TW200540269A - Novel transformant - Google Patents

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, this invention is to provide 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

200540269 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a novel plastid for the production of high 3HH composition polyester, a transform body containing the plastid, and a high 3HH composition polymer using the plastid. • How to make esters. [Prior art] So far, many microorganisms have been known to accumulate polyester in bacteria cells for energy storage. A representative example of this is poly 3-hydroxybutyric acid (hereinafter referred to as P (3HB)), which is a homopolymer of 3-hydroxybutyric acid (hereinafter referred to as 3HB). It was first established in 1925 in Bacillus megaterium). p (3HB) is a thermoplastic polymer, which has attracted attention because it can be biodegraded in the natural environment, and it is environmentally friendly plastic. However, p (3HB) is hard and brittle due to its high crystallinity, and its practical application is limited. Therefore, the improvement of its nature becomes the purpose of research. Among them, a method for producing a copolymer containing 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter referred to as P (3HB-CO-3HV)) has been disclosed (for example, refer to Patent Document 1 and Patent Reference 2). This p (3HB_co-3HV) is more flexible than P (3HB) and can be used in a wide range of applications. However, as far as P (3HB-CO-3HV) is concerned, even if the molar ratio of 3HV is increased, the resulting changes in physical properties are still insufficient, especially the flexibility required for film processing and other aspects cannot be improved. It can be used in the field of shampoo bottles or hard shaped bodies such as disposable razors and handles. In recent years, research has been conducted on the two-component copolymerized polyester (hereinafter referred to as p (3HB-co-3HH)) of 3HB and 3-hydroxyhexanoic acid (hereinafter referred to as 3ηη) and its manufacturer 100136.doc 200540269 method (refer to patent literature 3 and Patent Document 4). These reported methods for producing P (3HB-CO-3HH) are produced by fermenting fatty acids such as oleic acid or fatty acids such as olive oil using Ae romonas caviae, a guinea pig isolated from the soil. The properties of P (3HB-co-3HH) have also been studied (see Non-Patent Document 1). In this report, A. guinea pig Aeromonas was cultured with fatty acids with 12 or more carbons as the sole carbon source, and the 3HH composition was fermented to produce p (3hb_CO-3HH) of 19 to 19 mole%. As the composition of P (3HB-CO-3HH) increases with the increase of 3HH, the hard and brittle nature of P (3HB) gradually exhibits a soft nature, which significantly improves the flexibility of P (3HB-CO-3HH). That is, P (3HB_C0-3HH) has a wide range of physical properties that can be applied to hard polymers and even soft polymers by changing the composition of 3HH, so it can be expected to be applied to those who require hardness such as television casings to requirements such as films Wide range of soft people. However, since the production volume of the bacterial cells in this manufacturing method is 4 g / L 'and the polymer content is 30% and the polymer productivity is low, it is still not a production method that can be moved to practical use, so it is still to be explored for practical use. And more productive methods. The research and improvement are aimed at the industrial production of P (3HB-co-3HH). In the culture using Aeromonas hydrophila, a 43-hour fed_batch culture was performed with O. sylvestris as a slave source, and the production cell was 95.7 g / L. 4 (3HB-CO-3HH) having a polymer content of 45.2% and a 3HH composition of 17% (see Non-Patent Document 2). In addition, Aeromonas hydrophila was cultured using saccharose and lauric acid as carbon sources, and the amount of Dali was 50% of the old gL polymer (see non-patent literature. However, since Aeromonas hydrophila Bacillus is pathogenic to humans (see Non-Patent Document 4), so 100136.doc 200540269 is not a suitable product for industrial production. Χ, because a high-priced carbon source is used in such cultivation and production, from the viewpoint of manufacturing costs Therefore, it is also necessary to seek for the use of cheap carbon sources. Φ Therefore, research and improvement are aimed at the use of safe host production and productivity improvement. Synthesis of polyhydroxy acid (PHa) from Aeromonas hydrophila Selection of enzyme genes (refer to Patent Document 5 and Non-Patent Document 5). This gene was used to introduce the Eutrophopathic eUtr〇pha, the old name of Alcaligenes eutróphus. The transformed body produced POHB-codHH). As a result, the bacterial production amount was 4 g / L, and the polymer content was 30 /. . Furthermore, the transformant was cultured using a vegetable oil as a carbon source. As a result, the bacterial content was 4 g / L in Dallas and the polymer content was 80% (refer to non-patent literature). The cheap vegetable oil was used as a carbon source. Its polymer productivity is low, and its 3HH composition is 5%, which has hard and brittle physical properties. In addition, although fructose can be used as a carbon source to build R. eutropha capable of producing p (3HB_c0_3HH), the polymer productivity of this strain is low and it is not suitable for practical production (see Non-Patent Document 7) . A p (3HB_co-3HH) production strain can also be constructed with coliform as a host. A strain was constructed that introduced the PHA synthetase gene of Aeromonas genus, and the NADP-acetamidinyl co-A-reducing enzyme gene of R. eutropha into the coliform strain. Using dodecane as a carbon source, the same coliform was cultured for 40.8 hours, and the fruit amount was 79 g / L, the polymer content was 27.2%, and the composition of 3%-was 10.8 ° /. (See Non-Patent Document 8). It is also possible to construct coliforms that introduce the PHA synthetase gene, acetyl CoA hydratase, and fluorenyl coA dehydrogenase gene from Aeromonas guinea pigs. 100136.doc 200540269 If the same coliform is cultured in a medium containing lauric acid, the polymer content is 16 ° /. The 3HH composition was 16% (see Non-Patent Document 9). The use of these coliform bacteria has low productivity and is difficult to apply to industrial production. On the other hand, with the goal of improving the productivity of P (3HB-co-3HH) and controlling the 3HH group, artificial changes of PHA synthesis enzymes are performed (Non-Patent Document 10 and Non-Patent Document 11). Among the variants of PHA synthetic enzymes derived from Aeromonas aeromonas, 149 was a variant enzyme in which aspartic acid was replaced by serine, and aspartic acid as in 171 was Glycine-substituted mutant enzymes have been shown to increase the activity of PHA synthesis enzymes and 3HH composition in coliforms. In addition, the mutant enzymes with amino acid phenylalanine replaced by isoleucine and 214 have been reported. The mutant enzyme in which the amino acid oxalic acid was replaced by glycine was not long; PH A synthesizes enzyme activity and polymer content in large intestinal pain. However, due to their low content of polymer using special coliform bacteria, further improvement is needed to utilize the characteristics of these mutant enzymes for industrial production. T. Fukui et al. Used p. 3HB-c0-3HH as the host to produce pHA synthetic enzymes expressing plastids.

PJRDEE32, PJRDEE32dl3, etc., in which Co-A hydratase gene and the like were introduced into pJRD215 (ATCC 37533) (see Patent Document 5). Although the bacteria content of this strain is low, only 4 g / L, but by using vegetable oil as a stone anaerobe to improve the culture conditions of the halogen strain, the bacteria content can be increased to 4 5 g / L, and the polymer content to 62 _ 5%, 3 HH composition to 8.1%; A method of producing P (3HB-co-3HH) with a high 3 HH composition according to culture conditions has also been studied (see Patent Document 6) 〇100136.doc 200540269 Also, it has been disclosed A method for controlling the physical properties of P (3HB_co-3HH) (see Patent Document 6). By using at least two kinds of fats and / or fatty acids with different carbon numbers as carbon sources, 3HH composition can be produced from 1 to 40 mole% 2P (3HB_c0-3HH), so P (3HB- C0_3HH). However, in this manufacturing method, in order to control the 3HH composition, it is necessary to add hexanoic acid or caprylic acid with higher valence, and hexanoic acid exhibiting cytotoxicity as a result of two agricultural products, resulting in a reduction in bacterial productivity. Furthermore, the production equipment may become complicated and expensive due to the addition of a multi-component carbon source. [Patent Document 1] JP 57-150393 [Patent Document 2] JP 59-220192 [Patent Document 3] JP 5-93049 [Patent Document 4] JP 7-265065 Gazette [Patent Document 5] JP 10-108682 [Patent Document 6] JP 2001-340078 [Non-Patent Document 1] Y. Doi, S. Kitamura, H. Abe, Macromolecules 28, 4822- 4823 (1995) [Non-patent document 2] Biotechnology and Bioengineering, vol 67, 240 (2000) [Non-patent document 3] Appl · Microbiol. Biotechnol ·, vol 57, 50 (2001) [Non-patent document 4] National infection of Japan Institute for Disease Management, pathogens, and other safety regulations, Schedule 1 to Schedule 1 (1999) [Non-Patent Document 5] T. Fukui, Y. Doi, J. Bacteriol, 179, 15, 4821-4830 (1997) 100136.doc 200540269 [ Non-Patent Literature 6] T. Fukui et al., Appl. Microbiol. Biotechnol 49, 333 (1998) [Non-Patent Literature 7] T. Fukui et al., Biomolecules, vol 3, 618 (2002) [Non-Patent Literature 8] S. Park et al., Biomacromolecules, vol 2, 248 (2001) [Non-Patent Document 9] X. Lu et al., FEMS Microbiology Letters, vol 221, 97 (2003) [ Non-Patent Document 10] T. Kichise et al., Hachiman 1. £ 1 ^ 1 * 〇11.] ^ (^ 〇1 ^ 〇1 · 68, 2411-2419 (2002) [Non-Patent Document 11] A. Amara App. Microbiol. Biotechnol., Vol 59, 477 (2002) [Disclosure of the Invention] Problems to be Solved by the Invention From the viewpoints of safety, high productivity, and availability of inexpensive carbon sources, true cultivation can be expected. Ralstonia eutr〇pha is the most suitable host for industrial production of P (3HB-co-3HH). However, compared with low 3HH constitutive P (3HB-C0-3HH) production system, the use of The safe and inexpensive production system of high 3HH composition type p (3HB-co-3HH) suitable for processing into films and the like due to its flexible properties has not yet been established, so it is still expected to develop its high production system. Means to solve this problem: The present author: Intensive review in order to solve the above problems, construct a new polyacetate containing a novel phaC gene derived from guinea pig Aeromonas 100136.doc 10 200540269 into enzyme expression plastids . A transformant was produced by introducing the expressed plastid into a host such as R. eutropha, and cultivating it with cheap vegetable oil as the sole carbon source. As a result, it was found that the 3HH composition with high productivity was 12 mol%. P (3HB-co-3HH) above. That is, the present invention relates to a polyester synthetic enzyme expressing plastid which can produce high 3HH composition type P (3HB-co-3HH) which is excellent in processability such as processing into a film. Polyester synthetic ability transform body, and safe and cheap manufacturing method of high 3HH composition polyester using the same. In summary, the present invention relates to high 3HH constitutive polyester synthetic enzymes expressing plastids, namely, the polymorphic synthetic enzymes expressing plastids contained in PHB-4 / pJRDdTc + 149NS171DG (FERM BP-10259 and FIRDI BCRC 940479). The miniaturized polyester synthetic enzyme expressing plastid (II), and the above-mentioned high 3HH constitutive polyester synthetic enzyme expressing the transforming plastid and

PHB-4 / pJRDdTc + 149NS171DG (FERM BP-1 0259 and FIRDI BCRC 940479), and a polyester manufacturing method using a turn body. The above-mentioned manufacturing method is preferably a safe and inexpensive manufacturing method of the high 3HH composition type polyester using the above-mentioned transformed body, in which the polyester is the following formula (1) Γ U, Γ η u Γ ch3 Ί r c3h7 η Η — 1 —OC Η CC | —OCHCC —OH (1) h2 om 丨 h2 on (where m and η represent integers greater than or equal to 1) copolymerization containing 3-light butyric acid and 3-hydroxyhexanoic acid Polyester P (3HB-co-3HH). [Embodiment] Hereinafter, the present invention will be described in detail. The polyester synthetic enzyme expression plastid (I) of the present invention is contained in 100136.doc 11 200540269

PHB-4 / pJRDdTc + 149NS171DG (FERM BIM0259 and FIRDI BCRC 940479) can synthesize the performance plastid of high 3HH constitutive p (3HB_ co_3HH) enzyme with high productivity. Production of expression plastids • First, the production of the polyester synthetic enzyme expression plastids of the present invention will be described. All genetic manipulations can be performed according to the method described in Molecular Cloning (ceid pH Harbor Laboratory Press, (1989)). In addition, the base φ can be purchased from suppliers in the market due to the enzymes and breeding hosts used in the operation, and used in accordance with its instructions. The enzyme is not particularly limited as long as it can be used for a genetic operator. The selection host is not particularly limited, and may be, for example, a coliform DH5a strain. In the present invention, as a vector, a derivative of pJRD215 (ATCC 37533), PJRDdTc (described in WO04 / 074476), which is one of a wide host range vector, can be used. The sequence of the vector used in the construction of the plastid is shown in sequence number 4. • Polyester synthetic enzyme gene, which can modulate the N149S / D171G double mutant gene fragment from Aeromonas caviae shown in sequence number 3 as the restriction enzyme EcoRI fragment, the same restriction as inserted into the vector Constructed by enzyme parts. Furthermore, the N149S mutant gene and the di7ig mutant gene, as described in Appl. Environ. Microbiol. 68, 2411-2419 (2002) by T. Kichise, etc., were each used asparagine of amino acid No. 149 Serine acid substitution, aspartic acid No. 171, amino acid Aspartic acid replaced with glycine, a polyester synthetic enzyme gene from Aeromonas guinea pigs. Figure ^ shows the sequence of constructing the polyester synthetic enzyme-expressing plastid (I) of the present invention. 100136.doc 12 200540269 In addition to the construction gene, as long as it has a promoter and a terminator, it has an expression unit that has energy in the host bacteria. Furthermore, in the polyester synthetic enzyme expression plastid, one or more of the above expression units may be present. Furthermore, the present invention's polyenzyme-representing protoplasts (I) included in PHB-4 / pJRDdTc + 149NS171DG (FERM BP-10259 and FIRDI BCRC 940479) are represented by "pJRDdTc + 149NS171DG" in this specification. Next, the production of the polyester synthetic enzyme expression plastid (II) of the present invention will be explained. The polyester synthetic enzyme expression plastid (II) of the present invention is a miniaturized polyester synthetic enzyme expression plastid (I) included in PHB-4 / pJRDdTc + 149NS171DG (FERM BP-10259 and FIRDI BCRC 940479). Miniaturization of plastids is carried out by making the expression of the polyester synthase gene and losing unnecessary parts of the plastids replication. For example, if there are two or more anti-biotolerance genes in the plastid used, any one of them can be deleted. In the case of the plastid of the present invention, although a kanamycin gene or a streptomycin resistance gene can be deleted, it is preferable to delete a streptomycin resistance gene. As a method for losing a specific gene, a method using a restriction enzyme site or a method using PCR can be used. In addition, a polyester synthetic enzyme lacking part or all of the ability to transmit conjugation and lacking a streptomycin resistance gene may be appropriately used to express plastids. The polyester synthetic enzyme expressing plastids (II) of the present invention can reduce the transformation rate when the plastids are introduced into the host. Production of Transformers Next, the production of Transformers will be explained. The transformation of Ben Sheming > 胄 is a person who expresses plastids by the above-mentioned polyacetate synthetic enzyme. In short, the transformed body of the present invention is obtained by introducing any one of the above-mentioned polyacetate-synthetic enzyme-expressing plastids to (Π) into a host suitable for the plastid. • There are no special restrictions on the host. Microorganisms that are isolated from nature, or microorganisms that are deposited with strain deposit agencies (such as IFO or ATCC) can be used. Specifically, a can be used, for example, Ralstonia, Aeromonas, Escherichia, Alcaligenes, or pseudous Omonas) is a non-polyacetic acid producing bacterium. From the standpoint of safety and productivity, Ralstonia is preferred and Ralstonia eutropha is more preferred, especially by genetic modification by mutation treatment or homologous recombination Treatment and • Inactivation of polyester synthase by R. eutropha (eg, R. eutropha

PHB_4 strain, etc.) is particularly good. R. eutropha PHB-4 strain can be obtained from, for example, DSMZ nettles. The introduction of polyester synthetic enzyme-expressing plastids into microorganisms can be performed according to a known method. For example, the electroporation method (Current Prococois h Molecular Biology, Vol. 1, p. H84, 1994), or the calcium method (Lederberg · E · M · et al., J. Baceteriol. 119. 1072 (1974)) and so on. A preferred transformant of the present invention is, for example, a transformant of R. eutropha that introduces a polyester synthetic enzyme-expressing plastid (I) as a host. Specifically, such as 100136.doc -14- 200540269. To import pJRDdTc + 149NS171DG to true

Rabbiella PHB-4 / pJRDdTc + 149NS171DG (Reservation No .: FERM BP-10259, Retention date: February 23, 2007). In addition, the transformation system was filed at the Patent Biostorage Center of the 6th Independent Administrative Corporation Industrial Technology Research Institute in the central area of East 1-chome, Tsukuba City, Ibaraki Prefecture, Japan. The international mailing was carried out in accordance with the Budapest Treaty.

The name of PHB-4 / pJRDdTc + 149NS171DG was deposited with the Institute of Food Industry Development (FIRDI) of the Republic of China (Taiwan) on June 23, 2005, and its deposit number is bcrc 940479 〇P (3HB-co -3HH) Production Next, the production of P (3HB-co-3HH) is explained. The production method of the present invention is to use the above-mentioned transformed body. The above polyester has the following formula (1)

Η Γ ch3] Γ C3H7 Λ 1 1-0-CH-C-C--O — CH — c — C — h2 〇m H2 〇 • OH (1) (where m and η are integers of 1 or more) The copolymerized polyester P (3HB_c0_3HH) containing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid represented is more preferable, and the copolymerized polyester p (3HB_c0_3HH) with a higher 3HH composition is more preferable . The composition ratio of each monomer unit constituting the high 3HH composition P (3HB-co-3HH) is not particularly limited, but from the viewpoint of good softness, the 3HH unit is 8 mol. /. It is more preferably 50 mol% or less, and 10 mol% or more and 40 mol%. /. The following is more preferable, and it is more preferably 12 mol% or more and 100136.doc -15-200540269 30 mol% or less. In the production of P (3HB-CO-3HH), sugar, oil, or fatty acids are given as a carbon source, and a culture medium containing nitrogen sources other than carbon sources, inorganic salts, and other organic nutrient sources is used to cultivate the above. Transforming body. For example, 'used to cultivate a transformant obtained by using a microorganism belonging to the genus Ralstonia, Aeromonas, Escherichia, Alcaligenes, or Pseudomonas as a host As the culture medium, a culture medium capable of providing a broken source available to microorganisms and restricting any of a nitrogen source, an inorganic salt, and an organic nutrient source as appropriate may be used. For example, the use of a nitrogen source is limited to 0.01 to 0.00%. Medium. The sugar may be a carbohydrate such as glucose or fructose. Fats and oils are mostly oils containing saturated or unsaturated fatty acids having a carbon number of 10 or more, such as coconut oil, palm oil, palm kernel oil, and the like. The fatty acid may be a saturated or unsaturated fatty acid such as hexanoic acid, caprylic acid, capric acid, lauric acid, oleic acid, palmitic acid, linseed oil ^, linolenic acid, or linolenic acid, or the like Fatty acid derivatives such as esters or salts. The nitrogen source may be, for example, an ammonium salt such as ammonia, gasified ammonium, ammonium sulfate, or ammonium phosphate, or a peptone, a meat extract, or a yeast extract. The inorganic salt may be, for example, potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium linate, magnesium sulfate, sodium gas, or the like. Other organic nutrition sources may be, for example, amino acids such as glycine, alanine, serine, alanine, or proline, or vitamins such as vitamin B 1, vitamin B 12, or vitamin C. 100136.doc -16- 200540269 It is also possible to add an antibiotic (conamycin) corresponding to the antibiotic resistance gene present in the plastid of the present invention to the culture medium. • The culture temperature should only be the temperature at which the bacteria can multiply, but 20 ° C to 40 ° C is preferred. The culture time is not particularly limited, but it is preferably about 1 to 7 days *. Then, p (3hb_c0_3hh) may be recovered from the obtained cultured bacterial cells. In the present invention, recovery of p (3HB_c0_3HH) from the bacterial cells can be performed according to, for example, the following Φ method. After the completion of the culture, the bacteria are separated from the culture solution by a centrifuge or the like, and the bacteria are washed with distilled water, methanol, or the like, and dried. P (3HB-CO-3HH) was extracted from the dried cells using an organic solvent such as chloroform. From the organic solvent solution containing the P (3HB-C0-3HH), the bacteria are removed by filtration and the like, and a weak solvent such as methanol or hexane is added to the filtrate to precipitate P (3HB-CO-3HH). . In addition, the supernatant was removed by filtration and centrifugation, and the p (3HB-co-3HH) was dried and recovered. The weight average molecular weight (Mw) of the obtained P (3HB-CO-3HH) and the analysis of the 3HH group (mol%) can be performed by, for example, gas chromatography or nuclear magnetic resonance. Alternatively, a simple method of confirming the production of polyisocyanate can be confirmed by a dyeing method using Nilered. That is, Nile red was added to the agar medium in which the recombinant bacteria were propagated, and the recombinant bacteria were cultured for 7 days. By observing whether the recombinant bacteria became red ', the presence or absence of polyester production could be confirmed. [Effects of the Invention] As described above, by using a novel polyester synthetic enzyme to express plastid-derived transformants of R. eutropha, it can be safely and highly cultivated even under a single carbon source such as cheap vegetable oils and fats. Produce P (3HB-co-3HH) with a high 3HH composition suitable for processing into a thin film 100136.doc -17- 200540269. [Best Mode for Carrying Out the Invention] Hereinafter, the present invention will be described in detail by way of examples. However, the present invention does not limit its technical scope to these embodiments. (Example 1) Construction of expression plastids The expression plastids for the production of high 3HH composition P (3HB-co-3HH) are as follows. The vector used for the construction of pJRDdTc + 149NS171DG can be constructed using the DNA code shown in SEQ ID NO: 4 Carrier. This vector can be constructed in the same manner as pJRDdTc described in WO04 / 〇74476. The Nl49S / DmG double mutant gene fragment from a guinea pig aeromonas that is a polyester synthetic enzyme gene can be made by the pcR method. The N149S and D171G variants were replaced by serine and asparagine of amino acid No. 149, PHA synthetic enzyme from Guinea pig aeromonas, respectively. The acid is replaced with glycine. Therefore, the N149S mutant gene from Aeromonas guinea pigs described in Non-Patent Document 10 was used to segment the EcoRI site of PUC19 in sections, and the synthetic DNAs shown in SEQ ID NOs: 1 and 2 were used as primers. For PCR. its

The conditions are (1) 94 ° C for 2 minutes, (2) 94 ° C for 30 seconds, (3) 55 ° for 30 seconds, (4) 72 ° C for 2 minutes, 25 cycles from (2) to (4), and (5) 72 ° C for 5 minutes; LA Taq polymerase (manufactured by Po Sang) was used as the polymerase system. It was cut with restriction enzyme EcoRI, and the N149S / D171G fragment shown in SEQ ID NO: 3 was adjusted, and this vector was inserted into the site cut with the same enzyme to construct pjRDdTc + 149NS171DG, which expresses plastids. 100136.doc -18- 200540269 (Example 2) Production of a transfectant The electropulse method was used to produce a transformant of R. religiella PHB-4 strain containing a plastid that expresses plastid obtained in Example 1. In short, the gene introduction device is a gene interpreter (Geneparser) manufactured by Biorad Corporation, and a cuvette (Bioap Corporation) 0.2 cm is used in the same manner. A competent cell (competent cell) of 400 μ1 and a performance plastid of 20 μ were injected into a colorimetric tube and installed in a pulse device. The electric pulse was performed under the conditions of an electrostatic capacity of 25 pF, a voltage of 1.5 kV, and a resistance value of 800 Ω. . After the pulse, the bacterial solution in the cuvette was

NutrientBroth medium (manufactured by DIFCO) was shake-cultured at 30 ° C for 3 hours, and then cultured on a selection plate (NutrientAgar medium (manufactured by DIFC0), kanamycin 100 mg / L) and cultured at 30 ° C On the 2nd, the transformation was obtained. (Example 3) Selection of transformants The transformant obtained in Example 2 was inoculated into a medium containing Nile Red (disodium hydrogen phosphate · 12 hydrate 9 g, potassium dihydrogen phosphate L 5 g, and ammonium vaporized). 5 g, 0.02 g of magnesium sulfate, 7 hydrate, 0.5 g of fructose, cobaltated gas, 6 hydrate, 25 ppm, vaporized iron (in), 6 hydrate 16, gaseous polonium, 2 hydrate 10 3 ppm, nickel vaporized 0.16 ppm hexahydrate, copper sulfate 0.16 ppm hydrate, 0.5 mg Nile Red 0.5 mg and agar 15 g / 1 L), and cultured at 30 (> c One week. As a result, the accumulation of polyester in the bacteria was confirmed from the redness of the colony, and the colony was selected for the production of P (3HB-CO-3HH). The transformant was registered with PHB_4 / pJRDdTc + 149NS171DG (registered number: FERM BP-10259, deposited Day: February 23, 2007), the name is deposited in the Patent Biostorage Center of the Institute of Industrial Technology Research Institute of the Independent Administrative Corporation 6th, 1st, 1st, 1st, 1st, 1st, 1st, Tsukuba, Ibaraki Prefecture, Japan, and is internationally placed under the Budapest Treaty. In addition, the applicant of this case 100136.doc -19- 200540269 also named jRak / oe / a PHB-4 / pJRDdTc + 149NS1 7 IDG in 2005. June 23 and then transferred the body to deposit the Republic of China (Taiwan) Institute of Food Industry (FIRDI), as its storage numbers BCRC 940479. (Example 4) P (3HB-co-3HH) of production and refining

The composition of the seed culture medium is 1 w / v% meat extract, 1 w / v ° / q trypton for bacterial culture (Bacto-Trypton), 0.2 w / v% yeast extract, 0.9 w / v% Na2P04 · 12H20 and 0.15 w / v% KH2P04, and adjusted to pH 6.8. The composition of the pre-culture medium was 1.1 w / v% Na2P04 12H20, 0.19 w / v ° / 〇ΚΗ2Ρ〇4, 1.29 w / v% (NH4) 2S04, 0.1 w / v% MgS04 · 7Η20, 2.5 w / v% palm oil with oleic acid, 0.5 v / v trace metal salt solution (1.6 w / v% FeCl3 · 6Η20, 1 w / v% CaCl2 · 2Η20, 0.02 w / v% CoCl2 · 6H20 in 0.1N hydrochloric acid , 0.016 w / v% CuS04 · 5H20 and 0.012 w / v% NiCl2 · 6H20 dissolved) and 5xl0_6w / v% kanamycin. The composition of P (3HB-co-3HH) production medium is 0.385 w / v% Na2P04 · 12H20, 0.067 w / v% KH2P〇4, 0-291 w / v% (NH4) 2S04, 0.1 w / v% MgS04 · 7H20, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl3 · 6H2〇, 0.1 w / v ° / 〇CaCl2 · 2H20, 0.02 w / v% CoCl2 · 6H20 dissolved in 0.1N hydrochloric acid 0.016 w / v% CuS04 · 51120 and 0.012 stomach / ¥% offenders (: 12-6} 120 persons), 0.05 \ ¥ / ¥% 3108? 1; 11 ugly \ 200K (defoaming agent, Cognis Japan Co., Ltd.) and 5xl (T6w / v% kanamycin. The carbon source is palm kernel oleic acid oil which is used to separate palm kernel oil from the low melting point as the sole carbon source. During the whole cultivation period, the specific substrate supply rate is 0.08 to 0.1 (fat (g)) x (dry cell weight (g))-lx (hour (h))-1 100136.doc • 20- 200540269 flow port. PHB-4 / Glycerol stock (50 μ149) of pJRDdTc + 149NS171DG transformant was inoculated in seed mother medium (10 ml), and cultured for 24 hours, and then 1.0 v / v was inoculated into 3 of a 1.8 L pre-culture medium L tank fermentor (Jar fermentor, made by Marubishi Bioengi, MDL-300) The operating conditions were an incubation temperature of 30 ° C, an agitation speed of 500 rpm, an aeration volume of 1.8 L / min, and a pH control between 6.7 and 6.8, while incubating for 28 hours. The pH control system used a 7% ammonium hydroxide aqueous solution. P (3HB -co_3HH) Production culture, inoculating the pre-cultured seed mother at 5.0 v / v %% into a 10L tank-type fermentation tank (manufactured by Marubei Biotech, MDL-1000) containing 6L of production medium. The operating conditions are: The culture temperature is 28 ° C, the stirring speed is 400 rpm, the aeration rate is 3.6 L / min, and the pH is controlled between 6.7 and 6.8. The pH control system uses a 7% ammonium hydroxide aqueous solution. The culture is performed for about 60 hours. The bacteria were recovered by centrifugation, washed with methanol, and then freeze-dried to measure the weight of the dried bacteria. 100 g of chloroform was added to 1 g of the obtained dried bacteria, and the mixture was stirred at room temperature for one day and night to extract P in the bacteria. (3HB-co-3HH). After filtering the bacterial cell residue and concentrating it with an evaporator to a total volume of 30 ml, about 90 ml of hexane was slowly added and slowly stirred while being left for 1 hour. The precipitated P (3HB-co-3HH) was filtered, and then dried under vacuum at 50 ° C for 3 hours. The weight of dry P (3HB-co-3HH) was measured, and the content of the cells was calculated. Results The P (3HB-co-3HH) content of the PHB-4 / pJRDdTc + 149NS171DG transformant was "high content" of 66.1 (wt%) at 62 hours. (Example 5) Analysis of the 3HH composition of P (3HB-co-3HH) 100136.doc -21-200540269 Analysis of the 3HH composition of P (3HB-co-3HH) produced by the transformant PHB-4 / pJRDdTc + 149NS171DG The measurement was performed using gas chromatography as described below. Add 2 ml of sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform to about 20 mg of dry P (3HB-co-3HH), seal, and heat at 100 ° C for 140 minutes to obtain P (3HB- co-3HH) The decomposition of the former S purpose. After cooling, a small amount of 1.5 g of sodium bicarbonate was successively added to the mixture for neutralization, and the mixture was left to stand until the occurrence of the dioxide breakdown. After 4 ml of diisopropyl ether was added and mixed well, the mixture was centrifuged, and the monomer unit composition of the P (3HB-co-3HH) decomposition product in the supernatant was analyzed by capillary gas chromatography. Gas chromatography was performed using Shimadzu GC-17, and capillary tube system was NEUTRA BOND -1 manufactured by GL Science (a column length of 25 m, an inner diameter of the column of 0.25 mm, and a liquid film thickness of 0.4 μm). He was used as the carrier gas, the inlet pressure of the column was adjusted to 100 kPa, and 1 μΐ of the sample was injected. The temperature condition is that the temperature rises at a rate of 8 ° C / min from the initial temperature of 100 ° C to 200 ° C, and further rises at a rate of 30 ° C / min from 200 ° C to 290 ° C. As a result of the analysis under the above conditions, the 3HH composition of P (3HB-co-3HH) at the end of the 62-hour culture was 14.7 (mole%), which was a high 3HH composition suitable for processing into a thin film. As described above, by using novel polyester synthetic enzymes to express plastid-derived R. eutropha transformants, even when cultivated under a single carbon source such as cheap vegetable oils and fats, it is possible to produce safe and highly productive Processed into a film-like high 3HH composition type P (3HB-co_3HH). [Brief description of the diagram] Fig. 1 is a construction diagram of pJRDdTc + 149NS171DG (FERM BP-10259 and FIRDI BCRC 940479) polyester synthetic enzymes expressing plastids of the present invention. 100136.doc -22- 200540269 Sequence Listing < 11 < 12 'Branch Company (KANEKA CORPORATION) < 130 > B040191TW01 < 140) 094111212 < 141) 2005-04-08 < 150 > JP2004-115642 < 151) 2004-4-9 < 160 > 4 < 210 > 1 < 211 > 26 < 212 > DNA < 213> Artificial sequence

< 220 > < 223> primer < 400 > 1 accctggagt ccggcggcca gaacct 26 < 210 > 2 < 211 > 26 < 212 > DNA < 213> artificial sequence < 220> < 223> primer < 400 > 2 aggttctggc cgccggactc cagggt 26

< 210〉 3 < 211〉 594 < 212〉 PRT < 213 > Guinea Pig Aeromonas < 220〉 < 223〉 Polyhydroxyalkanoate Synthase Mutant < 400〉 9

Met Ser Gin Pro Ser Tyr Gly Pro Leu Phe Glu Ala Leu Ala His Tyr 15 10 15

Asn Asp Lys Leu Leu Ala Met Ala Lys Ala Gin Thr Glu Arg Thr Ala 20 25 30 6ln Ala Leu Leu Gin Thr Asn Leu Asp Asp Leu Gly Gin Val Leu Glu 100136.doc 200540269 35 40 45

Gin Gly Ser Gin Gin Pro Trp Gin Leu lie Gin Ala Gin Met Asn Trp 50 55 60

Trp Gin Asp 6ln Leu Lys Leu Met Gin His Thr Leu Leu Lys Ser Ala 65 70 75 80

Gly Gin Pro Ser Glu Pro Val lie Thr Pro Glu Arg Ser Asp Arg Arg 85 90 95

Phe Lys Ala Glu Ala Trp Ser Glu Gin Pro lie Tyr Asp Tyr Leu Lys 100 105 110

Gin Ser Tyr Leu Leu Thr Ala Arg His Leu Leu Ala Ser Val Asp Ala 115 120 125

Leu Glu Gly Val Pro Gin Lys Ser Arg Glu Arg Leu Arg Phe Phe Thr 130 135 140

Arg Gin Tyr Val Ser Ala Met Ala Pro Ser Asn Phe Leu Ala Thr Asn 145 150 155 160

Pro Glu Leu Leu Lys Leu Thr Leu Glu Ser Gly Gly Gin Asn Leu Val 165 170 175

Arg Gly Leu Ala Leu Leu Ala Glu Asp Leu Glu Arg Ser Ala Asp Gin 180 185 190

Leu Asn lie Arg Leu Thr Asp Glu Ser Ala Phe Glu Leu Gly Arg Asp 195 200 205

Leu Ala Leu Thr Pro Gly Arg Val Val Gin Arg Thr Glu Leu Tyr Glu 210 215 220

Leu lie Gin Tyr Ser Pro Thr Thr Glu Thr Val Gly Lys Thr Pro Val 225 230 235 240

Leu lie Val Pro Pro Phe lie Asn Lys Tyr Tyr lie Met Asp Met Arg 245 250 255

Pro 6ln Asn Ser Leu Val Ala Trp Leu Val Ala Gin Gly Gin Thr Val 100136.doc 200540269 260 265 270

Phe Met lie Ser Trp Arg Asn Pro Gly Val Ala Gin Ala Gin lie Asp 275 280 285

Leu Asp Asp Tyr Val Val Asp Gly Val lie Ala Ala Leu Asp Gly Val 290 295 300 6lu Ala Ala Thr Gly 6lu Arg Glu Val His Gly lie Gly Tyr Cys lie 305 310 315 320

Gly Gly Thr Ala Leu Ser Leu Ala Met Gly Trp Leu Ala Ala Arg Arg 325 330 335

Gin Lys Gin Arg Val Arg Thr Ala Thr Leu Phe Thr Thr Leu Leu Asp 340 345 350

Phe Ser Gin Pro Gly Glu Leu Gly lie Phe lie His Glu Pro lie lie 355 360 365

Ala Ala Leu Glu Ala Gin Asn Glu Ala Lys Gly lie Met Asp Gly Arg 370 375 380

Gin Leu Ala Val Ser Phe Ser Leu Leu Arg Glu Asn Ser Leu Tyr Trp 385 390 395 400

Asn Tyr Tyr lie Asp Ser Tyr Leu Lys Gly Gin Ser Pro Val Ala Phe 405 410 415

Asp Leu Leu His Trp Asn Ser Asp Ser Thr Asn Val Ala Gly Lys Thr 420 425 430

His Asn Ser Leu Leu Arg Arg Leu Tyr Leu Glu Asn Gin Leu Val Lys 435 440 445

Gly Glu Leu Lys lie Arg Asn Thr Arg lie Asp Leu Gly Lys Val Lys 450 455 460

Thr Pro Val Leu Leu Val Ser Ala Val Asp Asp His lie Ala Leu Trp 465 470 475 480

Gin Gly Thr Trp Gin Gly Met Lys Leu Phe Gly Gly Glu Gin Arg Phe 100136.doc 200540269 485 490 495

Leu Leu Ala Glu Ser 6ly His lie Ala Gly lie lie Asn Pro Pro Ala 500 505 510

Ala Asn Lys Tyr Gly Phe Trp His Asn Gly Ala Glu Ala Glu Ser Pro 515 520 525

Glu Ser Trp Leu Ala Gly Ala Thr His Gin Gly Gly S, er Trp Trp Pro 530 535 540

Glu Met Met Gly Phe lie Gin Asn Arg Asp Glu Gly Ser Glu Pro Val 545 550 555 560

Pro Ala Arg Val Pro Glu Glu Gly Leu Ala Pro Ala Pro Gly His Tyr 565 570 575

Val Lys Val Arg Leu Asn Pro Val Phe Ala Cys Pro Thr Glu Glu Asp 580 585 590

Ala Ala < 210 > 4 < 211 > 9141

< 212 > DNA < 213 > pJRDdTc < 220>

≪ 223> PJRDdTc of mutant < 400 > 4 60 120 180 240 300 360 420 480 aactgcacat tcgggatatt tctctatatt cgcgcttcat cagaaaactg aaggaacctc cattgaatcg aactaatatt ttttttggtg aatcgcattc tgactggttg cctgtcagag gcggagaatc tggtgatttt gtttttcgac gtggtgacgg gcatgccttc gcgaaaatcg cacctgcttc ccgccgcggt gagctcgctg gagagcgtga ccgcctcatt tggctcaaag gtcgaggtgt ggcttgcccc gaggtcatca actggcagga ggaacaggag ggtgcatgct tggtgataac ggcaattccg ggagtaccgg cggctgatct gtctggagcg gatttgctca aagcgtggcc gtcaatgggg cagcaacttg gcgctgttca cagcctatcg gttgatcaat gtccgtttga gcgcaggctg tcgcgaatgt tcggacgcgc cgttgatgtg gtgtcccgca atgccgtcaa tcccgacttc ttaccggacg aggacaagag tacgccgcag ctcgatcttt 100136.doc -4- 540 600 600

200540269 tggctcgtgt cgaacgagag ctaccggtgc ggctcgacca agagcgcacc gatatggttg tttgccatgg tgatccctgc atgccgaact tcatggtgga ccctaaaact cttcaatgca cgggtctgat cgaccttggg cggctcggaa cagcagatcg ctatgccgat ttggcactca tgattgctaa cgccgaagag aactgggcag cgccagatga agcagagcgc gccttcgctg tcctattcaa tgtattgggg atcgaagccc ccgaccgcga acgccttgcc ttctatctgc gattggaccc tctgacttgg ggttgatgtt catgccgcct gtttttcctg ctcattggca cgtttcgcaa cctgttctca ttgcggacac cttttccagc ctcgtttgga aagtttcatt gccagacggg actcctgcaa tcgtcaaggg attgaaacct atagaagaca ttgctgatga actgcgcggg gccgactatc tggtatggcg caatgggagg ggagcagtcc · ggttgctcgg tcgtgagaac aatctgatgt tgctcgaata tgccggggag cgaatgctct ctcacatcgt tgccgagcac ggcgactacc aggcgaccga aattgcagcg gaactaatgg cgaagctgta tgccgcatct gaggaacccc tgccttctgc ccttctcccg atccgggatc gctttgcagc tttgtttcag cgggcgcgcg atgatcaaaa cgcaggttgt caaactgact acgtccacgc ggcgattata gccgatcaaa tgatgagcaa tgcctcggaa ctgcgtgggc tacatggcga tctgcatcat gaaaacatca tgttctccag tcgcggctgg ctggtgatag atcccgtcg g tctggtcggt gaagtgggct ttggcgccgc caatatgttc tacgatccgg ctgacagaga cgacctttgt ctcgatccta gacgcattgc acagatggcg gacgcattct ctcgtgdgct ggacgtcgat ccgcgtcgcc tgctcgacca ggcgtacgct tatgggtgcc tttccgcagc ttggaacgcg gatggagaag aggagcaacg cgatctagct atcgcggccg cgatcaagca ggtgcgacag acgtcatact agatatcaag cgacttctcc tatcccctgg gaacacatca atctcaccgg agaatatcgc tggccaaagc cttagcgtag gattccgccc cttcccgcaa acgaccccaa acaggaaacg cagctgaaac gggaagctca acacccactg acgcatgggt tgttcaggca gtacttcatc aaccagcaag gcggcacttt cggccatccg ccgcgcccca cagctcgggc agaaaccgcg acgcttacag ctgaaagcga ccaggtgctc ggcgtggcaa gactcgcagc gaacccgtag aaagccatgc tccagccgcc cgcattggag aaattcttca aattcccgtt gcacatagcc cggcaattcc tttccctgct ctgccataag cgcagcgaat gccgggtaat actcgtcaac gatctgatag agaagggttt gctcgggtcg gtggctctgg taacgaccag tatcccgatc ccggctggcc gtcctggccg ccacatgagg catgttccgc 100136.doc 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220

200540269 gtccttgcaa tactgtgttt acatacagtc tatcgcttag cggaaagttc ttttaccctc agccgaaatg cctgccgttg ctagacattg ccagccagtg cccgtcactc ccgtactaac tgtcacgaac ccctgcaata actgtcacgc ccccctgcaa taactgtcac gaacccctgc aataactgtc acgcccccaa acctgcaaac ccagcagggg cgggggctgg cggggtgttg gaaaaatcca tccatgatta tctaagaata atccactagg cgcggttatc agcgcccttg tggggcgctg ctgcccttgc ccaatatgcc cggccagagg ccggatagct ggtctattcg ctgcgctagg ctacacaccg ccccaccgct gcgcggcagg gggaaaggcg ggcaaagccc gctaaacccc acaccaaacc ccgcagaaat acgctggagc gcttttagcc gctttagcgg cctttccccc tacccgaagg gtgggggcgc gtgtgcagcc ccgcagggcc tgtctcggtc gatcattcag cccggctcat ccttctggcg tggcggcaga ccgaacaagg cgcggtcgtg gtcgcgttca aggtacgcat ccattgccgc catgagccga tcctccggcc actcgctgct gttcaccttg gccaaaatca tggcccccac cagcaccttg cgccttgttt cgttcttgcg ctcttgctgc tgttcccttg cccgcacccg ctgaatttcg gcattgattc gcgctcgttg ttcttcgagc ttggccagcc gatccgccgc cttgttgctc cccttaacca tcttgacacc ccattgttaa tgtgctgtct cgtaggctat caaggaggca cagcggcggc aatcccgacc ctactttgta gcctaacggc cacccttcgg gcggtgcgct ctccgagggc cattgcatgg agccgaaaag caaaagcaac agcgaggcag catggcgatt tatcacctta cggcgaaaac cggcagcagg tcgggcggcc aatcggccag ggccaaggcc gactacatcc agcgcgaagg caagtatgcc cgcgacatgg atgaagtctt gcacgccgaa tccgggcaca tgccggagtt cgtcgagcgg cccgccgact actgggatgc tgccgacctg tatgaacgcg ccaatgggcg gctgttcaag gaggtcgaat ttgccctgcc ggtcgagctg accctcgacc agcagaaggc gctggcgtcc gagttcgccc agcacctgac cggtgccgag cgcctgccgt atacgctggc catccatgcc ggtggcggcg agaacccgca ctgccacctg atgatctccg agcggatcaa tgacggcatc gagcggcccg ccgctcagtg gttcaagcgg tacaacggca agaccccgga gaagggcggg gcacagaaga ccgaagcgct caagcccaag gcatggcttg agcagacccg cgaggcatgg gccgaccatg ccaaccgggc attagagcgg gctggccacg acgcccgcat tgaccacaga acacttgagg cgcagggcat cgagcgcctg cccggtgttc acctggggcc gaacgtggtg gagatggaag gccggggcat ccgcaccgac cgggcagacg tggccctgaa 100136.doc 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3 840 3900 200540269

catcgacacc tgaacgcaat aaccgctggc cccagcaggc cggcatgggt cgcagagcgc tggcttccat cgccactgac caggactttg cggcgtcagg gctccagaac gcccgccgag ggatgacatg ctatcaggca ccggacgctg ccgcttggcg gtgggtgctg gcaggctggc cagcctcgaa ccgcagcgag tatcgccgcg ggccgaggcc gcgcaccgtc acgggcaccg gtgcttgcgt tatggaatac ttggttgccc cgctgatatg gccaacgccc cgacagagtg ccagagcatg cagcgaggcg ggagctggcc ccggagcgcg gatgcctacg aaccgaggac caggccatag gacgccacca acgccatggc caggagcggc aaagccgagg tgggtcaagg gccagcgagt ggcttcacca ctgcgtgaat cagcagatcg ctgcccgagc atggccgggc cagaagctgg agcccagcgc agcaaggtca gcaccccgcc tggtactcac gaaaaaagcg ggtggctttg gccagcctta agatcatcga aagaaatcca gcgacactgg caggcggcgg aaagagttgc tcgcgggggt gtggtgctgc ggctcgatct gccgacagct ccggccagat tcaagcggat atggtctggt gccgggagcc tggccgacgc acgacgccga accgcaagga ccaagggcaa agcaggccca ggcagcttag tggtcaagcg ccagccgggg tggcagagcg tgggtctgcc agcgaggcat gcctgttata cttcagggtc gttatacgtc accttgacgg cttacaggaa gaggcatcaa ccgacgaagc agtggctgaa gggaggtagc ggcactggaa tgatcgcatc ggctgcgctt caaggccatg gatgaaccgg gaatgcccag gctggtggac tgccctggta cgcaggcggt cccggccagc caagcacacc gaccgccacc gcggcagcag ccgccaccgg cttcggtgat ccgcagtgcc caagcccggc cagcgtccag ggacaggggc ctatgagtac ggtctacctg aaacaaggcc ctgcaccttg taccgggagg cgagttagcg ccggcaggtc agcccagcgc cgccgaggtg gctatggcta gttgctcttg ggtggcccga ggctgtgagc gaatggtcag ggcaatgacg gacctcagcg gtggaaacca gaacttcggg gccgacagcc acccgcgccg gctggcccgg gagaaggccc cgcacggcgc gabctcagca gaggaaatcg cacgaagcgg cttgcgcggg gggccagatt tcacgcacag atcaaaagtg gaggctggcc tccttgttcc caatagacca gagcagatcg atgagccaga cagacagggg agcagcgccg accgtgatgc ctcgacctga tgaagaacga gcttcgatat ccgccgaagt tgtatatcag agtttgacct gcccgaagaa ggcagattgc gccactatgg gttatcagcc cgctggtgca gcaggctggc tggacgagta agtgcgactt gcaaggccat attacatcga ccgagctggc tcagcatgta aagggggttt acaagggcta gcttttcagt gcgaagacaa 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 100136.doc

200540269 gcctttcggc cccggcaagt ttctcggtga ctgatatgaa agaccaaaag gacaagcaga ccggcgacct gctggccagc cctgacgctg tacgccaagc gcgatatgcc gagcgcatga aggccaaagg gatgcgtcag cgcaagttct ggctgaccga cgacgaatac gaggcgctgc gcgagtgcct ggaagaactc agagcggcgc agggcggggg tagtgacccc gccagcgcct aaccaccaac tgcctgcaaa ggaggcaatc aatggctacc cataagccta tcaatattct ggaggcgttc gcagcagcgc cgccaccgct ggactacgtt ttgcccaaca tggtggccgg tacggtcggg gcgctggtgt cgcccggtgg tgccggtaaa tccatgctgg ccctgcaact ggccgcacag attgcaggcg ggccggatct gctggaggtg ggcgaactgc ccaccggccc ggtgatctac ctgcccgccg aagacccgcc caccgccatt catcaccgcc tgcacgccct tggggcgcac ctcagcgccg aggaacggca agccgtggct gacggcctgc tgatccagcc gctgatcggc agcctgccca acatcatggc cccggagtgg ttcgacggcc tcaagcgcgc cgccgagggc cgccgcctga tggtgctgga cacgctgcgc cggttccaca tcgaggaaga aaacgccagc ggccccatgg cccaggtcat cggtcgcatg gaggccatcg ccgccgatac cgggtgctct atcgtgttcc tgcaccatgc cagcaagggc gcggccatga tgggcgcagg cgaccagcag caggccagcc ggggcagctc ggtactggtc gataacatcc gctggcagtc ctacctgtcg agcatgacca gcgccgaggc cgaggaatgg ggtgtggacg acgaccagcg ccggttcttc gtccgcttcg gtgtgagcaa ggccaactat ggcgcaccgt tcgctgatcg gtggttcagg cggcatgacg gcggggtgct caagcccgcc gtgctggaga ggcagcgcaa gagcaagggg gtgccccgtg gtgaagccta agaacaagca cagcctcagc cacgtccggc acgacccggc gcactgtctg gcccccggcc tgttccgtgc cctcaagcgg ggcgagcgca agcgcagcaa gctggacgtg acgtatgact acggcgacgg caagcggatc gagttcagcg gcccggagcc gctgggcgct gatgatctgc gcatcctgca agggctggtg gccatggctg ggcctaatgg cctagtgctt ggcccggaac ccaagaccga aggcggacgg cagctccggc tgttcctgga acccaagtgg gaggccgtca ccgctgatgc catggtggtc aaaggtagct atcgggcgct ggcaaaggaa atcggggcag aggtcgatag tggtggggcg ctcaagcaca tacaggactg catcgagcgc ctttggaagg tatccatcat cgcccagaat ggccgcaagc ggcaggggtt tcggctgctg tcggagtacg ccagcgacga ggcggacggg cgcctgtacg tggccctgaa ccccttgatc gcgcaggccg tcatgggtgg cggccagcat gtgcgcatca 100136.doc 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7 200 7260

200540269 gcatggacga ggtgcgggcg ctggacagcg aaaccgcccg cctgctgcac cagcggctgt gtggctggat cgaccccggc aaaaccggca aggcttccat agataccttg tgcggctatg tctggccgtc agaggccagt ggttcgacca tgcgcaagcg ccgccagcgg gtgcgcgagg cgttgccgga gctggtcgcg ctgggctgga cggtaaccga gttcgcggcg ggcaagtacg acatcacccg gcccaaggcg gcaggctgac cccccccact ctattgtaaa caagacattt ttatctttta tattcaatgg cttattttcc tgctaattgg taataccatg aaaaatacca tgctcagaaa aggcttaaca atattttgaa aaattgccta ctgagcgctg ccgcacagct ccataggccg ctttcctggc tttgcttcca gatgtatgct cttctgctcc tgcagttcgg gggcatggat gcgcggatag ccgctgctgg tttcctggat gccgacggat ttgcactgcc ggtagaactc cgcgaggtcg tccagcctca ggcagcagct gaaccaactc gcgaggggat cgagcccggg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgcctcga ggcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc ctagtatagt ctatagtccg tggaattatt atatttatct ccgacgatat tctcatcagt 100136.doc 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8 880 8940 200540269 gaaatccagg ttatcacagt atcgtcatcc ctgccgggcc ggaattctca tgtttgacag taaattgcta acgcagtcag tcggcaccgt caccctggat tcttgcggga tggtggg tgat tgat tgat tgat tgat tgat tgat

100136.doc 10-

Claims (1)

200540269 10. Scope of patent application: 1. A poly-S synthetic enzyme expression plastid, which is included in pHB_4 / pjRDdTc + 149NS171DG (FERM BP-10259 and FIRDI BCRC 940479). 2. A polyester synthetic enzyme expressing a plastid, which miniaturizes the plastid of the request item 丨 0 3 · A transformant ', which is transformed by the plastid of the request item 1 or 2. 4. The transgenic body of item 3 as specified, wherein the host is a non-polyester-producing Ralstonia. 5. The transformant of claim 3 or 4, wherein the host is a non-polyester-producing R. eutropha. 6. The transformant of any one of claims 3 to 5, wherein the host is a R. eutropha PHB-4 strain. 7 ·-A type of transformant, which is PHB-4 / PJRDdTC + 149NS17lDG (PERM BP-10259 and FIRDI BCRC 940479). 8. A method for producing a polyester, which uses the transformation body of any one of claims 3 to 7. ~ 9 · The method for producing a polyester according to claim 8, wherein the polyester is ch3 Γ c3h7 ^ 1 1 —〇—CH — C-• C-0-CH —C-c-L H2 〇 Copolymerized polyester represented by m L · h2 o • OH (wherein m and η represent integers of 1 or more), hydroxybutyric acid and 3-hydroxyhexanoic acid. 100136.doc