JP2005333933A - New expression plasmid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an ultrahigh molecular weight polyester P (3HB-co-3HH) excellent in characteristics of being processed into a fiber, etc., capable of safely producing the polyester at a low cost with high productivity, to provide an expression plasmid used for the same, and to provide a transformant. <P>SOLUTION: This expression plasmid in which a part or all of conjugation transfer ability is deleted and which is used for producing the ultrahigh molecular weight polyester, the transformant transformed by the expression plasmid, and the method for producing the ultrahigh molecular weight polyester by using the transformant are provided, respectively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a novel expression plasmid for producing ultrahigh molecular weight polyester, a transformant thereof, and a method for producing ultrahigh molecular weight polyester using the same.

To date, it has been known that polyesters accumulate in cells as an energy storage substance in many microorganisms. 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). In 1925, Bacillus megaterium (Bacillus megaterium) First discovered. P (3HB) is a thermoplastic polymer and is biologically decomposed in the natural environment, and thus has attracted attention as an environmentally friendly plastic. However, since P (3HB) has high crystallinity, it has a hard and brittle nature, so its practical application range is limited. For this reason, research aimed at improving this property has been conducted.

Among them, a method for producing a copolymer composed of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter abbreviated as P (3HB-co-3HV)) is disclosed (Patent Literature). 1 and 2). Since this P (3HB-co-3HV) is more flexible than P (3HB), it was considered that it could be applied to a wide range of applications. In practice, however, P (3HB-co-3HV) increases the 3HV mole fraction, so that the change in physical properties associated therewith is poor, and the flexibility is not particularly improved. Therefore, it is difficult to use a hard shampoo bottle or disposable razor handle. It was used only in the field of molded products.

In recent years, research has been conducted on a two-component copolymerized polyester (hereinafter abbreviated as P (3HB-co-3HH)) of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH) and a production method thereof (Patent Document 3, 4). These reported methods for producing P (3HB-co-3HH) are fermentatively produced from fatty acids such as oleic acid and fats and oils such as olive oil using Aeromonas caviae isolated from soil. It was. Studies on the properties of P (3HB-co-3HH) have also been made (see Non-Patent Document 1). In this report, Aeromonas caviae is cultured using a fatty acid having 12 or more carbon atoms as a sole carbon source, and P (3HB-co-3HH) having a 3HH composition of 11 to 19 mol% is produced by fermentation. This P (3HB-co-3HH) gradually becomes more flexible from the hard and brittle nature of P (3HB) as the 3HH composition increases, and the flexibility exceeds P (3HB-co-3HV). It was made clear to show. That is, P (3HB-co-3HH) has a wide range of physical properties that can be applied from hard polyester to soft polyester by changing the 3HH composition. Thus, it can be expected to be applied to a wide range of fields, such as those requiring flexibility. However, in this production method, the productivity of the bacterial cells is 4 g / L, the polyester content is 30%, and the polyester productivity is low. Therefore, it must be said that the production method for practical use of the polyester is still insufficient. Thus, a method for obtaining higher productivity for practical use was sought.

Moreover, artificial modification | change of PHA synthase was performed aiming at the productivity improvement of P (3HB-co-3HH), and 3HH composition control (refer nonpatent literature 2, 3). Among the mutants of PHA synthase derived from Aeromonas caviae, the mutant enzyme in which the 149th amino acid asparagine is replaced with serine, and the mutant enzyme in which the 171st aspartic acid is replaced with glycine are It has been shown that PHA synthase activity and 3HH composition are improved, and the mutant enzyme in which the 518th phenylalanine is replaced with isoleucine and the mutant enzyme in which the 214th valine is replaced with glycine are It has been reported that PHA synthase activity and polyester content have been improved. However, it has been recognized that increasing the PHA synthase activity tends to reduce the molecular weight of the accumulated polyester. In addition, since special Escherichia coli is used as a host and the polyester content is still low, further improvements for industrial production utilizing the characteristics of these mutant enzymes are required.

T.A. The PHA synthase expression plasmid that Fukui et al. Produced P (3HB-co-3HH) using Ralstonia / eutropha as a host was pJRDEE32 in which a polyester synthase gene, a D-enoyl-CoA hydratase gene, etc. were introduced into pJRD215 (ATCC 37533). And pJRDEE32d13 (see Patent Document 5). Although the bacterial cell production amount of this strain was as low as 4 g / L, the bacterial cell production amount was 45 g / L, the polyester content was 62.5%, and the 3HH composition was 8.1 by improving the culture conditions of the strain using vegetable oil as a carbon source. Studies have also been conducted on the culture method so as to improve the percentage (see Patent Document 6).

On the other hand, regarding the production of P (3HB), a method of co-expressing phbA (β-ketothiolase) and phbB (acetoacetyl-CoA reductase) derived from Ralstonia eutropha which is a 3HB supply system in addition to PHB synthase is also disclosed (See Patent Documents 7, 8, and 9).
In addition, production studies of P (3HB-co-3HH) using an acyl-CoA dehydrogenase (fadE (also referred to as yafH)) gene derived from E. coli, which is an enzyme of the β-oxidation pathway, were also conducted. In E. coli into which the enzyme gene and (R) -enoyl-CoA hydratase derived from Aeromonas caviae and the polyester synthase gene were introduced, culturing using lauric acid as a carbon source resulted in about 1 g of cell production in 48 hours. / L, the polyester content is about 16% (see Non-Patent Document 4).
Furthermore, the production of P (3HB-co-3HH) by a strain in which an Escherichia coli-derived acyl-CoA dehydrogenase gene is introduced into Aeromonas hydrophila has also been studied (see Non-Patent Document 5). In this report, culturing using lauric acid and glucose as a carbon source has achieved a cell production of about 3 g / L and a polyester content of about 60% after 48 hours of culture.

In order to process the polyester into a fiber, it is desirable to have as high a molecular weight as possible. Therefore, ultra high molecular weight P (3HB) production has been studied, and a method for producing ultra high molecular weight P (3HB) having a weight average molecular weight exceeding 10 million has been shown by controlling pH and glucose concentration during culture. . Among them, it has been clarified that ultra-high molecular weight P (3HB) improves important physical properties (for example, tensile strength and re-extension property) during processing into a fibrous form (see Non-Patent Documents 6, 7, and 8). . In addition, in the production of P (3HB) in a test tube, it was also revealed that P (3HB) having a weight average molecular weight of 3 to 12 million can be produced by changing the PHB synthase concentration ( Non-patent document 9). Furthermore, in the production of P (3HB) by Escherichia coli using an expression vector having a phaC gene controlled by an inducible promoter, the amount of enzyme expressed by controlling the amount of the inducing agent added, the weight average molecular weight was 780,000 to It is disclosed that control is possible at 4 million (see Patent Document 10).
Regarding the production of P (3HB-co-3HH), P (3HB-co-3HH) having a weight average molecular weight of about 3.1 million was accumulated in the culture using relatively expensive octanoic acid as a single carbon source. (See Non-Patent Document 10). However, there is no report on the production of ultrahigh molecular weight P (3HB-co-3HH) in culture using cheap vegetable oils and fats as a carbon source.

By the way, pJRD215 is a derivative of RSF1010, which is a broad host range vector, and has conjugative transmissibility (see Non-Patent Document 11).
Conjugation transfer is a phenomenon that occurs when bacteria with different traits are mixed and cultured, and refers to the transfer of a part of a gene of a certain bacterium (donating bacterium) to another bacterium (receiving bacterium). The strength of the conjugation transfer ability is determined by the gene involved in conjugation transfer on the donor chromosome or on the plasmid of the donor. The genes involved in conjugation transmission include the self-transmitting gene tra and the coupled transfer gene mob, and the oriT sequence. The protein encoded by tra is involved in the interaction between donor and recipient bacteria. The protein encoded by mob has a function of nicking the oriT sequence and a function of stably carrying a single-stranded DNA. The oriT sequence consists of a recognition sequence for entering a nick site and a nick. Only when these three types coexist, junction transmission occurs.
In addition, since pJRD215 has a mob gene group and an oriT sequence among genes involved in conjugation transmission, in the unlikely event that a transformant carrying pJRD215 leaks during the production of polyester on an industrial scale, for example, TRA4 When contacted with a microorganism having a gene-containing plasmid, conjugation transfer may occur, which has a safety problem such as containment of recombinants.

Furthermore, Ralstonia / eutropha is expected to be the most suitable host for industrial production of P (3HB-co-3HH) from the viewpoints of safety, high productivity, and availability of inexpensive carbon sources. However, since there is no safe and inexpensive production system capable of producing ultra-high molecular weight P (3HB-co-3HH) suitable for processing into fibers and the like utilizing flexible properties, development of the production system is desired. It was.

JP-A-57-150393 JP 59-220192 A JP-A-5-93049 JP 7-265065 A Japanese Patent Laid-Open No. 10-108682 JP 2001-340078 A US Pat. No. 5,663,063 US Pat. No. 5,661,026 US Pat. No. 5,798,235 US Pat. No. 5,811,272 Y. Doi, S .; Kitamura, H .; Abe, Macromolecules 28, 4822-4823 (1995). T.A. Kichise et al., Appl. Environ. Microbiol. 68, 2411-2419 (2002) A. Amara et al. App. Microbiol. Biotechnol. , Vol 59, 477 (2002) Xiaoyun et al., FEMS Microbiology Letters, vol. 221, p97, (2003) Lu et al., Appl. Microbiol. Biotechnol. , Vol. 64, p41, (2004) Kusaka et al., Appl. Microbiol. Biotechnol. , Vol. 47, p140, (1997) Kusaka et al. M.M. S. -PURE APPL. CHEM. , Vol. A35, p319, (1998) Kusaka et al., International Journal of Biological Macromolecules, vol. 25, p87, (1999) Gerngross et al., Proc. Natl. Acad. Sci. , Vol. 92, 6279, (1995) Kichise et al., International Journal of Biological Macromolecules, vol. 25, p69, (1999) J. et al. Davison, M.D. Heestersprite et al., Gene, 51, 275-280 (1987)

An object of the present invention is to provide an ultra-high molecular weight polyester P (3HB-co-3HH) having excellent processing characteristics to fibers and the like, which can be produced at low cost, safely and with high productivity, and to this method. An expression plasmid and a transformant are provided.

The present inventor has intensively studied to solve the above problems, and has developed a novel expression plasmid for producing ultra-high molecular weight polyester lacking part or all of the junction transmission ability, in particular, β-ketothiolase derived from Ralstonia eutropha A novel (phbA) gene and an acetoacetyl-CoA reductase (phbB) gene, an acyl-CoA dehydrogenase (fadE (also referred to as yafH)) gene from E. coli, and a polyester synthase (phaC) gene from Aeromonas caviae A polyester synthase expression plasmid was constructed. Moreover, as a result of producing a transformant in which the expression plasmid was introduced into Ralstonia and Eutropha and culturing it under a single carbon source using inexpensive vegetable oils and fats, ultra high molecular weight P (3HB) having a weight average molecular weight (Mw) of 3.5 million or more was obtained. It was found that -co-3HH) can be produced safely and with high productivity.

That is, the present invention relates to a polyester synthase expression plasmid for producing an ultrahigh molecular weight polyester P (3HB-co-3HH) having excellent processing characteristics into fibers and the like; an ultrahigh molecular weight polyester synthesis comprising the expression plasmid The present invention relates to a safe and inexpensive method for producing an ultrahigh molecular weight polyester using the transformant.

That is, the present invention relates to an expression plasmid for producing an ultrahigh molecular weight polyester that lacks part or all of the junction transmission ability; a transformant transformed with the above expression plasmid for producing an ultrahigh molecular weight polyester; (1)

(Wherein m and n represent an integer of 1 or more), the above expression plasmid or trait, which is a copolymerized polyester P (3HB-co-3HH) composed of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid The present invention relates to a method for producing ultrahigh molecular weight polyester using a converter.

The present invention is described in detail below.
The expression plasmid for producing ultra-high molecular weight polyester lacking part or all of the junction transmission ability of the present invention is a polyester synthase expression plasmid used for producing ultra-high molecular weight polyester with high safety and high productivity. is there.
In other words, the expression plasmid for producing ultra-high molecular weight polyester that lacks part or all of the conjugation transfer ability of the present invention means that the polyester synthase gene is inserted into a vector that lacks part or all of the join transfer ability. A polyester synthase expression plasmid for producing ultrahigh molecular weight polyester.

Overall genetic manipulation can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press, (1989)). In addition, enzymes, cloning hosts, etc. used for gene manipulation can be purchased from market suppliers and used according to the explanation.
The enzyme is not particularly limited as long as it can be used for gene manipulation. The cloning host is not particularly limited, and examples thereof include Escherichia coli DH5α strain.

<Preparation of expression plasmid>
First, the production of a vector with improved safety by deleting some or all of the conjugation transferability will be described.
The vector used in the present invention is not particularly limited as long as it is a broad host range vector, but a derivative of pJRD215 (ATCC 37533), particularly a vector modified to be non-conjugative, can be preferably used. The base sequence of pJRD215 is shown in SEQ ID NO: 1.

To reduce or delete conjugative transmissibility from a vector, the gene region involved in conjugative transmissibility is reduced or deleted by performing full deletion, partial deletion, base insertion, base substitution, etc. be able to.

For example, in the case of pJRD215, all deletions, partial deletions, base insertions, base substitutions, and the like are performed on the mobA, mobB, mobC, and oriT sequences. The function of any of the mobA, mobB, mobC, and oriT sequences may be deleted. In particular, the above operation is performed on the oriT sequence including the mobA gene essential for mobility or the nick site. It is preferable. Further, the number of genes whose function is reduced or deleted may be one, but two or more are more preferable.

Note that pJRD215 is a derivative of RSF1010. Scholz et al., Gene, Vol. 75, p271 (1989), the definition of RSF1010 can be cited as it is. That is, the oriT sequence of pJRD215 is 3081 to 3169 of SEQ ID NO: 1, and the nick site can be defined between 3138 and 3139. The mobA gene can be defined as 3250-4407 of SEQ ID NO: 1, and the mobB gene can be defined as 3998-4411 of SEQ ID NO: 1.

Examples of a vector in which a part or all of the conjugation transmission ability is deleted by deletion or mutation of the mob gene region include, for example, a vector in which p15RD-4075 of pJRD215 (SEQ ID NO: 1) has been deleted; A vector in which 4378 is deleted; a vector in which 4000 to 4378 in pJRD215 is deleted; a vector in which adenine in 3323 in pJRD215 is substituted with thymine, and the like.

Examples of a vector in which a part or all of the conjugation transmission ability is deleted by deletion of the oriT region include, for example, a vector in which 3132-3145 of pJRD215 (SEQ ID NO: 1) is deleted; 3132-3169 of pJRD215 And a vector in which is deleted.

Furthermore, as a vector in which part or all of the conjugation transmission ability is deleted by deletion of the mob gene region and the oriT region, for example,
a vector in which the deletion of the mob gene region is a deletion of 3215-4075 of pJRD215 (SEQ ID NO: 1) and the deletion of the oriT region is a deletion of 3132-3145 of pJRD215 (SEQ ID NO: 1);
a vector in which the deletion of the mob gene region is a deletion of 3215-4075 of pJRD215 and the deletion of the oriT region is a deletion of 3132-3169 of pJRD215;
a vector in which the deletion of the mob gene region is a deletion of 3215-4075 of pJRD215 and the deletion of the oriT region is a deletion of 3132-3178 of pJRD215;
a vector in which the deletion of the mob gene region is a deletion of 3215-4075 of pJRD215 and the deletion of the oriT region is a deletion of 3132-3214 of pJRD215;
Examples include a vector in which the deletion of the mob gene region is deletion of 3215-4075 of pJRD215, and the deletion of the oriT region is deletion of 3095-3214 of pJRD215.

Here, deletion of the mob gene region can be carried out by deletion of a restriction enzyme fragment containing the mob gene portion, deletion using PCR, or the like. Further, mutation of the mob gene region can be performed by site-specific mutagenesis or the like. Furthermore, the deletion of the oriT region can be performed by deletion of a restriction enzyme fragment containing the oriT portion, deletion using PCR, or the like, as in the case of deletion of the mob gene region.

Further, in order to reduce the size of the vector for improving the transformation efficiency, it is possible to delete a portion unnecessary for the expression of the polyester synthase gene and the vector replication. For example, if the vector to be used has two or more antibiotic resistance genes, one of them can be deleted.
For example, in the case of pJRD215, it is possible to delete the kanamycin resistance gene or the streptomycin resistance gene, but it is preferable to delete the streptomycin resistance gene from the viewpoint of drug effectiveness.

Examples of the vector in which the streptomycin resistance gene region has been deleted include a vector in which 206-1690 of pJRD215 (SEQ ID NO: 1) has been deleted.

In the case of pJRD215, for the purpose of reducing the size of the vector, a multicloning site, a cos region, etc. that are not involved in the replication of the vector can be deleted. Multiple cloning sites and lambda phage derived cos regions are not clearly defined. Davison et al., Gene, vol. 51, p275 (1987), it can be said that the vicinity of 9680 to 10130 of SEQ ID NO: 1 is a multicloning site and the vicinity of 9260 to 9660 is a cos region.

Examples of the vector from which the cos region has been deleted include a vector from which 9237 to 10127 of pJRD215 (SEQ ID NO: 1) have been deleted; a vector from which 8915 to 10055 of pJRD215 have been deleted, and the like.

Deletion of the streptomycin resistance gene region, deletion of the cos region, and deletion of the multicloning site can be performed in the same manner as the deletion of the mob gene region.

In addition, a vector that lacks part or all of the conjugation transfer ability and that lacks the streptomycin resistance gene can also be suitably used. In addition, the said vector can be obtained by combining the deletion of part or all of the above-mentioned conjugation transmission ability and the deletion of a streptomycin resistance gene.

Furthermore, it is possible to combine the deletion of the cos region and the deletion of the multicloning site, for example, a vector in which part or all of the conjugation transfer ability is deleted and the cos region and / or the multicloning site is deleted. A vector lacking the streptomycin resistance gene and lacking the cos region and / or the multicloning site; deleting part or all of the conjugation transfer ability, lacking the streptomycin resistance gene, and the cos region And / or a vector lacking a multicloning site.

Next, the polyester synthase gene in the present invention will be described.
As the polyester synthase gene, those derived from Aeromonas caviae are preferable. For example, the gene fragments EE32 and EE32d13 derived from Aeromonas caviae described in JP-A-10-108682 can be used.
T. Kichise et al., Appl. Environ. Microbiol. 68, 2411-2419 (2002), a polyester synthase gene (N149S mutant gene) derived from Aeromonas caviae in which the asparagine of the 149th amino acid is substituted with serine, and the aspartic acid of the 171st amino acid. Polyester synthase gene (D171G mutant gene) derived from Aeromonas caviae in which is substituted with glycine, or a program that can identify useful amino acid mutations based on the three-dimensional structure or predicted three-dimensional structure of the enzyme on a computer For example, the polyester synthase gene derived from Aeromonas caviae in which the 353rd amino acid phenylalanine is substituted with threonine (F353T mutant residue designed by Shrike (Japanese Patent Laid-Open No. 2001-184831)) Children), or it can be preferably used the any two or more polyester synthase gene amino acid substitutions is derived from Aeromonas caviae in combination of the substitution (mutant gene combines the mutation) or the like.

The polyester synthase gene only needs to have an expression unit that functions in the host fungus, such as a promoter and a terminator, in addition to the structural gene. In the polyester synthase expression plasmid, one or more of the above expression units may be present.

In addition to the polyester synthase gene, the polyester synthase expression plasmid for producing ultrahigh molecular weight polyester of the present invention further contains a β-ketothiolase gene, an acetoacetyl-CoA reductase gene and an acyl-CoA dehydrogenase gene. Is preferred.
By using a polyester synthase expression plasmid containing these genes, a polyester having a higher molecular weight can be produced.

The β-ketothiolase (phbA) gene and acetoacetyl-CoA reductase (phbB) gene, which are substrate (3HB) supply genes in the polyester synthesis system of Ralstonia and Eutropha, have already been cloned, for example, pBS-phbAB _Re (T Kichise et al., Appl.Environ.Microbiol.68, 2411-2419 (2002)) can be excised as a restriction enzyme BamHI fragment. The phbAB _Re fragment can be inserted into the same restriction enzyme site as the pJRD215 derivative.

In addition, the acyl-CoA dehydrogenase (fadE) gene, which is the rate-limiting enzyme in the β-oxidation pathway that is the fatty acid metabolism system of Escherichia coli, has already been cloned, and its base sequence is already known (Campbell et al., J. Bacteriol. Vol. , P3759, (2002), DDBJ AF486265). Therefore, a gene containing the promoter sequence and the like can be amplified by PCR or the like, prepared as an appropriate restriction enzyme fragment, and inserted into a pJRD215 derivative or the like.

The expression plasmid for producing ultra-high molecular weight polyester that lacks part or all of the conjugation transfer ability of the present invention comprises inserting the polyester synthase gene into a vector lacking part or all of the conjugation transfer ability. Can be produced. As a result, the resulting expression plasmid also lacks part or all of the conjugative transmissibility.
As an example, a polyester synthase expression plasmid can be constructed by preparing a D171G mutant gene fragment as a restriction enzyme EcoRI fragment and inserting it into the same restriction enzyme site of the vector.

Preferable specific examples of the expression plasmid include, for example, a vector from which the oriT region and the cos region are deleted, a phbAB _Re fragment derived from Ralstonia eutropha, a D171G mutant gene fragment that is a polyester synthase gene, and an E. coli-derived fadE gene Examples include pJRDdTcES + phbAB + 171DG + fadE prepared using the fragment.

The “ultra high molecular weight” in the “ultra high molecular weight polyester” means that the weight average molecular weight (Mw) of the polyester is 3.5 million or more. Preferably it is 4 million or more and 22 million or less, More preferably, it is 4.2 million or more and 20 million or less.
The weight average molecular weight can be measured by a gel permeation chromatography (GPC) method.
Moreover, as polyester, the copolymer polyester P (3HB-co-3HH) which consists of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is preferable.

<Production of transformant>
Next, preparation of a transformant will be described.
The transformant of the present invention is transformed with the above polyester synthase expression plasmid. That is, the transformant of the present invention can be obtained by introducing the polyester synthase expression plasmid obtained above into a host compatible with the expression plasmid.

Although there is no restriction | limiting in particular as a host, The microorganisms isolate | separated from nature, the microorganisms deposited in the depository organization (for example, IFO, ATCC, etc.), etc. can be used. Specifically, non-polyester-producing bacteria of bacteria such as Ralstonia genus, Aeromonas genus, Escherichia genus, Alcaligenes genus, and Pseudomonas genus can be used. From the viewpoint of safety and productivity, Ralstonia is preferable, and Ralstonia eutropha is more preferable.

Introduction of a polyester synthase expression plasmid into a microorganism can be performed by a known method. For example, the electroporation method (Current Protocols in Molecular Biology, Volume 1, 1.8.4, 1994), the calcium method (Lederberg. EM et al., J. Bacteriol. 119.1072 (1974). )) Etc. can be used.

A preferable transformant used in the present invention includes, for example, a transformant in which the above-mentioned polyester synthase expression plasmid is introduced into Ralstonia eutropha as a host.
Specifically, PHB-4 / pJRDdTcES + phbAB + 171DG + fade (accession number FERM P-19767, accession date: March 31, 2004), which is a transformant obtained by introducing the expression plasmid pJRDdTcES + phbAB + 171DG + fadE into Ralstonia utropha PHB-4 strain Can be mentioned. This transformant has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, located at 1st, 1st, 1st East, Tsukuba, Ibaraki, Japan.

<Polyester production>
Next, production of polyester will be described.
The method for producing the polyester of the present invention uses the transformant of the above expression plasmid, and uses the following formula (1):

(Wherein m and n represent an integer of 1 or more), a method for producing an ultrahigh molecular weight copolymer polyester P (3HB-co-3HH) comprising 3-hydroxybutyric acid and 3-hydroxyhexanoic acid It is.

In the production of polyester, sugar, fat or fatty acid is given as a carbon source, and the transformant is cultured using a medium containing a nitrogen source, inorganic salts, and other organic nutrient sources other than the carbon source. be able to.

For example, as a medium for cultivating transformants obtained using bacteria such as microorganisms belonging to the genus Ralstonia, Aeromonas, Escherichia, Alkagenes or Pseudomonas, a carbon source that can be assimilated by the microorganism is given. In addition, a medium in which any one of a nitrogen source, an inorganic salt, and an organic nutrient source is limited, for example, a medium in which the nitrogen source is limited to 0.01 to 0.1% can be used.

Examples of the sugar include carbohydrates such as glucose and fructose. Examples of the fats and oils include fats and oils containing a large amount of saturated / unsaturated fatty acids having 10 or more carbon atoms, such as coconut oil, palm oil, and palm kernel oil. Examples of fatty acids include saturated and 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 fatty acid derivatives such as esters and salts of these fatty acids. Can be mentioned.
Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like.
Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.

Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, and vitamin C.
Moreover, you may add antibiotics (kanamycin etc.) corresponding to the drug resistance gene which exists in an expression plasmid in a 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 10 days.
Then, what is necessary is just to collect | recover polyester from this obtained cultured microbial cell.

In the present invention, the polyester can be recovered from the cells by, for example, the following method. After completion of the culture, the cells are separated from the culture solution with a centrifuge, and the cells are washed with distilled water, methanol, and the like and dried. Polyester is extracted from the dried cells using an organic solvent such as chloroform. The bacterial cell component is removed from the organic solvent solution containing the polyester by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the polyester. Further, the supernatant is removed by filtration or centrifugation, and dried to recover the polyester.

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 can be confirmed by adding Nilered to the agar medium where the recombinant bacteria are grown, culturing the recombinant bacteria for 1 to 7 days, and observing whether the recombinant bacteria turn red.

The weight average molecular weight (Mw) of the ultrahigh molecular weight copolymer polyester P (3HB-co-3HH) obtained by the above production method is preferably 3.5 million or more, more preferably 4 million or more and 22 million or less, and still more preferably Is 4.2 million or more and 20 million or less.
The weight average molecular weight can be measured by a gel permeation chromatography (GPC) method.

The 3HH composition of the ultrahigh molecular weight copolymer polyester P (3HB-co-3HH) obtained by the above production method is preferably 1 to 20 mol%, more preferably 2 to 10 mol%.
The analysis of the 3HH composition can be performed by, for example, a gas chromatography method.

<Evaluation of joint transmission ability>
Next, the evaluation of joint transmission ability will be described.
The junction transfer ability of the produced expression plasmid can be evaluated by the following method, but is not limited to this method.

First, an expression plasmid to be evaluated is transformed into a bacterium having a tra gene, and this is used as a donor bacterium. As a bacterium having a tra gene, for example, E. coli strain S17-1 can be used. As the recipient bacteria, any bacteria can be used as long as it can co-culture with the donor bacteria and the expression plasmid to be evaluated can autonomously replicate in the cells, but can be separated from the donor bacteria after the mixed culture. Those having antibiotic resistance different from the donor bacteria are desirable.

Next, each of the donor bacterium and the recipient bacterium is cultured in an appropriate medium and increased in advance. After mixing each of them, mixed culture is performed by inoculating the appropriate medium.
The medium used at this time may be either solid or liquid, and no antibiotics are added. The culture temperature of the mixed culture may be any temperature at which donor and recipient bacteria can grow, but is preferably 20 ° C to 40 ° C. The culture time is not particularly limited but is preferably 5 to 20 hours.

The evaluation of the junction transmission ability is performed by calculating the junction transmission frequency per recipient bacterium, but is not limited to this method. The method for calculating the junction transmission frequency will be described in detail in the following examples.

By using a transformant of Ralstonia / eutropha with a novel polyester synthase expression plasmid, ultra-high molecular weight polyester P (3HB-co-3HH) having excellent processing properties to fibers and the like can be obtained by using simple materials such as inexpensive vegetable oils and fats. Even in culture under a single carbon source, it has become possible to produce it safely and with high productivity.

Hereinafter, the present invention will be described in detail by way of examples. However, the technical scope of the present invention is not limited to these examples.

Reference Example 1 The movA, mobB partial deletion vector pJRD215 was digested with the restriction enzyme Van91I to obtain a DNA fragment of about 9.5 kb from which a part of the mobA and B genes were deleted. This is referred to as DNA Ligation Kit Ver. 1 (Takara Shuzo Co., Ltd.) was used for self-ligation to obtain pJRDdm (FIG. 1). The vector pJRDdm is obtained by deleting 3215-4075 of pJRD215.

Reference Example 2 A cos region-deleted and oriT region-deleted vector pJRD215, which had been cut with restriction enzymes SpeI and BglII, was blunt-ended and self-ligated using DNA Blunting Kit (Takara Shuzo Co., Ltd.). This is designated as pJRDdc (FIG. 2). The pJRDdc is obtained by deleting 8915 to 10055 of pJRD215.

Next, PCR reaction was performed using pJRDdc as a template and the primers shown in SEQ ID NO: 4 and SEQ ID NO: 6 to obtain a DNA fragment of about 0.5 kb. Similarly, a PCR reaction was performed using pJRDdc as a template and the primers shown in SEQ ID NO: 5 and SEQ ID NO: 7 to obtain a DNA fragment of about 2.4 kb. The overlapping PCR method was performed using the fact that the obtained two fragments had overlapping portions. Pyrobest (manufactured by Takara Shuzo Co., Ltd.) was used as the polymerase. A fragment of about 2.8 kb obtained by this overlap PCR method was cleaved with restriction enzymes EcoO109I and AflIII, and ligated with the above-mentioned pJRDdc that had been cleaved with EcoO109I and AflIII. Thereby, pJRDdTc was obtained (FIG. 2). The vector pJRDdTc is obtained by deleting 3132 to 3169 of pJRD215 and deleting the cos region and the like (8915 to 10055).

Similarly, PCR reaction was performed using pJRDdc as a template and the primers shown in SEQ ID NO: 4 and SEQ ID NO: 8 to obtain a DNA fragment of about 0.5 kb. Similarly, a PCR reaction was carried out using pJRDdc as a template and the primers shown in SEQ ID NO: 5 and SEQ ID NO: 9 to obtain a DNA fragment of about 2.4 kb. The overlapping PCR method was performed using the fact that the obtained two fragments had overlapping portions. Pyrobest was used as the polymerase. A fragment of about 2.8 kb obtained by this overlap PCR method was cleaved with restriction enzymes EcoO109I and AflIII and ligated with pJRDdc similarly cleaved with restriction enzymes EcoO109I and AflIII. Thereby, pJRDdnc was obtained (FIG. 2). The vector pJRDdnc is obtained by deleting 3132-3145 of pJRD215 and deleting the cos region and the like (8915-10055).

Reference Example 3 Cos region deletion and mob gene region deletion or mutation introduction vector pJRDdm was cut from pJRDdm with restriction enzymes SpeI and BglII, and blunt-ended using DNA Blunting Kit (Takara Shuzo Co., Ltd.). , Self-linked. This is designated as pJRDdcm (FIG. 3).

Next, PCR reaction was performed using pJRDdcm as a template and the primers shown in SEQ ID NO: 5 and SEQ ID NO: 10. The obtained DNA fragment was cleaved with restriction enzymes Van91I and AflIII and ligated to pJRDdcm similarly cleaved with restriction enzymes Van91I and AflIII to obtain vector pJRDdcm4380 (FIG. 3).

On the other hand, PCR reaction was performed using pJRD215 as a template and the primers shown in SEQ ID NOs: 11 and 12. Pyrobest was used as the polymerase. The obtained DNA fragment of about 1.5 kb was cleaved with restriction enzymes XhoI and PstI and inserted into a pSTV28 vector (used by Takara Shuzo Co., Ltd.) cleaved with restriction enzymes XhoI and PstI to prepare pSTVmob. Next, pSTVmob was amplified using SEQ ID NO: 13 and SEQ ID NO: 14 as PCR primers in order to delete the second Van91I site among the three Van91I sites in pSTVmob. Pfu (manufactured by Stratagene) was used as the polymerase. After the PCR reaction, the vector using the restriction enzyme DpnI as a template was cleaved and transformed into Escherichia coli JM109 strain to obtain pSTVmob-delVan from which one Van91I site was deleted from the transformant (FIG. 4).

Next, PCR reaction was performed using pSTVmob-delVan as a template and SEQ ID NO: 15 and SEQ ID NO: 16 as primers. Pyrobest was used as the polymerase. The amplified fragment was cleaved with the restriction enzyme Van91I, and ligated with pJRDdcm4380 treated with Van91I to obtain pJRDdcm163 (FIG. 4). The vector pJRDdcm163 is obtained by deleting 3737-4378 of pJRD215 and deleting the cos region and the like (8915-10055).

Similarly, PCR reaction was performed using pSTVmob-delVan as a template and SEQ ID NO: 15 and SEQ ID NO: 17 as primers. Pyrobest was used as the polymerase. The amplified fragment was cleaved with the restriction enzyme Van91I, and ligated with pJRDdcm4380 treated with Van91I to obtain pJRDdcmB (FIG. 4). The vector pJRDdcmB is obtained by deleting 4000-4378 of pJRD215 and deleting the cos region and the like (8915-10055).

Next, in order to replace 3323 adenine of pJRD215 (SEQ ID NO: 1) with thymine, that is, to replace the 25th amino acid residue of the mobA gene from tyrosine to phenylalanine, the following operation was performed. Amplification was performed using pSTVmob-delVan as a template and SEQ ID NO: 18 and SEQ ID NO: 19 as PCR primers. Pfu was used as the polymerase. After the PCR reaction, the restriction enzyme DpnI was added to cleave the plasmid as a template and transformed into E. coli strain JM109, and pSTVmob25YF was obtained from the transformant (FIG. 5). A fragment of about 0.9 kb obtained by treating pSTVmob25YF with Van91I was ligated to pJRDdc cleaved with Van91I to obtain pJRDdcm25YF (FIG. 5). The vector pJRDdcm25YF is obtained by replacing the adenine at 3323 of pJRD215 with thymine and deleting the cos region and the like (8915 to 10055).

(Reference Example 4) Streptomycin resistance gene deletion pJRD215 and vectors pJRDdm, pJRDdTc, pJRDdnc, pJRDdcm163, pJRDdcmB, and pJRDdcm25YF obtained in Reference Examples 1 to 3 were cleaved with the restriction enzymes EcoICRI and PshAI to give streptomycin (stA). And about 1.5 kb of the strB gene), a blunt-ended DNA fragment of about 8.0 kb was obtained. This was self-ligated to obtain vectors pJRDds, pJRDdms, pJRDdTcs, pJRDdncs, pJRDdscm163, pJRDdscmB, and pJRDdscm25YF (FIG. 1). These are obtained by deleting 206-1690 of pJRD215 from the vector used.

(Reference Example 5) Deletion of cos region and multicloning site from pJRDdms The vector pJRDdms obtained in Reference Example 4 was cleaved with restriction enzymes NheI and EcoRI. SEQ ID NO: 2 (forward chain) and SEQ ID NO: 3 (reverse chain) ) And the synthetic DNA sequence shown in FIG. As a result, about 0.9 kb containing the cos sequence and the multicloning site was completely deleted by replacing it with synthetic DNA 45 bp to obtain a vector pJRDdmsc of about 7.1 kb (FIG. 1).

(Reference Example 6) Preparation of mob region and oriT region deletion vector Next, a PCR reaction was performed using pJRD215 as a template using primers shown in SEQ ID NO: 20 and SEQ ID NO: 21 to obtain a DNA fragment of about 1 kb. Pyrobest was used as the polymerase. This DNA fragment was cleaved with restriction enzymes SfiI and Van91I, and then ligated with pJRDdm and pJRDdms which were also cleaved with restriction enzymes SfiI and Van91I, respectively, thereby obtaining vectors pJRDdmT1 and pJRDdmsT1 (FIG. 6).

Similarly, a PCR reaction was carried out using pJRD215 as a template using the primers shown in SEQ ID NO: 20 and SEQ ID NO: 22 to obtain a DNA fragment of about 1 kb. This DNA fragment was cleaved with restriction enzymes SfiI and Van91I, and then similarly restricted. The vectors pJRDdmT2 and pJRDdmsT2 were obtained by ligation with pJRDdm and pJRDdms cut with the enzymes SfiI and Van91I, respectively (FIG. 6).

Similarly, a PCR reaction was performed using pJRD215 as a template using the primers shown in SEQ ID NO: 20 and SEQ ID NO: 23 to obtain a DNA fragment of about 1 kb. This DNA fragment was cleaved with restriction enzymes SfiI and Van91I. The vectors pJRDdmT3 and pJRDdmsT3 were obtained by ligating with pJRDdm and pJRDdms cut with the restriction enzymes SfiI and Van91I, respectively (FIG. 6).

(Example 1) Construction of expression plasmid An expression plasmid for producing ultrahigh molecular weight polyester was constructed as follows. PJRDdTc prepared in Reference Example 2 was cleaved with EcoRI, and a synthetic DNA consisting of SEQ ID NOs: 24 and 25 was inserted into the same site to give a cloning site, to obtain pJRDdTcES. The phbAB _Re fragment derived from Ralstonia eutropha has already been cloned, and was prepared as a restriction enzyme BamHI fragment from pBS-phbAB _Re (T. Kichise et al., Appl. Environ. Microbiol. 68, 2411-2419 (2002)). pJRDdTcES + phbAB was constructed by inserting the phbAB _Re fragment into the BamHI site of pJRDdTcES (FIG. 7).

A D171G mutant gene fragment, which is a polyester synthase gene, was prepared by PCR. In the D171G mutation, glycine is substituted for aspartic acid, which is the 171st amino acid of PHA synthase derived from Aeromonas caviae. Therefore, the gene fragment EE32d13 derived from Aeromonas caviae described in JP-A-10-108682 is once subcloned into the EcoRI site of pUC19 (manufactured by Takara Shuzo) and synthesized as shown in SEQ ID NO: 26 and SEQ ID NO: 27. PCR was performed using DNA as a primer. The conditions are (1) 94 ° C. for 2 minutes, (2) 94 ° C. for 30 seconds, (3) 55 ° C. for 30 seconds, (4) 72 ° C. for 2 minutes, (2) to (4) for 25 cycles, (5) 5 minutes at 72 ° C, and Ex Taq polymerase (manufactured by Takara Bio Inc.) was used as the polymerase. A D171G fragment was prepared by digestion with the restriction enzyme EcoRI, and pJRDdTcES + phbAB was inserted into the site cleaved with the enzyme to produce pJRDdTcES + phbAB + 171DG (FIG. 7).

Next, for the E. coli fadE gene, PCR was carried out using the chromosomal gene of E. coli K-12 as a template and SEQ ID NOs: 28 and 29 as primers to amplify the first half of the structural gene containing its promoter region. PCR was carried out using primers 31 and 31 to amplify a fragment containing the latter half of the structural gene. PCR conditions were (1) 94 ° C. for 2 minutes, (2) 94 ° C. for 30 seconds, (3) 55 ° C. for 1 minute, (4) 68 ° C. for 3 minutes, (2) to (4) for 30 cycles, (5) It was 10 minutes at 68 ° C., and KOD plus-polymerase (manufactured by Toyobo) was used as the polymerase. Each fragment was then cleaved with ClaI and EcoRI and subcloned into the EcoRI site of pUC19. From this, a fadE gene fragment was prepared as an NheI fragment and inserted into the NheI site of pJRDdTcES + phbAB + 171DG to complete the expression plasmid pJRDdTcES + phbAB + 171DG + fadE (FIG. 8).

Example 2 Preparation of Transformant A transformant of Ralstonia eutropha PHB-4 strain containing the expression plasmid pJRDdTcES + phbAB + 171DG + fadE prepared in Example 1 was prepared by an electric pulse method. That is, the gene introduction apparatus used was a gene pulser manufactured by Biorad, and the cuvette used was a gap of 0.2 cm manufactured by Biorad. 400 μl of competent cells and 20 μl of expression plasmid were injected into a cuvette and set in a pulse device, and an electric pulse was applied under the conditions of electrostatic capacity 25 μF, voltage 1.5 kV, and resistance value 800Ω. After pulsing, the bacterial solution in the cuvette was cultured with shaking on Nutrient Broth medium (manufactured by DIFCO) at 30 ° C. for 3 hours. By culturing for a day, transformant PHB-4 / pJRDdTcES + phbAB + 171DG + fadE was obtained.
This transformant (PHB-4 / pJRDdTcES + phbAB + 171DG + fadE, accession number FERM P-19767, accession date March 31, 2004) has been deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary.

(Example 3) Selection of transformant The transformant obtained in Example 2 was treated with a Nilered-containing medium (9 g of disodium hydrogen phosphate · 12 hydrate, 1.5 g of potassium dihydrogen phosphate, 1.5 ml of ammonium chloride). 05g, Magnesium sulfate, heptahydrate 0.02g, Fructose 0.5g, Cobalt chloride, hexahydrate 0.25ppm, Iron (III) chloride, hexahydrate 16ppm, Calcium chloride, dihydrate 10.3ppm, Nickel chloride -6 hydrate 0.12 ppm, copper sulfate pentahydrate 0.16 ppm, Nilered 0.5 mg, agar 15 g / 1 L), and cultured at 30 ° C. for 1 week. As a result, since the colony turned red, it was confirmed that polyester was accumulated in the cells, and the colony was selected to produce polyester.

Example 4 Polyester Production and Purification Seed medium composition is 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / v% Na ₂ PO ₄ · 12H ₂ O, 0.15 w / v% KH ₂ PO ₄ (pH 6.8).
The composition of the preculture medium is 1.1 w / v% Na ₂ PO ₄ · 12H ₂ O, 0.19 w / v% KH ₂ PO ₄ , 1.29 w / v% (NH ₄ ) ₂ SO ₄ , 0.1 w _{/ v% MgSO 4 · 7H 2} O, 2.5w / v% palm W olein oil, 0.5 v / v% trace metal salt solution (1.6 w in 0.1N HCl _{/ v% FeCl 3 · 6H 2} O, 1 w / v% CaCl ₂ .2H ₂ O, 0.02 w / v% CoCl ₂ .6H ₂ O, 0.016 w / v% CuSO ₄ .5H ₂ O, 0.012 w / v% NiCl ₂ .6H ₂ O It was taken as 5 × 10 ⁻⁶ w / v% kanamycin.

The composition of the polyester production medium is 0.385 w / v% Na ₂ PO ₄ .12H ₂ O, 0.067 w / v% KH ₂ PO ₄ , 0.291 w / v% (NH ₄ ) ₂ SO ₄ , 0.1 w. _{/ v% MgSO 4 · 7H 2} O, 0.5v / v% trace metal salt solution (1.6 w in 0.1N HCl _{/ v% FeCl 3 · 6H 2} O, 1w / v% CaCl 2 · 2H 2 O, 0.02 w / v% CoCl ₂ .6H ₂ O, 0.016 w / v% CuSO ₄ .5H ₂ O, 0.012 w / v% NiCl ₂ .6H ₂ O.), 0.05 w / v % BIOSSPUREX200K (antifoaming agent: manufactured by Cognis Japan), 5 × 10 ⁻⁶ w / v% kanamycin.
As a carbon source, palm kernel oil olein, which is a low melting point fraction obtained by fractionating palm kernel oil, is used as a single carbon source. Throughout the entire culture, the specific substrate supply rate is 0.08 to 0.1 (g oil). g Net dry cell weight) ^-1 x (h) ^-1 was fed.

A glycerol stock (50 μl) of the transformant PHB-4 / pJRDdTcES + phbAB + 171DG + fadE obtained in Example 2 was inoculated into a seed medium (10 ml), cultured for 24 hours, and a 3 L jar containing 1.8 L of a preculture medium. Fermenter (manufactured by Maruhishi Bioengine, MDL-300 type) was inoculated at 1.0 v / v%. The operating conditions were a culture temperature of 30 ° C., a stirring speed of 500 rpm, an aeration rate of 1.8 L / min, and the culture was continued for 28 hours while controlling the pH between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control.

In the polyester production culture, a 10 L jar fermenter (manufactured by Maruhishi Bioengine, Model MDL-1000) containing 6 L of production medium was inoculated with 5.0 v / v% of the preculture seed. The operating conditions were a culture temperature of 28 ° C., a stirring speed of 400 rpm, an aeration rate of 3.6 L / min, and a pH controlled between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control. Culturing was carried out for about 64 hours, and after culturing, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured to be 126.4 g / L.

100 ml of chloroform was added to about 1 g of the obtained dried microbial cells, and stirred overnight at room temperature to extract the polyester in the microbial cells. The bacterial cell residue was filtered off and concentrated with an evaporator until the total volume reached about 30 ml. Then, about 90 ml of hexane was gradually added, and the mixture was allowed to stand for 1 hour with slow stirring. The precipitated polyester was filtered off and then vacuum dried at 50 ° C. for 3 hours. The weight of the dried polyester was measured, and the polyester content in the cells was calculated. As a result, the polyester content of the transformant PHB-4 / pJRDdTcES + phbAB + 171DG + fadE was as high as 73.6 (wt%) after culturing for 64 hours.

(Example 5) Analysis of 3HH composition (mol%) of polyester The 3HH composition of polyester produced by transformant PHB-4 / pJRDdTcES + phbAB + 171DG + fadE was measured by gas chromatography as follows.
To about 20 mg of the dried polyester, 2 ml of a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added and sealed, and heated at 100 ° C. for 140 minutes to obtain a methyl ester of a polyester degradation product. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize it, and the mixture was allowed to stand until generation of carbon dioxide gas ceased. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged and the monomer unit composition of the polyester degradation product in the supernatant was analyzed by capillary gas chromatography. The gas chromatograph used was Shimadzu Corporation GC-17A, and the capillary column used was GL Science's NEUTRA BOND-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). He was used as the carrier gas, the column inlet pressure was set to 100 kPa, and 1 μl of the sample was injected. As temperature conditions, the temperature was raised from the initial temperature of 100 to 200 ° C. at a rate of 8 ° C./min, and further from 200 to 290 ° C. at the rate of 30 ° C./min. As a result of analysis under the above conditions, the 3HH composition of the polyester at the end of the 64-hour culture was 2.5 (mol%).

(Example 6) Weight average molecular weight (Mw) analysis of polyester Weight average molecular weight (Mw) analysis of polyester was performed by gel permeation chromatography. About 15 mg of the extracted polyester was dissolved in 10 ml of chloroform, filtered through a 0.2 μm filter to obtain a measurement sample, and 0.05 ml of the sample was analyzed. The measurement system was SLC-10A (manufactured by Shimadzu Corporation), and the column was connected with two Shodex GPC K-806L (manufactured by Showa Denko) in series, and measurements were made at 40 ° C. The moving layer was chloroform at 1.0 ml / min, and an RI detector (RID-10A, manufactured by Shimadzu Corporation) was used. As a standard product, similarly treated polystyrene (manufactured by Showa Denko, weight average molecular weight: about 7 million, about 1.70 million, 150,000, 30,000) was used, and the weight average molecular weight of the polyester was calculated from a calibration curve. As a result, the weight average molecular weight (Mw) of the polyester produced by the transformant PHB-4 / pJRDdTcES + phbAB + 171DG + fadE was about 4.2 million and an ultra high molecular weight.

(Example 7) Evaluation of junction transmissibility of transformant The junction transmissibility of the expression plasmid pJRDdTcES + phbAB + 171DG + fadE described in Example 1 was evaluated. The vector pJRD215 was used as a positive control.
Escherichia coli S17-1 transformed with each of these strains was used as a donor bacterium, and Escherichia coli XL10-Gold strain was used as a recipient bacterium. Each of the donor and recipient bacteria was inoculated into a TB medium and cultured overnight at 37 ° C. A sterilized nitrocellulose filter was placed on the LB plate, and each culture solution, 10 μl of donor bacteria and 10 μl of recipient bacteria were mixed and inoculated on the nitrocellulose filter. After culturing at 37 ° C. for 18 hours, the nitrocellulose filter was transferred to a tube containing 1 ml of physiological saline and suspended sufficiently to allow the bacteria on the nitrocellulose filter to float.

The suspension of the mixed bacteria thus obtained was diluted 10 ⁰ to 10 ⁷ times, an LB plate (Km + Cm) containing kanamycin (Km) 50 μg / ml and chloramphenicol (Cm) 20 μg / ml, and chloramphenic 100 μl each was plated on an LB plate containing only 20 μg / ml of call and cultured at 37 ° C. overnight. After culturing, the number of appearance colonies was counted, and the results shown in Table 1 were obtained.

E. coli strain S17-1 is Cm sensitive and Km sensitive. On the other hand, E. coli XL10-Gold strain is Cm resistant and Km sensitive. Since the expression plasmid and vector used for transformation of E. coli strain S17-1 contain a Km resistance gene, the bacteria carrying the expression plasmid or vector become Km resistant. Therefore, the total number of E. coli XL10-Gold can be detected on the LB plate containing only Cm 20 μg / ml, and the expression plasmid or the vector resulting from the conjugation transfer of the expression plasmid or vector on the LB plate containing Km 50 μg / ml and Cm 20 μg / ml. -Gold bacteria can be detected.
Therefore, the conjugation transmission frequency per recipient cell is obtained by dividing the number of E. coli XL10-Gold strains, which are contained in 100 μl of the cell suspension, to which the expression plasmid or vector has been transferred by conjugation transfer, by the total number of E. coli XL10-Gold strains. Calculated. The actual calculated values are shown below.

From the results of Table 1, the conjugation transmission frequency per recipient when the expression plasmid pJRDdTcES + phbAB + 171DG + fadE coexists with the tra gene was 14/162 × 10 ⁴ = 8.64 × 10 ⁻⁶ . Similarly, when calculating the conjugal transfer frequency of pJRD215, it was ^{153 × 10 1/91 × 10} 4 = 1.68 × 10 -3. Therefore, it was found that the junction transmission frequency of pJRDdTcES + phbAB + 171DG + fadE was reduced to about 1/200 times that of pJRD215.

By using a transformant of Ralstonia / eutropha with a novel polyester synthase expression plasmid, ultra-high molecular weight polyester P (3HB-co-3HH) having excellent processing properties to fibers and the like can be obtained by using simple materials such as inexpensive vegetable oils and fats. Even in culture under a single carbon source, it has become possible to produce it safely and with high productivity.

FIG. 2 is a construction diagram of a mob region deletion vector and a streptomycin resistance gene deletion vector. FIG. 2 is a construction diagram of a cos region oriT region deletion vector. FIG. 4 is a construction diagram of a mob gene region partial deletion cos region deletion vector. FIG. 4 is a construction diagram of a mob gene region partial deletion cos region deletion vector. FIG. 4 is a construction diagram of a mobA gene mutation cos region deletion vector. FIG. 2 is a construction diagram of a mob region and oriT region deletion vector. It is an expression plasmid construction figure for ultra high molecular weight polyester production. It is an expression plasmid construction figure for ultra high molecular weight polyester production.

Claims (13)

An expression plasmid for producing ultra-high molecular weight polyester that lacks some or all of its ability to transfer junctions. The expression plasmid for producing ultrahigh molecular weight polyester according to claim 1, wherein the vector contained in the expression plasmid is a pJRD215 (SEQ ID NO: 1) derivative. The expression plasmid for producing ultrahigh molecular weight polyester according to claim 1 or 2, wherein the polyester synthase gene contained in the expression plasmid is one or more of the following genes (1) to (5):
(1) a polyester synthase gene derived from Aeromonas caviae,
(2) a polyester synthase gene derived from Aeromonas caviae in which the asparagine of the 149th amino acid is substituted with serine,
(3) a polyester synthase gene derived from Aeromonas caviae in which the 171st amino acid aspartic acid is substituted with glycine;
(4) a polyester synthase gene derived from Aeromonas caviae in which phenylalanine of the 353rd amino acid is substituted with threonine;
(5) A polyester synthase gene derived from Aeromonas caviae, which is a combination of any two or more of the substitutions (2) to (4) above. The expression plasmid for producing ultrahigh molecular weight polyester according to any one of claims 1 to 3, further comprising a β-ketothiolase gene, an acetoacetyl-CoA reductase gene and an acyl-CoA dehydrogenase gene in addition to the polyester synthase gene. The expression plasmid for producing ultrahigh molecular weight polyester according to any one of claims 1 to 4, which is pJRDdTcES + phbAB + 171DG + fadE. 6. The ultrahigh molecular weight polyester production according to any one of claims 1 to 5, wherein the deletion of part or all of the junction transmission ability is deletion or mutation of the mob gene region and / or deletion of the oriT region. Expression plasmid. Furthermore, the expression plasmid for ultrahigh molecular weight polyester production according to any one of claims 1 to 6, wherein the streptomycin resistance gene is deleted. Furthermore, the expression plasmid for ultra-high molecular weight polyester production according to any one of claims 1 to 7, wherein the cos region is deleted. A transformant transformed with the expression plasmid for producing ultrahigh molecular weight polyester according to any one of claims 1 to 8. The transformant according to claim 9, wherein the host is Ralstonia eutropha. The transformant according to claim 9 or 10, which is PHB-4 / pJRDdTcES + phbAB + 171DG + fadE (FERM P-19676). The polyester has the following formula (1)
(Wherein m and n represent an integer of 1 or more), which is a copolyester P (3HB-co-3HH) composed of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid. A method for producing an ultrahigh molecular weight polyester using the expression plasmid or transformant according to any one of the above. The method for producing an ultrahigh molecular weight polyester according to claim 12, wherein the polyester has a weight average molecular weight of 3.5 million or more.