WO2005098001A1

WO2005098001A1 - Novel transformant

Info

Publication number: WO2005098001A1
Application number: PCT/JP2005/006922
Authority: WO
Inventors: Tetsuya Nagaoka
Original assignee: Kaneka Corporation
Priority date: 2004-04-09
Filing date: 2005-04-08
Publication date: 2005-10-20
Also published as: JPWO2005098001A1; TW200540269A

Abstract

From the viewpoints of its safety, high productivity, availability as an economical carbon source and so on, Ralstonia eutropha is expected as a host that is most suitable for the industrial production of P(3HB-co-3HH). Compared with a low-3HH composition P(3HB-co-3HH) production system, however, there has been established no safe and economical production system of high-3HH composition P(3HB-co-3HH) appropriate for processing into films and the like by taking advantage of its high flexibility. Thus, it has been required to develop a high production system thereof. Namely, a novel polyester synthase-expressing plasmid for producing high-3HH composition P(3HB-co-3HH) showing a high flexibility and thus being appropriate for processing into films and the like; a transformant capable of synthesizing high-3HH composition P(3HB-co-3HH) which contains the above plasmid; and a safe and economical method of producing high-3HH composition P(3HB-co-3HH) by using the above transformant.

Description

Specification

New transformant

Technical field

The present invention relates to a novel plasmid for producing a polyester having a high 3HH composition, a transformant thereof, and a method for producing a polyester having a high 3HH composition using the same.

Background art

[0002] It has been known that many microorganisms accumulate polyester in cells as an energy storage substance. A typical example is poly-3-hydroxybutyric acid (hereinafter abbreviated as P (3HB)), which is a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB), and was first introduced in 1925 in Bacillus megaterium (Bacillus megaterium). Was discovered in P (3HB) is a thermoplastic polymer that is biologically degraded in the natural environment and is attracting attention as an environmentally friendly plastic. However, P (3HB) has high crystallinity and is hard and brittle, so its practical application is limited. For this reason, research aimed at improving this property has been made.

[0003] Among them, a method for producing a copolymer (hereinafter, abbreviated as P (3HB-CO-3HV)) that is also powerful with 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) is disclosed! (For example, see Patent Document Patent Document 2). this? ! ^ ^ ー! ^) Is considered to be applicable to a wide range of applications because it is more flexible than P (3HB). In fact, P (3HB-CO-3HV) is not required to change its physical properties even if the 3HV mole fraction is increased. It was not used in the field of hard moldings, such as shampoo bottles and handles for throwing razors.

In recent years, research has been conducted on a two-component copolymer polyester (hereinafter abbreviated as P (3HB-CO-3HH)) of 3HB and 3hydroxyhexanoic acid (hereinafter abbreviated as 3HH) and a method for producing the same. (See Patent Documents 3 and 4). The method for producing P (3HB-co-3HH) reported in these reports is based on fermentative production of fatty acids such as oleic acid and fatty oils such as olive oil using aeromonas caviae isolated from soil. there were. Also, studies on the properties of P (3HB co 3HH) have been made (see Non-Patent Document 1). This Reported that aeromonas capillarum was cultured using fatty acids having 12 or more carbon atoms as the sole carbon source, and fermentatively produced P (3HB-co-3HH) with a 3HH composition of 11 to 19 mol%! . This P (3HB—co—3HH) becomes harder and more brittle than P (3HB) as the 3HH composition increases, and the P (3HB—co—3HV) becomes more flexible than P (3HB—co—3HV). It was revealed to show. In other words, P (3HB—co—3HH) has a wide range of physical properties that can be applied to soft polymers by changing the 3HH composition. It can be expected to be applied to a wide range of fields, such as those requiring flexibility, such as. However, this production method has a low cell productivity of 4 g ZL, a polymer content of 30%, and a low polymer productivity, making it inadequate as a production method for the practical use of this polymer. As a result, we searched for ways to achieve even higher productivity for practical use.

[0005] Efforts have also been made toward industrial production of P (3HB—co—3HH). In the culture using Aeromonas hydrophila, in a 43-hour fed-batch culture using oleic acid as a carbon source, a P cell with a cell mass of 95.7 g / L, a polymer content of 45.2%, and a 3HH composition of 17% was obtained. (3HB—co—3HH) was produced (see Non-Patent Document 2). Further, Aeromonas' hydrofila was cultured using glucose and lauric acid as carbon sources to achieve a cell amount of 50 gZL and a polymer content of 50% (see Non-Patent Document 3). However, Aeromonas hydrofila is pathogenic to humans (see Non-Patent Document 4), and thus cannot be said to be a species suitable for industrial production. In addition, since these culture productions use expensive carbon sources, it is required to use inexpensive carbon sources from the viewpoint of production costs.

[0006] For this reason, efforts have been made to improve production in a safe host and improve productivity.

A polyhydroxyalkanoic acid (PHA) synthase gene was cloned from Aeromonas cinerea (see Patent Document 5, Non-patent Document 5). Production of P (3HB—co—3HH) using a transformant in which this gene was introduced into Ralstonia eutropha (formerly Alcaligenes eutrophas) resulted in a cell productivity of 4 gZL and polymer content. Was 30%. Furthermore, as a result of culturing the transformant using vegetable oils and fats as a carbon source, a cell content of 4 gZL and a polymer content of 80% were achieved (see Non-Patent Document 6). However, the polymer productivity is low, although cheap vegetable oils and fats are used as carbon sources. The 3HH composition of 5% was hard, brittle, and had physical properties.

[0007] In addition, Ralstonia トロ -tropha capable of producing P (3HB-co-3HH) using fructose as a carbon source has also been constructed. The polymer productivity of this strain is low, and it is not suitable for actual production. (See Non-Patent Document 7).

[0008] A P (3HB-co-3HH) producing strain using Escherichia coli as a host has also been constructed. A strain was constructed in which Escherichia coli introduced a PHA synthase gene of the genus Aeromonas and a NADP-acetoacetyl Co-A reductase gene of Ralstonia eutopha. As a result of culturing the Escherichia coli for 40.8 hours using dodecane as a carbon source, the bacterial mass was 79 gZL, the polymer content was 27.2%, and the 3HH composition was 10.8% (see Non-Patent Document 8).

[0009] An Escherichia coli transfected with the PHA synthase gene of Aeromonas' rabies, the enol CoA hydratase gene and the acyl-CoA dehydrogenase gene was also constructed. When the Escherichia coli was cultured in a medium containing lauric acid, the polymer content was about 16% and the 3HH composition was about 16% (Non-Patent Document 9). The productivity of these Escherichia coli was low and it was difficult to apply them to industrial production.

[0010] On the other hand, in order to improve the productivity of P (3HB-co-3HH) and control the composition of 3HH, artificial modification of PHA synthase was performed (see Non-Patent Documents 10 and 11). ). Among the PHA synthase mutants derived from A. aeruginosa, a mutant enzyme in which the amino acid aspargin at position 149 has been replaced with serine and a mutant enzyme in which glycine has been substituted for position 171 aspartic acid have been found in Escherichia coli. PHA synthase activity and 3HH composition were also improved, and mutants in which 518th phenylalanine was replaced with isoleucine and 214th in which palin was replaced with glycine It has been reported that the enzyme has improved PHA synthase activity and polymer content in Escherichia coli. However, since they use special E. coli as their host and still have a low polymer content, further improvement was required for industrial production utilizing the characteristics of these mutant enzymes.

[0011] T. Fukui et al. Produced a PHA synthase expression plasmid that produced P (3HB—co—3HH) using Ralstonia eutopha as a host, and the polyester synthase gene and D-enol PJRDEE32 and pJRDEE32dl3 into which a Co—A hydratase gene or the like was introduced (see Patent Document 5). Is the cell content of this strain as low as 4 gZL? As a result, the cell content was increased to 45 g ZL, the polymer content was increased to 62.5%, and the 3HH was increased to 8.1% by improving the culture conditions of the same strain using vegetable oil as a carbon source. A method for producing P (3HB-co-3HH) has also been studied (see Patent Document 6). Also, a method for controlling the physical properties of P (3HB-co-3HH) has been disclosed (see Patent Document 6). By using at least two kinds of fats and oils having different carbon numbers and Z or fatty acids as a carbon source, it becomes possible to produce P (3HB-co-3HH) having a 3HH composition of 1 to 40 mol%, and various physical properties can be obtained. (3HB-co-3HH) can be produced. However, in this production method, it is necessary to add relatively expensive hexanoic acid, octanoic acid, etc. to control the 3HH composition, and high concentrations of hexanoic acid show cytotoxicity. The result is a decrease. In addition, the addition of multi-component carbon sources can make the production facilities complex and expensive.

Patent Document 1: JP-A-57-150393

Patent Document 2: JP-A-59-220192

Patent Document 3: JP-A-5-93049

Patent Document 4: JP-A-7-265065

Patent Document 5: JP-A-10-108682

Patent Document 6: Japanese Patent Application Laid-Open No. 2001-340078

Non-Patent Document 1: Y. Doi, S. Kitamura, H. Abe, Macromolecules 28, 4822-4 823 (1995)

Non-patent document 2: Biotechnology and Bioengineering, vol67, 240 (2000) Non-patent document 3: Appl.Microbiol.Biotechnol., Vol57, 50 (2001)

Non-Patent Document 4: National Institute of Infectious Diseases, Regulations for Safety Management of Pathogens, Appendix 1 Appendix 1 (1999) Non-Patent Document 5: T. Fukui, Y. Doi, J. Bacteriol, 179, 15, 4821—4830 (1997) Non-patent document 6: T. Fukui et al., Appl. Microbiol. Biotecnol. 49, 333 (1998) Non-patent document 7: T. Fukui et al., Biomolecules, vol3, 618 (2002)

Non-Patent Document 8: S. Park et al. Biomacromolecules, vol2, 248 (2001)

Non-Patent Document 9: X. Lu et al., FEMS Microbiology Letters, vol221, 97 (2003) Non-Patent Document 10: T. Kichise et al., Appl.Environ.Microbiol. 68, 2411-2419 (2 002)

Non-Patent Document 11: A. Amara et al. App.Microbiol.Biotechnol., Vol59, 477 (200 2)

Disclosure of the invention

Problems to be solved by the invention

[0013] As described above, Ralstonia's eutopha is the most suitable host for industrial production of P (3HB-co-3HH) from the viewpoints of safety, high productivity, availability of an inexpensive carbon source, and the like. Is expected. However, compared to the low 3HH composition P (3HB-co-3HH) production system, safe and inexpensive production of high 3HH composition P (3HB-co-3HH) suitable for processing into films, etc., making use of its flexible properties Since the system was established, the development of a high production system was desired.

Means for solving the problem

[0014] The present inventors have conducted intensive studies in order to solve the above problems, and have constructed a novel polyester synthase expression plasmid containing a novel phaC derivative gene derived from Aeromonas' biebie. A transformant in which the expression plasmid was introduced into a host such as Ralstonia-eutropha was prepared and cultured under a single carbon source using inexpensive vegetable oils and fats. As a result, P (3HB-co-3HH) with a 3HH composition of 12 mol% or more was obtained. Can be produced with high productivity.

[0015] That is, the present invention provides a polyester synthase expression plasmid capable of producing P (3HB-co-3HH) having a high 3HH composition excellent in processability into a film or the like, and a high 3HH composition polyester containing the plasmid. The present invention relates to a transformant having an ability and a safe and inexpensive method for producing a high 3HH composition polyester using the transformant.

[0016] That is, the present invention provides a polyester synthase expression plasmid (I) contained in PHB-4 / pJRDdTc + 149NS17IDG (FERM BP-10259), which is a high 3HH composition polyester synthase expression plasmid; Polyester synthase expression plasmid (II) obtained by miniaturizing the plasmid; a transformant transformed with the above-mentioned high 3HH composition polyester synthase expression plasmid and PHB-4ZpJRDdTc + 149NS171DG (FERM BP-10259); The present invention relates to a method for producing a polyester. In the above production method, preferably, the polyester is represented by the following formula (1) [0017] [Formula 1]

H-OH (1)

(Wherein m and n each represent an integer of 1 or more), which is a copolymerized polyester P (3HB—co—3HH) having 3-hydroxybutyric acid and 3-hydroxyhexanoic acid power. This is a safe and inexpensive method for producing a high 3HH composition polyester using a transformant.

Hereinafter, the present invention will be described in detail.

The polyester synthase expression plasmid (I) of the present invention is contained in PHB-4ZpJRDdTc + 149N S171DG (FERM BP-10259), and has a high productivity and high 3HH composition P (3HB-co-3HH). This is an expression plasmid for the enzyme to be synthesized.

Preparation of Expression Plasmid

First, the production of the polyester synthase expression plasmid (I) of the present invention will be described. Global genetic manipulations can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press, (1989)). Enzymes, Cloying hosts, etc. used for genetic manipulation can be purchased from market suppliers and used according to the description. The enzyme is not particularly limited as long as it can be used for genetic manipulation. The Clawing host is not particularly limited, and examples include Escherichia coli DH5α strain.

In the present invention, a pJRD215 (ATC C 37533) derivative pJRDdTc (described in WO04Z074476), which is one of the broad host range vectors, can be used as the vector. SEQ ID NO: 4 shows the sequence of the vector used in the construction of this expression plasmid.

As a polyester synthase gene, an N149SZD171G double mutant gene fragment derived from Aeromonas capillaris shown in SEQ ID NO: 3 is prepared as a restriction enzyme EcoRI fragment, and inserted into the same restriction enzyme site of the above vector to construct.

The N149S mutant gene and the D171G mutant gene are described in T. Kichise et al., Appl. Environ. Microbiol. 68, 2411-2419 (2002) [this description describes that asparagine at the 149th amino acid is serine. The 171st amino acid aspartic acid is This is a polyester synthase gene derived from Aeromonas capillaris, substituted for syn.

FIG. 1 shows the procedure for constructing the polyester synthase expression plasmid (I) of the present invention.

[0022] The polyester synthase gene only needs to have an expression unit that functions in the host bacterium, such as a promoter and a terminator, in addition to the structural gene. Note that the polyester synthase expression plasmid may contain a plurality of expression units as long as at least one expression unit is present.

The polyester synthase expression plasmid (I) contained in PHB-4ZpJRDdTc + 149NS171DG (FERM BP-10259) of the present invention may be referred to as “pJRDdTc + 149NS171DG” in this specification.

Next, the production of the polyester synthase expression plasmid (II) of the present invention will be described.

The polyester synthase expression plasmid (Π) of the present invention is obtained by miniaturizing the polyester synthase expression plasmid (I) contained in PHB-4ZpJRDdTc + 149 NS171DG (FERM BP-10259).

[0024] The size of the plasmid can be reduced by deleting portions unnecessary for expression of the polyester synthase gene and plasmid replication. For example, if the plasmid used has two or more antibiotic resistance genes, either of them can be deleted. In the case of the expression plasmid of the present invention, it is possible to delete the kanamycin resistance gene or the streptomycin resistance gene, but it is preferable to delete the streptomycin resistance gene. As a method for deleting a specific gene as described above, a method using a restriction enzyme site or a method using PCR can be used.

[0025] Also, a polyester synthase expression plasmid lacking part or all of the conjugation transfer ability and lacking the streptomycin resistance gene can be suitably used.

[0026] The polyester synthase expression plasmid (本) of the present invention can improve the transformation rate when the expression plasmid is introduced into a host by miniaturizing the plasmid.

[0027] Fabrication of the form

Next, production of a transformant will be described.

The transformant of the present invention comprises at least one of the above-mentioned polyester synthase expression plasmids (I) to (II). It has been transformed by this. That is, the transformant of the present invention can be obtained by introducing any of the polyester synthase expression plasmids (I) to (I) obtained above into a host compatible with the plasmid.

[0028] The host is not particularly limited, but a microorganism isolated from nature, a microorganism deposited at a strain depository (eg, IFO, ATCC, etc.), and the like can be used. Specifically, non-polyester-producing bacteria of bacteria such as Ralstonia, Aeromonas, Escherichia, Alcaligenes, and Pseudomonas can be used. From the viewpoint of safety and productivity, Ralstonia is preferable, Ralstonia eutropha is more preferable, and polyester synthase is inactivated by mutagen treatment or gene disruption treatment based on homologous recombination. Ralstonia's Eutropha, for example, Ralstonia's Eutropha PHB-4 strain. Ralstonia's Eutropha PHB-4 strain is available from institutional sources such as D SMZ.

[0029] Introduction of a polyester synthase expression plasmid into a microorganism can be performed by a known method. For example, the electo-portion method (Current Protocols in Morecular Biology, vol. 1, page 1.8.4, 1994), the calcium method (Lederberg. EM et al., J. Bacteriol. 119. 1072 (1974)), etc. Can be used.

[0030] Preferable transformants of the present invention include, for example, a transformant in which the polyester synthase expression plasmid (I) is introduced into Ralstonia's utop as a host. Specific examples include the following transformants.

PHB-4 / pJRDdTc + 149NS171DG (Accession number: FERM BP-10259, accession date: February 23, 2005), which is a transformant in which pJRDdTc + 149NS 171DG was introduced into Ralstonia eutropha PHB-4 strain. This transformant has been deposited internationally under the Budapest Treaty at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, located at 1-1, Tsukuba-Higashi, Ibaraki, Japan. .

[0031] P (3HB—co—3HH) production

Next, the production of P (3HB-co-3HH) will be described.

The method for producing a polyester of the present invention uses the above transformant. As the above polyester, the following formula (1)

[0032] [Formula 2]

H-OH (1)

[0033] Copolymerized polyester P (3HB—co—3HH), which also has 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, represented by the formula (where m and n represent integers of 1 or more) is preferred. Higher 3HH copolymer polyester P (3HB-co-3HH) is more preferred!

[0034] The composition ratio of each monomer unit constituting the high 3HH composition P (3HB—co—3HH)! Although not particularly limited, from the viewpoint of good flexibility, 3HH units are preferably at least 8 mol% and at most 50 mol%, more preferably at least 10 mol% and at most 40 mol%, particularly at most 12 mol%. % Or more and 30 mol% or less.

[0035] In the production of P (3HB-co-3HH), sugars, fats and oils or fatty acids are given as carbon sources and include nutrients other than carbon sources, such as nitrogen sources, inorganic salts, and other organic nutrients. The transformant can be cultured using a medium.

For example, as a medium for culturing a transformant obtained by using a microorganism such as a microorganism belonging to the genus Ralstonia, Aeromonas, Escherichia, Alcaligenes or Pseudomonas as a host, a carbon source that can be assimilated by the microorganism is used. In some cases, a medium in which any one of nitrogen source, inorganic salts and organic nutrients is restricted, for example, a medium in which the nitrogen source is restricted to 0.01 to 0.1%, etc. can be used. .

[0037] Examples of the sugar include carbohydrates such as glucose and fructose.

Examples of fats and oils include fats and oils rich in saturated 'unsaturated fatty acids having 10 or more carbon atoms, such as coconut oil, palm oil, palm kernel oil and the like.

Fatty acids include saturated unsaturated fatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid and myristic acid, and esters and salts of these fatty acids. Fatty acid derivatives are mentioned.

[0038] Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate and the like, as well as peptone, meat extract, yeast extract and the like. Be [0039] Examples of the inorganic salts include potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium salt and the like.

[0040] Other organic nutrients include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; and vitamins such as vitamin B1, vitamin B12, and vitamin C.

Further, an antibiotic (such as kanamycin) corresponding to the antibiotic resistance gene present in the expression plasmid of the present invention may be added to the culture solution.

The culture temperature may be any temperature at which the bacteria can grow, but is preferably 20 ° C to 40 ° C. The culture time is not particularly limited, but may be about 1 to 7 days.

Thereafter, P (3HB-CO-3HH) should be recovered from the obtained cultured cells.

[0042] In the present invention, P (3HB-co-3HH) can be recovered from cells by, for example, the following method. After completion of the culture, the cells are separated from the culture using a centrifuge or the like, and the cells are washed with distilled water, methanol and the like, and dried. From the dried cells, p (3HB-co-3HH) is extracted using an organic solvent such as black form. Cell components are removed from the organic solvent solution containing P (3HB—co—3HH) by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to form P (3HB co—3HH). Let it settle. Further, the supernatant is removed by filtration or centrifugation, and dried to recover P (3HB-co-3HH).

The analysis of the weight average molecular weight (Mw) and the 3HH composition (mol%) of the obtained P (3HB-co-3HH) can be performed by, for example, a gas chromatography method or a nuclear magnetic resonance method. Alternatively, as a simple method for confirming polyester production, a dyeing method using Nilered can be used. That is, the presence or absence of polyester production is determined by culturing Nilered on an agar medium on which the recombinant bacterium grows, culturing the recombinant bacterium for 1 to 7 days, and observing whether the recombinant bacterium turns red. You can check.

The invention's effect

[0044] As described above, the use of a transformant of Ralstonia 'Uto ora by using a novel polyester synthase expression plasmid enables the formation of a film or the like even when cultured under a single carbon source such as inexpensive vegetable oils and fats. P (3HB—co—3HH) with high 3HH composition suitable for It has become possible to produce safely and with high productivity.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention does not limit the technical scope to these examples.

(Example 1) Construction of expression plasmid

An expression plasmid for production of high 3HH composition P (3HB-co-3HH) was constructed as follows.

As a vector for constructing pJRDdTc + 149NS171DG, a vector encoded by the DNA shown in SEQ ID NO: 4 was used. This vector can be constructed in the same manner as pJRDd Tc described in WO04Z074476.

The N149SZD171G double mutant gene fragment derived from Aeromonas radiata, which is a polyester synthase gene, was prepared by PCR. In the N149S mutation and the D171G mutation, asparagine, which is the 149th amino acid of PHA synthase derived from Aeromonas' rabies, is substituted with serine, and aspartic acid, which is the 171st amino acid, is substituted with glycine. Therefore, the N149S mutant gene derived from Aeromonas serrata described in Non-Patent Document 10 was subcloned at the EcoRI site of pUC19, and the synthetic DNAs represented by SEQ ID NO: 1 and SEQ ID NO: 2 were used as primers. Was used for PCR. The conditions were (1) 2 minutes at 94 ° C, (2) 30 seconds at 94 ° C, (3) 30 seconds at 55 ° C, (4) 2 minutes at 72 ° C, (2) to (4) ) For 25 cycles, (5) 5 minutes at 72 ° C, and LA Taq polymerase (Takara Bio) was used as the polymerase. The N149S ZD171G fragment shown in SEQ ID NO: 3 was prepared by digestion with the restriction enzyme EcoRI, and this vector was inserted into the site digested with the same enzyme to construct the expression plasmid pJRDdTc + 149NS171DG.

(Example 2) Preparation of transformant

A transformant of Ralstonia'eutropha PHB-4 strain containing the expression plasmid obtained in Example 1 was prepared by an electric pulse method. That is, a gene pulsar manufactured by Biomd was used for the gene transfer device, and a cuvette having a gap of 0.2 cm manufactured by Biorad was used as the cuvette. Inject 400 μl of the competent cells and 20 μl of the expression plasmid into a cuvette, set them in a pulse device, and apply an electric pulse under the conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance of 800 Ω. I did. After the pulse, the bacterial solution in the cuvette is shake-cultured at 30 ° C for 3 hours in a Nutrient Broth medium (DIFCO), and is incubated at 30 ° C in a selection plate (NutrientAgar medium (DIFCO), kanamycin lOOmgZL). After culturing for 2 days, a transformant was obtained.

(Example 3) Selection of transformant

The transformant obtained in Example 2 was transformed into a Nilered-containing medium (9 g of disodium hydrogen phosphate · 12 hydrate, 1.5 g of potassium dihydrogen phosphate, 0.05 g of ammonium chloride, 0.05 g of magnesium sulfate, and 7 water of magnesium sulfate). 0.02 g of salt, 0.5 g of fructose, 0.5 g of Shii-Dani Conolt · 0.25 ppm of 6-hydrate salt, 16 ppm of Shi-i-Dai Tetsu (Π Ι) · 16 ppm of 6-hydrate, calcium chloride · 10.3 ppm of 2-hydrate, nickel chloride · Hexahydrate 0.12 ppm, copper sulfate pentahydrate 0.16 ppm, Nilered 0.5 mg, agar 15 gZlL), and cultured at 30 ° C for 1 week. As a result, it was confirmed that the colony turned red and that the mosquito also accumulated polyester inside the cells, and the colony was selected to produce P (3HB-co-3HH). This transformant was named PHB-4ZpJRDdTc + 149NS171DG (Accession number: FERM BP-10259, Accession date: February 23, 2005), 1-1 Tsukuba East, Ibaraki, Japan 1 Central No. 6 It has been internationally deposited under the Budapest Treaty at the Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology, Japan.

Example 4 Production and Purification of P (3HB—co—3HH)

The seed culture medium was composed of lwZv% Meat-extract, lw / v% Bacto-Trypton, 0.2 w / v% Yeast -extract, 0.9 w / v% Na PO12Η, 0.15 w / v % KH

2 4 2 2

PO, pH 6.8.

Four

[0049] The composition of the preculture medium was 1. lw / v% Na PO · 12Η 0, 0.19 w / v% KH PO, 1

2 4 2 2 4

29w / v% (NH) SO, 0.lw / v% MgSO-7H 0, 2.5w / v% Palm W

4 2 4 4 2

Oil, 0.5vZv% trace metal salt solution (1.6w / v% FeCl · 6N in 0.1N hydrochloric acid)

3 2

0, lw / v% CaCl2Η 0, 0.02w / v% CoCl-6H 0, 0.016w / v% CuS

2 2 2 2

O · 5Η 0, 0.012w / v% NiCl-6H O dissolved. ), 5 X 10 " ⁶ w / v% power

4 2 2 2

Namycin.

[0050] The composition of the P (3HB—co—3HH) production medium was 0.385 w / v% Na PO · 12Η0, 0.06.

2 4 2

7w / v% KH PO, 0-291w / v% (NH) SO, 0.1lw / v% MgSO-7H 0,

2 4 4 2 4 4 2

0.5v / v% trace metal salt solution (0.6w / v% FeCl in 0.6N HCl, 6Η0, lw / v % CaCl2Η 0, 0.02w / v% CoCl-6H 0, 0.016w / v% CuSO-5H O

2 2 2 2 4 2

0.021w / v% NiCl-6HO dissolved. ), 0.05w / v% BIOSPUREX20

twenty two

OK (antifoaming agent: Cognis Japan), 5 x 10 " ⁶ w / v% kanamycin. The carbon source is palm kernel oil olein, a low melting point fraction of palm kernel oil, used as a single carbon source. Throughout the entire culture, feeding was performed so that the specific substrate supply rate was 0.08 to 0.1 (oil () X (net dry cell weight (g) Γ ¹ X (time (h)) ^_1 ) ^_1 ).

[0051] A glycerol stock (50 µl) of the PHB-4ZpJRDdTc + 149NS171DG transformant was inoculated into a seed culture medium (10 ml), cultured for 24 hours, and a 3 L jaw amenter (circle) containing 1.8 L of preculture medium was added. 1.0vZv% was inoculated into Ryo Bioenge's MDL-300). The operating conditions were a culture temperature of 30 ° C., a stirring speed of 500 rpm, an aeration rate of 1.8 LZmin, and culture for 28 hours while controlling the pH between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control.

[0052] P (3HB—co—3HH) production culture is a 10L jar armamentar containing 6L of production medium.

(MDL-1000 manufactured by Marubishi Biohenge) was inoculated with 5.0 vZv% of the precultured seed. The operating conditions were a culture temperature of 28 ° C, a stirring speed of 400 rpm, an aeration rate of 3.6 LZmin, and a pH of 6.7 to 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control. The culture was performed for about 60 hours. After completion of the culture, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured.

[0053] To about lg of the dried cells obtained above, 100 ml of black-mouthed form was added and stirred at room temperature for 24 hours to extract P (3HB-co-3HH) in the cells. After filtering off the bacterial cell residue, the mixture was concentrated by an evaporator until the total volume became about 30 ml, then about 90 ml of hexane was gradually added, and the mixture was left for 1 hour while stirring slowly. After the precipitated P (3HB—co—3HH) was filtered off, it was vacuum dried at 50 ° C. for 3 hours. Dry? ! ^-^-! ! ^ Was weighed and the intracellular content was calculated. As a result, PHB - 4 / pJRDdTc + 149NS 171DG transformants P (3HB- co- 3 HH) content was high content of 6 in 62 hours ⁶ 1 (wt%)..

(Example 5) 31111 yarn analysis of (3118—.0—31111)

The 3HH composition analysis of P (3HB-co-3HH) produced by the PHB-4 / pJRDdTc + 149NS171 DG transformant was measured by gas chromatography as follows. [0055] Approximately 20 mg of dry P (3HB-co-3HH) was added with 2 ml of a mixture of sulfuric acid and methanol (15:85) and 2 ml of a form of lip, sealed and heated at 100 ° C for 140 minutes. As a result, a methyl ester of a P (3HB-co-3HH) decomposition product was obtained. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize, and the mixture was allowed to stand until carbon dioxide gas generation was stopped. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged, and the monomer unit composition of the P (3HB-co-3HH) degradation product in the supernatant was analyzed by canary gas chromatography. The gasket mat used was Shimadzu GC-17A, and the one-shot ram was NEUTRA BOND-1 (column length: 25 m, column inner diameter: 0.25 mm, liquid film thickness: 0.4 / zm). He was used as the carrier gas, the column inlet pressure was 100 kPa, and 1 μl of the sample was injected. The temperature was raised to the initial temperature of 100 to 200 ° C at a rate of 8 ° CZ, and further to 200 to 290 ° C at a rate of 30 ° CZ. As a result of analysis under the above conditions, the 3HH yarn of P (3HB-co-3HH) at the end of the 62-hour culture was 14.7 (mol%), a high 3HH yarn suitable for application to a finolem. It was a success.

Industrial applicability

[0056] As described above, the use of a transformant of Ralstonia 'Uto lipophora with a novel polyester synthase expression plasmid enables the formation of a film or the like even when cultured under a single carbon source such as inexpensive vegetable oils and fats. It has become possible to produce P (3HB-co-3HH) with a high 3HH composition suitable for the processing of ash safely and with high productivity.

Brief Description of Drawings

FIG. 1 is a drawing showing the construction of a plasmid for expressing pJRDdTc + 149NS171DG (FERM BP-10259) polyester synthase of the present invention.

Claims

The scope of the claims

[1] A plasmid for expressing a polyester synthase contained in PHB-4 / pJRDdTc + 149NS171DG (FERM BP-10259).

[2] A plasmid for expressing a polyester synthase obtained by miniaturizing the plasmid according to claim 1.

[3] A transformant transformed by the plasmid according to claim 1 or 2.

4. The transformant according to claim 3, wherein the host is a non-polyester-producing genus Ralstonia.

[5] The transformant according to [3] or [4], wherein the host is Ralstonia eutropha which does not produce polyester.

[6] The transformant according to any one of claims 3 to 5, wherein the host is Ralstonia'eutropha PHB-4 strain.

[7] A transformant which is PHB-4 / pJRDdTc + 149NS171DG (FERM BP-10259)

[8] A method for producing a polyester using the transformant according to any one of claims 3 to 7!

[Chemical 1]

9. The method for producing a polyester according to claim 8, which is a copolymerized polyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid represented by the formula (where m and n represent integers of 1 or more).