 130 135 140 Lys Gly Gln Phe Gly Gln Thr Asn Tyr Ser Thr Ala Lys Ala Gly Leu 145 150 155 160 His Gly Phe Thr Met Ala Leu Ala Gln Glu Val Ala Thr Lys Gly Val 165 170 175 Thr Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Asp Met Val Lys 180 185 190 Ala Ile Arg Gln Asp Val Leu Asp Lys Ile Val Ala Thr Ile Pro Val 195 200 205 Lys Arg Leu Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys Ala Trp Leu 210 215 220 Ser Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala Asp Phe Ser Leu Asn 225 230 235 240 Gly Gly Leu His Met Gly 245 <210> 6 <211> 741 <212> DNA <213> Ralstonia eutropha TB64 <220> <221> CDS <222> (1) ... (741) Acetoacetyl CoA reductase encoding sequence <400> 6 atgactcagc gcattgcgta tgtgaccggc ggcatgggtg gtatcggaac cgccatttgc 60 cagcggctgg ccaaggatgg ctttcgtgtg gtggccggtt gcggccccaa ctcgccgcgc 120 cgcgaaaagt ggctggagca gcagaaggcc ctgggcttcg atttcattgc ctcggaaggc 180 aatgtggctg actgggactc gaccaagacc gcattcgaca aggtcaagtc cgaggtcggc 240 gaggttgatg tgctgatcaa caacgccggt atcacccgcg acgtggtgtt ccgcaagatg 300 acccgcgccg actgggatgc ggtgatcgac accaacctga cctcgctgtt caacgtcacc 360 aagcaggtga tcgacggcat ggccgaccgt ggctggggcc gcatcgtcaa catctcgtcg 420 gtgaacgggc agaagggcca gttcggccag accaactact ccaccgccaa ggccggcctg 480 catggcttca ccatggcgct ggcgcaggaa gtggcgacca agggcgtgac cgtcaacacg 540 gtttctccgg gctatatcgc caccgacatg gtcaaggcga tccgccagga cgtgctcgac 600 aagatcgtcg cgacgatccc ggtcaagcgc ctgggcctgc cggaagagat cgcctcgatc 660 tgcgcctggt tgtcgtcgga ggagtccggt ttctcgaccg gcgccgactt ctcgctcaac 720 ggcggcctgc atatgggctg a 741 < 210> 7 <211> 32 <212> DNA <220> <223> Primer sequence for PCR <400> 7 gagagaggat ccaatcatgg cgaccggcaa ag 32 <210> 8 <211> 31 <212> DNA <220> <223> Primer sequence for PCR <400> 8 gactggtgga tccaggccgg caggtcagcc c 31

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/00 C12N 9/10 9/02 C12P 7/62 9/10 21/02 C C12P 7/62 C12N 15/00 ZNAA 21/02 5/00 A (72) Inventor Tsukasa Honma 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Sakae Suda 3-30 Shimomaruko, Ota-ku, Tokyo No. 2 F term in Canon Inc. (reference) 4B024 AA03 BA80 CA03 EA04 GA11 4B050 CC01 CC03 LL05 4B064 AD83 CA02 CA19 CC24 DA16 4B065 AA01Y AA26X AB01 AC14 BA02 CA12 CA60

Claims (17)

[Claims] 1. A polyhydroxybutyrate synthase having the amino acid sequence represented by SEQ ID NO: 1. 2. The method according to claim 2, wherein the amino acid in the amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence deleted, substituted or added within a range not impairing the polyhydroxybutyrate synthase activity based on the amino acid sequence. Polyhydroxybutyrate synthase. 3. A polyhydroxybutyrate synthase gene comprising a DNA encoding the amino acid sequence according to claim 1 or 2. 4. The polyhydroxybutyrate synthase gene according to claim 3, wherein the DNA encoding the polypeptide having polyhydroxybutyrate synthase activity is represented by SEQ ID NO: 2. 5. A β-ketothiolase having the amino acid sequence represented by SEQ ID NO: 3. 6. The amino acid in the amino acid sequence represented by SEQ ID NO: 3 has a deleted, substituted or added amino acid sequence as long as the β-ketothiolase activity based on the amino acid sequence is not impaired. Β
-Ketothiolase. 7. A β-ketothiolase gene comprising a DNA encoding the amino acid sequence according to claim 5 or 6. 8. The β-ketothiolase gene according to claim 7, wherein the DNA encoding the polypeptide having β-ketothiolase activity is represented by SEQ ID NO: 4. 9. An acetoacetyl-CoA reductase having the amino acid sequence represented by SEQ ID NO: 5. 10. An amino acid in the amino acid sequence represented by SEQ ID NO: 5 has a deleted, substituted or added amino acid sequence as long as the acetoacetyl-CoA reductase activity based on the amino acid sequence is not impaired. Acetoacetyl-CoA reductase. An acetoacetyl-CoA containing a DNA encoding the amino acid sequence according to claim 9 or 10.
Reductase gene. 12. The acetoacetyl-CoA reductase gene according to claim 11, wherein the DNA encoding the polypeptide having acetoacetyl-CoA reductase activity is represented by SEQ ID NO: 6. 13. A recombinant vector comprising the polyhydroxybutyrate synthase gene according to claim 3 or 4. 14. The recombinant vector according to claim 13, which comprises at least one of the β-ketothiolase gene according to claim 7 or 8 and the acetoacetyl-CoA reductase gene according to claim 11 or 12. 15. A transformant transformed by the recombinant vector according to claim 13 or 14. 16. A polyhydroxybutyrate synthase or polyhydroxybutyrate synthase, wherein the transformant according to claim 15 is cultured, and a polyhydroxybutyrate synthase or a group of polyhydroxybutyrate synthase enzymes is obtained from the obtained culture. A method for producing a hydroxybutyric acid synthesis enzyme group. 17. A method for producing polyhydroxybutyric acid, comprising culturing the transformant according to claim 15, and obtaining polyhydroxybutyric acid from the resulting culture.