WO2005098001A1 - 新規形質転換体 - Google Patents
新規形質転換体 Download PDFInfo
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- WO2005098001A1 WO2005098001A1 PCT/JP2005/006922 JP2005006922W WO2005098001A1 WO 2005098001 A1 WO2005098001 A1 WO 2005098001A1 JP 2005006922 W JP2005006922 W JP 2005006922W WO 2005098001 A1 WO2005098001 A1 WO 2005098001A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
Definitions
- the present invention relates to a novel plasmid for producing a polyester having a high 3HH composition, a transformant thereof, and a method for producing a polyester having a high 3HH composition using the same.
- P (3HB) poly-3-hydroxybutyric acid
- 3HB 3-hydroxybutyric acid
- Bacillus megaterium Bacillus megaterium
- P (3HB) is a thermoplastic polymer that is biologically degraded in the natural environment and is attracting attention as an environmentally friendly plastic.
- P (3HB) has high crystallinity and is hard and brittle, so its practical application is limited. For this reason, research aimed at improving this property has been made.
- P (3HB-CO-3HH) 3HB and 3hydroxyhexanoic acid
- 3HH 3hydroxyhexanoic acid
- Patent Documents 3 and 4 The method for producing P (3HB-co-3HH) reported in these reports is based on fermentative production of fatty acids such as oleic acid and fatty oils such as olive oil using aeromonas caviae isolated from soil. there were. Also, studies on the properties of P (3HB co 3HH) have been made (see Non-Patent Document 1).
- this production method has a low cell productivity of 4 g ZL, a polymer content of 30%, and a low polymer productivity, making it inadequate as a production method for the practical use of this polymer.
- Aeromonas hydrofila is pathogenic to humans (see Non-Patent Document 4), and thus cannot be said to be a species suitable for industrial production.
- these culture productions use expensive carbon sources, it is required to use inexpensive carbon sources from the viewpoint of production costs.
- PHA polyhydroxyalkanoic acid synthase gene was cloned from Aeromonas cinerea (see Patent Document 5, Non-patent Document 5).
- Production of P (3HB—co—3HH) using a transformant in which this gene was introduced into Ralstonia eutropha (formerly Alcaligenes eutrophas) resulted in a cell productivity of 4 gZL and polymer content.
- a cell productivity of 4 gZL and a polymer content of 80% were achieved (see Non-Patent Document 6).
- the polymer productivity is low, although cheap vegetable oils and fats are used as carbon sources.
- the 3HH composition of 5% was hard, brittle, and had physical properties.
- a P (3HB-co-3HH) producing strain using Escherichia coli as a host has also been constructed.
- a strain was constructed in which Escherichia coli introduced a PHA synthase gene of the genus Aeromonas and a NADP-acetoacetyl Co-A reductase gene of Ralstonia eutopha.
- the bacterial mass was 79 gZL
- the polymer content was 27.2%
- the 3HH composition was 10.8% (see Non-Patent Document 8).
- PHA synthase activity and 3HH composition were also improved, and mutants in which 518th phenylalanine was replaced with isoleucine and 214th in which palin was replaced with glycine It has been reported that the enzyme has improved PHA synthase activity and polymer content in Escherichia coli. However, since they use special E. coli as their host and still have a low polymer content, further improvement was required for industrial production utilizing the characteristics of these mutant enzymes.
- Patent Document 6 A method for producing P (3HB-co-3HH) has also been studied (see Patent Document 6). Also, a method for controlling the physical properties of P (3HB-co-3HH) has been disclosed (see Patent Document 6). By using at least two kinds of fats and oils having different carbon numbers and Z or fatty acids as a carbon source, it becomes possible to produce P (3HB-co-3HH) having a 3HH composition of 1 to 40 mol%, and various physical properties can be obtained. (3HB-co-3HH) can be produced. However, in this production method, it is necessary to add relatively expensive hexanoic acid, octanoic acid, etc. to control the 3HH composition, and high concentrations of hexanoic acid show cytotoxicity. The result is a decrease. In addition, the addition of multi-component carbon sources can make the production facilities complex and expensive.
- Patent Document 1 JP-A-57-150393
- Patent Document 2 JP-A-59-220192
- Patent Document 3 JP-A-5-93049
- Patent Document 4 JP-A-7-265065
- Patent Document 5 JP-A-10-108682
- Patent Document 6 Japanese Patent Application Laid-Open No. 2001-340078
- Non-Patent Document 1 Y. Doi, S. Kitamura, H. Abe, Macromolecules 28, 4822-4 823 (1995)
- Non-patent document 2 Biotechnology and Bioengineering, vol67, 240 (2000)
- Non-patent document 3 Appl.Microbiol.Biotechnol., Vol57, 50 (2001)
- Non-Patent Document 4 National Institute of Infectious Diseases, Regulations for Safety Management of Pathogens, Appendix 1 Appendix 1 (1999)
- Non-Patent Document 5 T. Fukui, Y. Doi, J. Bacteriol, 179, 15, 4821—4830 (1997)
- Non-patent document 6 T. Fukui et al., Appl. Microbiol. Biotecnol. 49, 333 (1998)
- Non-patent document 7 T. Fukui et al., Biomolecules, vol3, 618 (2002)
- Non-Patent Document 8 S. Park et al. Biomacromolecules, vol2, 248 (2001)
- Non-Patent Document 9 X. Lu et al., FEMS Microbiology Letters, vol221, 97 (2003)
- Non-Patent Document 10 T. Kichise et al., Appl.Environ.Microbiol. 68, 2411-2419 (2 002)
- Non-Patent Document 11 A. Amara et al. App.Microbiol.Biotechnol., Vol59, 477 (200 2)
- Ralstonia's eutopha is the most suitable host for industrial production of P (3HB-co-3HH) from the viewpoints of safety, high productivity, availability of an inexpensive carbon source, and the like. Is expected.
- P (3HB-co-3HH) production system safe and inexpensive production of high 3HH composition P (3HB-co-3HH) suitable for processing into films, etc., making use of its flexible properties Since the system was established, the development of a high production system was desired.
- the present inventors have conducted intensive studies in order to solve the above problems, and have constructed a novel polyester synthase expression plasmid containing a novel phaC derivative gene derived from Aeromonas' biebie.
- a transformant in which the expression plasmid was introduced into a host such as Ralstonia-eutropha was prepared and cultured under a single carbon source using inexpensive vegetable oils and fats.
- P (3HB-co-3HH) with a 3HH composition of 12 mol% or more was obtained. Can be produced with high productivity.
- the present invention provides a polyester synthase expression plasmid capable of producing P (3HB-co-3HH) having a high 3HH composition excellent in processability into a film or the like, and a high 3HH composition polyester containing the plasmid.
- the present invention relates to a transformant having an ability and a safe and inexpensive method for producing a high 3HH composition polyester using the transformant.
- the present invention provides a polyester synthase expression plasmid (I) contained in PHB-4 / pJRDdTc + 149NS17IDG (FERM BP-10259), which is a high 3HH composition polyester synthase expression plasmid; Polyester synthase expression plasmid (II) obtained by miniaturizing the plasmid; a transformant transformed with the above-mentioned high 3HH composition polyester synthase expression plasmid and PHB-4ZpJRDdTc + 149NS171DG (FERM BP-10259);
- the present invention relates to a method for producing a polyester.
- the polyester is represented by the following formula (1) [0017] [Formula 1]
- the polyester synthase expression plasmid (I) of the present invention is contained in PHB-4ZpJRDdTc + 149N S171DG (FERM BP-10259), and has a high productivity and high 3HH composition P (3HB-co-3HH). This is an expression plasmid for the enzyme to be synthesized.
- polyester synthase expression plasmid (I) of the present invention will be described.
- Global genetic manipulations can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press, (1989)).
- Enzymes, Cloying hosts, etc. used for genetic manipulation can be purchased from market suppliers and used according to the description.
- the enzyme is not particularly limited as long as it can be used for genetic manipulation.
- the Clawing host is not particularly limited, and examples include Escherichia coli DH5 ⁇ strain.
- pJRD215 ATC C 37533
- pJRDdTc a pJRD215 derivative pJRDdTc (described in WO04Z074476), which is one of the broad host range vectors, can be used as the vector.
- SEQ ID NO: 4 shows the sequence of the vector used in the construction of this expression plasmid.
- an N149SZD171G double mutant gene fragment derived from Aeromonas capillaris shown in SEQ ID NO: 3 is prepared as a restriction enzyme EcoRI fragment, and inserted into the same restriction enzyme site of the above vector to construct.
- the N149S mutant gene and the D171G mutant gene are described in T. Kichise et al., Appl. Environ. Microbiol. 68, 2411-2419 (2002) [this description describes that asparagine at the 149th amino acid is serine.
- the 171st amino acid aspartic acid is This is a polyester synthase gene derived from Aeromonas capillaris, substituted for syn.
- FIG. 1 shows the procedure for constructing the polyester synthase expression plasmid (I) of the present invention.
- the polyester synthase gene only needs to have an expression unit that functions in the host bacterium, such as a promoter and a terminator, in addition to the structural gene.
- the polyester synthase expression plasmid may contain a plurality of expression units as long as at least one expression unit is present.
- polyester synthase expression plasmid (I) contained in PHB-4ZpJRDdTc + 149NS171DG (FERM BP-10259) of the present invention may be referred to as “pJRDdTc + 149NS171DG” in this specification.
- the polyester synthase expression plasmid ( ⁇ ) of the present invention is obtained by miniaturizing the polyester synthase expression plasmid (I) contained in PHB-4ZpJRDdTc + 149 NS171DG (FERM BP-10259).
- the size of the plasmid can be reduced by deleting portions unnecessary for expression of the polyester synthase gene and plasmid replication.
- the plasmid used has two or more antibiotic resistance genes, either of them can be deleted.
- it is possible to delete the kanamycin resistance gene or the streptomycin resistance gene but it is preferable to delete the streptomycin resistance gene.
- a method for deleting a specific gene as described above a method using a restriction enzyme site or a method using PCR can be used.
- polyester synthase expression plasmid lacking part or all of the conjugation transfer ability and lacking the streptomycin resistance gene can be suitably used.
- the polyester synthase expression plasmid ( ⁇ ) of the present invention can improve the transformation rate when the expression plasmid is introduced into a host by miniaturizing the plasmid.
- the transformant of the present invention comprises at least one of the above-mentioned polyester synthase expression plasmids (I) to (II). It has been transformed by this. That is, the transformant of the present invention can be obtained by introducing any of the polyester synthase expression plasmids (I) to (I) obtained above into a host compatible with the plasmid.
- the host is not particularly limited, but a microorganism isolated from nature, a microorganism deposited at a strain depository (eg, IFO, ATCC, etc.), and the like can be used.
- non-polyester-producing bacteria of bacteria such as Ralstonia, Aeromonas, Escherichia, Alcaligenes, and Pseudomonas can be used.
- Ralstonia is preferable, Ralstonia eutropha is more preferable, and polyester synthase is inactivated by mutagen treatment or gene disruption treatment based on homologous recombination.
- Ralstonia's Eutropha for example, Ralstonia's Eutropha PHB-4 strain.
- Ralstonia's Eutropha PHB-4 strain is available from institutional sources such as D SMZ.
- Preferable transformants of the present invention include, for example, a transformant in which the polyester synthase expression plasmid (I) is introduced into Ralstonia's utop as a host. Specific examples include the following transformants.
- PHB-4 / pJRDdTc + 149NS171DG (Accession number: FERM BP-10259, accession date: February 23, 2005), which is a transformant in which pJRDdTc + 149NS 171DG was introduced into Ralstonia eutropha PHB-4 strain.
- This transformant has been deposited internationally under the Budapest Treaty at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, located at 1-1, Tsukuba-Higashi, Ibaraki, Japan. .
- the method for producing a polyester of the present invention uses the above transformant.
- the above polyester the following formula (1)
- Copolymerized polyester P (3HB—co—3HH), which also has 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, represented by the formula (where m and n represent integers of 1 or more) is preferred.
- Higher 3HH copolymer polyester P (3HB-co-3HH) is more preferred!
- composition ratio of each monomer unit constituting the high 3HH composition P (HB—co—3HH)!
- 3HH units are preferably at least 8 mol% and at most 50 mol%, more preferably at least 10 mol% and at most 40 mol%, particularly at most 12 mol%. % Or more and 30 mol% or less.
- sugars, fats and oils or fatty acids are given as carbon sources and include nutrients other than carbon sources, such as nitrogen sources, inorganic salts, and other organic nutrients.
- the transformant can be cultured using a medium.
- a medium for culturing a transformant obtained by using a microorganism such as a microorganism belonging to the genus Ralstonia, Aeromonas, Escherichia, Alcaligenes or Pseudomonas as a host
- a carbon source that can be assimilated by the microorganism is used.
- a medium in which any one of nitrogen source, inorganic salts and organic nutrients is restricted for example, a medium in which the nitrogen source is restricted to 0.01 to 0.1%, etc. can be used. .
- sugar examples include carbohydrates such as glucose and fructose.
- fats and oils examples include fats and oils rich in saturated 'unsaturated fatty acids having 10 or more carbon atoms, such as coconut oil, palm oil, palm kernel oil and the like.
- Fatty acids include saturated unsaturated fatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid and myristic acid, and esters and salts of these fatty acids. Fatty acid derivatives are mentioned.
- Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate and the like, as well as peptone, meat extract, yeast extract and the like.
- Examples of the inorganic salts include potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium salt and the like.
- organic nutrients include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; and vitamins such as vitamin B1, vitamin B12, and vitamin C.
- an antibiotic such as kanamycin
- an antibiotic corresponding to the antibiotic resistance gene present in the expression plasmid of the present invention may be added to the culture solution.
- the culture temperature may be any temperature at which the bacteria can grow, but is preferably 20 ° C to 40 ° C.
- the culture time is not particularly limited, but may be about 1 to 7 days.
- P (3HB-CO-3HH) should be recovered from the obtained cultured cells.
- P (3HB-co-3HH) can be recovered from cells by, for example, the following method. After completion of the culture, the cells are separated from the culture using a centrifuge or the like, and the cells are washed with distilled water, methanol and the like, and dried. From the dried cells, p (3HB-co-3HH) is extracted using an organic solvent such as black form. Cell components are removed from the organic solvent solution containing P (3HB—co—3HH) by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to form P (3HB co—3HH). Let it settle. Further, the supernatant is removed by filtration or centrifugation, and dried to recover P (3HB-co-3HH).
- the analysis of the weight average molecular weight (Mw) and the 3HH composition (mol%) of the obtained P (3HB-co-3HH) can be performed by, for example, a gas chromatography method or a nuclear magnetic resonance method.
- a dyeing method using Nilered can be used as a simple method for confirming polyester production. That is, the presence or absence of polyester production is determined by culturing Nilered on an agar medium on which the recombinant bacterium grows, culturing the recombinant bacterium for 1 to 7 days, and observing whether the recombinant bacterium turns red. You can check.
- pJRDdTc + 149NS171DG a vector encoded by the DNA shown in SEQ ID NO: 4 was used. This vector can be constructed in the same manner as pJRDd Tc described in WO04Z074476.
- the N149SZD171G double mutant gene fragment derived from Aeromonas radiata which is a polyester synthase gene, was prepared by PCR.
- asparagine which is the 149th amino acid of PHA synthase derived from Aeromonas' rabies
- aspartic acid which is the 171st amino acid
- the N149S mutant gene derived from Aeromonas serrata described in Non-Patent Document 10 was subcloned at the EcoRI site of pUC19, and the synthetic DNAs represented by SEQ ID NO: 1 and SEQ ID NO: 2 were used as primers.
- the conditions were (1) 2 minutes at 94 ° C, (2) 30 seconds at 94 ° C, (3) 30 seconds at 55 ° C, (4) 2 minutes at 72 ° C, (2) to (4) ) For 25 cycles, (5) 5 minutes at 72 ° C, and LA Taq polymerase (Takara Bio) was used as the polymerase.
- the N149S ZD171G fragment shown in SEQ ID NO: 3 was prepared by digestion with the restriction enzyme EcoRI, and this vector was inserted into the site digested with the same enzyme to construct the expression plasmid pJRDdTc + 149NS171DG.
- a transformant of Ralstonia'eutropha PHB-4 strain containing the expression plasmid obtained in Example 1 was prepared by an electric pulse method. That is, a gene pulsar manufactured by Biomd was used for the gene transfer device, and a cuvette having a gap of 0.2 cm manufactured by Biorad was used as the cuvette. Inject 400 ⁇ l of the competent cells and 20 ⁇ l of the expression plasmid into a cuvette, set them in a pulse device, and apply an electric pulse under the conditions of a capacitance of 25 ⁇ F, a voltage of 1.5 kV, and a resistance of 800 ⁇ . I did.
- the bacterial solution in the cuvette is shake-cultured at 30 ° C for 3 hours in a Nutrient Broth medium (DIFCO), and is incubated at 30 ° C in a selection plate (NutrientAgar medium (DIFCO), kanamycin lOOmgZL). After culturing for 2 days, a transformant was obtained.
- DIFCO Nutrient Broth medium
- DIFCO NutrientAgar medium
- kanamycin lOOmgZL kanamycin lOOmgZL
- the transformant obtained in Example 2 was transformed into a Nilered-containing medium (9 g of disodium hydrogen phosphate ⁇ 12 hydrate, 1.5 g of potassium dihydrogen phosphate, 0.05 g of ammonium chloride, 0.05 g of magnesium sulfate, and 7 water of magnesium sulfate).
- the seed culture medium was composed of lwZv% Meat-extract, lw / v% Bacto-Trypton, 0.2 w / v% Yeast -extract, 0.9 w / v% Na PO12 ⁇ , 0.15 w / v % KH
- composition of the preculture medium was 1. lw / v% Na PO ⁇ 12 ⁇ 0, 0.19 w / v% KH PO, 1
- composition of the P (3HB—co—3HH) production medium was 0.385 w / v% Na PO ⁇ 12 ⁇ 0, 0.06.
- the carbon source is palm kernel oil olein, a low melting point fraction of palm kernel oil, used as a single carbon source. Throughout the entire culture, feeding was performed so that the specific substrate supply rate was 0.08 to 0.1 (oil () X (net dry cell weight (g) ⁇ 1 X (time (h)) _1 ) _1 ).
- a glycerol stock (50 ⁇ l) of the PHB-4ZpJRDdTc + 149NS171DG transformant was inoculated into a seed culture medium (10 ml), cultured for 24 hours, and a 3 L jaw inconveniencer (circle) containing 1.8 L of preculture medium was added.
- 1.0vZv% was inoculated into Ryo Bioenge's MDL-300).
- the operating conditions were a culture temperature of 30 ° C., a stirring speed of 500 rpm, an aeration rate of 1.8 LZmin, and culture for 28 hours while controlling the pH between 6.7 and 6.8.
- a 7% aqueous ammonium hydroxide solution was used for pH control.
- P (3HB—co—3HH) production culture is a 10L jar armamentar containing 6L of production medium.
- the operating conditions were a culture temperature of 28 ° C, a stirring speed of 400 rpm, an aeration rate of 3.6 LZmin, and a pH of 6.7 to 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control. The culture was performed for about 60 hours. After completion of the culture, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured.
- the 3HH composition analysis of P (3HB-co-3HH) produced by the PHB-4 / pJRDdTc + 149NS171 DG transformant was measured by gas chromatography as follows. [0055] Approximately 20 mg of dry P (3HB-co-3HH) was added with 2 ml of a mixture of sulfuric acid and methanol (15:85) and 2 ml of a form of lip, sealed and heated at 100 ° C for 140 minutes. As a result, a methyl ester of a P (3HB-co-3HH) decomposition product was obtained. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize, and the mixture was allowed to stand until carbon dioxide gas generation was stopped.
- the mixture was centrifuged, and the monomer unit composition of the P (3HB-co-3HH) degradation product in the supernatant was analyzed by canary gas chromatography.
- the gasket mat used was Shimadzu GC-17A, and the one-shot ram was NEUTRA BOND-1 (column length: 25 m, column inner diameter: 0.25 mm, liquid film thickness: 0.4 / zm). He was used as the carrier gas, the column inlet pressure was 100 kPa, and 1 ⁇ l of the sample was injected.
- the temperature was raised to the initial temperature of 100 to 200 ° C at a rate of 8 ° CZ, and further to 200 to 290 ° C at a rate of 30 ° CZ.
- the 3HH yarn of P (3HB-co-3HH) at the end of the 62-hour culture was 14.7 (mol%), a high 3HH yarn suitable for application to a finolem. It was a success.
- FIG. 1 is a drawing showing the construction of a plasmid for expressing pJRDdTc + 149NS171DG (FERM BP-10259) polyester synthase of the present invention.
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Cited By (10)
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WO2007049716A1 (ja) * | 2005-10-27 | 2007-05-03 | Kaneka Corporation | 新規プラスミドベクター及びプラスミドを安定に保持する形質転換体 |
JP2007259708A (ja) * | 2006-03-27 | 2007-10-11 | Kaneka Corp | 新規生分解性ポリエステルの製造方法 |
WO2008010296A1 (fr) * | 2006-07-21 | 2008-01-24 | Kaneka Corporation | Micro-organisme doté d'un gène remplacé et procédé de production de polyester à l'aide dudit micro-organisme |
JP2008029218A (ja) * | 2006-07-26 | 2008-02-14 | Kaneka Corp | 酵素活性を低下させた微生物を用いる共重合ポリエステルの製造方法 |
US7384766B2 (en) | 2006-07-26 | 2008-06-10 | Kaneka Corporation | Gene-substituted microorganisms, and production method of polyesters using the same |
JPWO2006101176A1 (ja) * | 2005-03-24 | 2008-09-04 | 株式会社カネカ | 超高分子量ポリエステルを蓄積する微生物 |
WO2012102371A1 (ja) | 2011-01-27 | 2012-08-02 | 株式会社カネカ | 高分子量pha生産微生物 |
US9051589B2 (en) | 2005-10-27 | 2015-06-09 | Kaneka Corporation | Plasmid vector and transformant stably retaining plasmid |
CN104845927A (zh) * | 2006-07-21 | 2015-08-19 | 株式会社钟化 | 基因取代微生物及使用该微生物的聚酯制造方法 |
WO2020218565A1 (ja) * | 2019-04-26 | 2020-10-29 | 株式会社フューエンス | ポリヒドロキシアルカン酸及びその製造方法 |
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WO2003033707A1 (fr) * | 2001-10-10 | 2003-04-24 | Kaneka Corporation | Gene d'enzyme participant a la synthese de polyester et procede de production de polyester a l'aide dudit gene |
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- 2005-04-08 WO PCT/JP2005/006922 patent/WO2005098001A1/ja active Application Filing
- 2005-04-08 TW TW094111212A patent/TW200540269A/zh unknown
- 2005-04-08 JP JP2006512128A patent/JPWO2005098001A1/ja active Pending
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