JP2007259708A - New method for producing biodegradable polyester - Google Patents
New method for producing biodegradable polyester Download PDFInfo
- Publication number
- JP2007259708A JP2007259708A JP2006085314A JP2006085314A JP2007259708A JP 2007259708 A JP2007259708 A JP 2007259708A JP 2006085314 A JP2006085314 A JP 2006085314A JP 2006085314 A JP2006085314 A JP 2006085314A JP 2007259708 A JP2007259708 A JP 2007259708A
- Authority
- JP
- Japan
- Prior art keywords
- strain
- seq
- synthase gene
- gene
- host
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 62
- 229920000229 biodegradable polyester Polymers 0.000 title claims description 15
- 239000004622 biodegradable polyester Substances 0.000 title claims description 15
- 238000000034 method Methods 0.000 claims abstract description 31
- 244000005700 microbiome Species 0.000 claims abstract description 18
- 108010010718 poly(3-hydroxyalkanoic acid) synthase Proteins 0.000 claims description 52
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical group CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims description 49
- 235000001014 amino acid Nutrition 0.000 claims description 26
- 229920000728 polyester Polymers 0.000 claims description 26
- 150000001413 amino acids Chemical class 0.000 claims description 23
- 108020004705 Codon Proteins 0.000 claims description 16
- 239000000178 monomer Substances 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 10
- HPMGFDVTYHWBAG-UHFFFAOYSA-N 3-hydroxyhexanoic acid Chemical group CCCC(O)CC(O)=O HPMGFDVTYHWBAG-UHFFFAOYSA-N 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 7
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 6
- 235000018417 cysteine Nutrition 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 6
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 claims description 6
- 229920000903 polyhydroxyalkanoate Polymers 0.000 claims description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 5
- 235000004279 alanine Nutrition 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 3
- 241001528539 Cupriavidus necator Species 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 49
- 239000012634 fragment Substances 0.000 description 29
- 101150048611 phaC gene Proteins 0.000 description 24
- 229940024606 amino acid Drugs 0.000 description 22
- 239000002609 medium Substances 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 17
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 14
- 229910052799 carbon Inorganic materials 0.000 description 14
- 229930027917 kanamycin Natural products 0.000 description 14
- 229960000318 kanamycin Drugs 0.000 description 14
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 14
- 229930182823 kanamycin A Natural products 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 239000013598 vector Substances 0.000 description 13
- 238000012217 deletion Methods 0.000 description 12
- 230000037430 deletion Effects 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 210000000349 chromosome Anatomy 0.000 description 11
- 238000002744 homologous recombination Methods 0.000 description 11
- 230000006801 homologous recombination Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 239000013604 expression vector Substances 0.000 description 10
- 241000607516 Aeromonas caviae Species 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000000813 microbial effect Effects 0.000 description 9
- 108091008146 restriction endonucleases Proteins 0.000 description 9
- REKYPYSUBKSCAT-UHFFFAOYSA-N 3-hydroxypentanoic acid Chemical compound CCC(O)CC(O)=O REKYPYSUBKSCAT-UHFFFAOYSA-N 0.000 description 8
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
- 229930195729 fatty acid Natural products 0.000 description 6
- 239000000194 fatty acid Substances 0.000 description 6
- 150000004665 fatty acids Chemical class 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000003925 fat Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 235000019198 oils Nutrition 0.000 description 5
- 239000013600 plasmid vector Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- NDPLAKGOSZHTPH-UHFFFAOYSA-N 3-hydroxyoctanoic acid Chemical compound CCCCCC(O)CC(O)=O NDPLAKGOSZHTPH-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 206010064571 Gene mutation Diseases 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 125000004432 carbon atom Chemical group C* 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 4
- 235000019197 fats Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 101150025220 sacB gene Proteins 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 3
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 3
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 3
- 241000607528 Aeromonas hydrophila Species 0.000 description 3
- WWZKQHOCKIZLMA-UHFFFAOYSA-N Caprylic acid Natural products CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000005642 Oleic acid Substances 0.000 description 3
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 239000013611 chromosomal DNA Substances 0.000 description 3
- 229920001577 copolymer Polymers 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 3
- 239000003346 palm kernel oil Substances 0.000 description 3
- 235000019865 palm kernel oil Nutrition 0.000 description 3
- 230000000704 physical effect Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- XBUXARJOYUQNTC-UHFFFAOYSA-N ()-3-Hydroxynonanoic acid Chemical compound CCCCCCC(O)CC(O)=O XBUXARJOYUQNTC-UHFFFAOYSA-N 0.000 description 2
- CWSNHZHHWHLJIM-UHFFFAOYSA-N 3-Hydroxytridecanoic acid Chemical compound CCCCCCCCCCC(O)CC(O)=O CWSNHZHHWHLJIM-UHFFFAOYSA-N 0.000 description 2
- ATMSEJBABXCWDW-UHFFFAOYSA-N 3-hydroxy-pentadecanoic acid Chemical compound CCCCCCCCCCCCC(O)CC(O)=O ATMSEJBABXCWDW-UHFFFAOYSA-N 0.000 description 2
- FYSSBMZUBSBFJL-UHFFFAOYSA-N 3-hydroxydecanoic acid Chemical compound CCCCCCCC(O)CC(O)=O FYSSBMZUBSBFJL-UHFFFAOYSA-N 0.000 description 2
- MUCMKTPAZLSKTL-UHFFFAOYSA-N 3-hydroxylauric acid Chemical compound CCCCCCCCCC(O)CC(O)=O MUCMKTPAZLSKTL-UHFFFAOYSA-N 0.000 description 2
- FARPMBPKLYEDIL-UHFFFAOYSA-N 3-hydroxyundecanoic acid Chemical compound CCCCCCCCC(O)CC(O)=O FARPMBPKLYEDIL-UHFFFAOYSA-N 0.000 description 2
- 241000607534 Aeromonas Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 241000252867 Cupriavidus metallidurans Species 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 239000005639 Lauric acid Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000000908 ammonium hydroxide Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 239000003471 mutagenic agent Substances 0.000 description 2
- 231100000707 mutagenic chemical Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 235000021313 oleic acid Nutrition 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 101150044547 phbA gene Proteins 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000013587 production medium Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 235000003441 saturated fatty acids Nutrition 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 229910021654 trace metal Inorganic materials 0.000 description 2
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- GYSCBCSGKXNZRH-UHFFFAOYSA-N 1-benzothiophene-2-carboxamide Chemical compound C1=CC=C2SC(C(=O)N)=CC2=C1 GYSCBCSGKXNZRH-UHFFFAOYSA-N 0.000 description 1
- YBTWUESFQWFDMR-UHFFFAOYSA-N 3-Hydroxyhexadecanoic acid Natural products CCCCCCCCCCCCCC(O)CC(=O)OC YBTWUESFQWFDMR-UHFFFAOYSA-N 0.000 description 1
- OXSSIXNFGTZQMZ-UHFFFAOYSA-N 3-hydroxyheptanoic acid Chemical compound CCCCC(O)CC(O)=O OXSSIXNFGTZQMZ-UHFFFAOYSA-N 0.000 description 1
- UZGRZSHGRZYCQV-UHFFFAOYSA-N 4,6-dichloro-1,3-benzothiazol-2-amine Chemical compound C1=C(Cl)C=C2SC(N)=NC2=C1Cl UZGRZSHGRZYCQV-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000193033 Azohydromonas lata Species 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 241001528480 Cupriavidus Species 0.000 description 1
- 241000097442 Cupriavidus metallidurans CH34 Species 0.000 description 1
- ATRNZOYKSNPPBF-UHFFFAOYSA-N D-beta-hydroxymyristic acid Natural products CCCCCCCCCCCC(O)CC(O)=O ATRNZOYKSNPPBF-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Natural products CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 1
- 101100243766 Dictyostelium discoideum phbA gene Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001509401 Gordonia rubripertincta Species 0.000 description 1
- 241000271815 Hydrogenimonas Species 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 101710096706 Poly(3-hydroxyalkanoate) polymerase Proteins 0.000 description 1
- 101710159749 Poly(3-hydroxyalkanoate) polymerase subunit PhaC Proteins 0.000 description 1
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000232299 Ralstonia Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000736901 Thiocystis Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241001106476 Violaceae Species 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 125000000539 amino acid group Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940053200 antiepileptics fatty acid derivative Drugs 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- GONOPSZTUGRENK-UHFFFAOYSA-N benzyl(trichloro)silane Chemical compound Cl[Si](Cl)(Cl)CC1=CC=CC=C1 GONOPSZTUGRENK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- CBWALJHXHCJYTE-UHFFFAOYSA-N beta-hydroxyhexadecanoic acid Natural products CCCCCCCCCCCCCC(O)CC(O)=O CBWALJHXHCJYTE-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N n-hexanoic acid Natural products CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 101150046540 phaA gene Proteins 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 235000019871 vegetable fat Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Abstract
Description
本発明は、生分解性ポリエステルであるポリヒドロキシアルカン酸（ＰＨＡ）の商業的生産に有用な、改良された微生物株と、それを利用するＰＨＡの製造方法に関する。 The present invention relates to an improved microbial strain useful for the commercial production of polyhydroxyalkanoic acid (PHA), a biodegradable polyester, and a method for producing PHA using the same.
ポリヒドロキシアルカン酸（以下ＰＨＡと略す）は、広範な微生物によって生成されるポリエステル型有機分子ポリマーである。これらのポリマーは生分解性を有し、熱可塑性高分子であること、また、再生可能資源から産生されることから、環境調和型素材または生体適合型素材として工業的に生産し、多様な産業へ利用する試みが行われている。 Polyhydroxyalkanoic acid (hereinafter abbreviated as PHA) is a polyester-type organic molecular polymer produced by a wide range of microorganisms. Since these polymers are biodegradable, are thermoplastic polymers, and are produced from renewable resources, they are industrially produced as environmentally friendly materials or biocompatible materials, and are used in various industries. Attempts have been made to use.
このポリエステルを構成するモノマー単位は一般名３−ヒドロキシアルカン酸であって、具体的には３−ヒドロキシ酪酸、３−ヒドロキシ吉草酸、３−ヒドロキシヘキサン酸、３−ヒドロキシオクタン酸、あるいはよりアルキル鎖の長い３−ヒドロキシアルカン酸が単独重合もしくは共重合することにより、ポリマー分子が形成されている。３−ヒドロキシ酪酸（以下３ＨＢと略す）のホモポリマーであるポリ−３−ヒドロキシ酪酸（以下、Ｐ（３ＨＢ）と略す）は、１９２５年にバチルス・メガテリウム（Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ）で最初に発見されたが、このＰ（３ＨＢ）は結晶性が高いため、硬くて脆い性質を持っており、実用的には応用範囲が限られている。この為、この性質の改良を目的とした研究がなされてきた。 The monomer unit constituting this polyester is a general name 3-hydroxyalkanoic acid, specifically 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, or a more alkyl chain. A polymer molecule is formed by homopolymerizing or copolymerizing a long 3-hydroxyalkanoic acid. Poly-3-hydroxybutyric acid (hereinafter abbreviated as P (3HB)), a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB), was first discovered in 1925 in Bacillus megaterium. Since P (3HB) has high crystallinity, it has a hard and brittle nature, and its practical application range is limited. For this reason, research aimed at improving this property has been conducted.
その中で、３−ヒドロキシ酪酸（３ＨＢ）と３−ヒドロキシ吉草酸（３ＨＶ）とからなる共重合体（以下Ｐ（３ＨＢ−ｃｏ−３ＨＶ）と略す）の製造方法が開示されている（例えば、特許文献１、特許文献２参照）。このＰ（３ＨＢ−ｃｏ−３ＨＶ）はＰ（３ＨＢ）に比べると柔軟性に富むため、幅広い用途に応用できると考えられた。しかしながら、実際にはＰ（３ＨＢ−ｃｏ−３ＨＶ）は３ＨＶモル分率を増加させても、それに伴う物性の変化が乏しく、特に柔軟性が向上しないため、シャンプーボトルや使い捨て剃刀の取っ手等、硬質成型体の分野にしか利用されなかった。 Among them, a method for producing a copolymer comprising 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter abbreviated as P (3HB-co-3HV)) is disclosed (for example, (See Patent Document 1 and Patent Document 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.
また、アルキル鎖の炭素数が６〜１６の３−ヒドロキシアルカン酸で構成される中鎖ＰＨＡはＰ（３ＨＢ）やＰ（３ＨＢ−ｃｏ−３ＨＶ）よりも結晶性が低く、弾力に富むことが知られており（非特許文献１参照）、異なる分野への用途が期待されている。中鎖ＰＨＡの製造研究はシュードモナス属のＰＨＡ合成酵素遺伝子を、シュードモナス属、ラルストニア属、大腸菌に導入することによって行われてきたが、いずれも生産性が低く工業生産に適したものではなかった（非特許文献２、非特許文献３、非特許文献４参照）。 Further, medium chain PHA composed of 3-hydroxyalkanoic acid having 6 to 16 carbon atoms in the alkyl chain has lower crystallinity than P (3HB) or P (3HB-co-3HV), and may be rich in elasticity. It is known (see Non-Patent Document 1) and is expected to be used in different fields. Research on the production of medium chain PHA has been carried out by introducing a PHA synthase gene belonging to the genus Pseudomonas into Pseudomonas, Ralstonia, and Escherichia coli, but none of them are low in productivity and suitable for industrial production ( Non-patent document 2, Non-patent document 3, Non-patent document 4).
近年、３ＨＢと３−ヒドロキシヘキサン酸（以下、３ＨＨと略す）との２成分共重合ポリエステル（以下Ｐ（３ＨＢ−ｃｏ−３ＨＨ）と略す）およびその製造方法について研究がなされている（特許文献３、特許文献４参照）。前記先行文献におけるＰ（３ＨＢ−ｃｏ−３ＨＨ）の製造方法は、土壌より単離されたアエロモナス・キャビエ（Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ）を用いてオレイン酸等の脂肪酸やオリーブオイル等の油脂から発酵生産するものであった。また、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の性質に関する研究もなされている（非特許文献５参照）。前記先行文献では炭素数が１２個以上の脂肪酸を唯一の炭素源としてアエロモナス・キャビエを培養し、３ＨＨ組成が１１〜１９ｍｏｌ％のＰ（３ＨＢ−ｃｏ−３ＨＨ）を発酵生産している。このＰ（３ＨＢ−ｃｏ−３ＨＨ）は３ＨＨ組成の増加にしたがって、Ｐ（３ＨＢ）の硬くて脆い性質から次第に柔軟な性質を示すようになり、Ｐ（３ＨＢ−ｃｏ−３ＨＶ）を上回る柔軟性を示すことが明らかにされた。すなわちＰ（３ＨＢ−ｃｏ−３ＨＨ）は３ＨＨ組成を変えることで、硬質ポリエステルから軟質ポリエステルまで応用可能な幅広い物性を持つため、テレビの筐体等のように硬さを要求されるものからフィルム等のように柔軟性を要求されるものまで、幅広い分野への応用が期待できる。しかしながら、本製造方法では菌体生産量４ｇ／Ｌ、ポリエステル含量３０％とポリエステル生産性は低いものであり、本ポリエステルの実用化に向けた生産方法としては未だ不十分と言わざるを得なかったため、実用化に向けて更に高い生産性が得られる方法が探索されてきた。 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). , See Patent Document 4). The method for producing P (3HB-co-3HH) in the above-mentioned prior art is a method in which Aeromonas caviae isolated from soil is fermented and produced from fatty acids such as oleic acid and fats and oils such as olive oil. there were. Studies on the properties of P (3HB-co-3HH) have also been made (see Non-Patent Document 5). In the prior literature, 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. Therefore, a method for obtaining higher productivity for practical use has been searched.
Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の工業生産を目指した取組みもなされている。アエロモナス・ハイドロフィラ（Ａｅｒｏｍｏｎａｓ ｈｙｄｒｏｐｈｉｌａ）を用いた培養では、オレイン酸を炭素源とした４３時間の流加培養において、菌体生産量９５．７ｇ／Ｌ、ポリエステル含量４５．２％、３ＨＨ組成１７％のＰ（３ＨＢ−ｃｏ−３ＨＨ）が生産された（非特許文献６参照）。また、アエロモナス・ハイドロフィラを炭素源としてグルコース及びラウリン酸を用いて培養し、菌体生産量５０ｇ／Ｌ、ポリエステル含量５０％を達成した（非特許文献７参照）。しかしながら、アエロモナス・ハイドロフィラはヒトに対して病原性を有することから（非特許文献８参照）、工業生産に適した種とはいえない。また、これらの培養生産では高価な炭素源を使用するため、製造コストの観点より安価な炭素源の利用も求められている。 Efforts aimed at industrial production of P (3HB-co-3HH) have also been made. In the culture using Aeromonas hydrophila, the cell production amount is 95.7 g / L, the polyester content is 45.2%, the 3HH composition is 17% in the fed-batch culture for 43 hours using oleic acid as a carbon source. Of P (3HB-co-3HH) was produced (see Non-Patent Document 6). In addition, Aeromonas hydrophila was cultured using glucose and lauric acid as a carbon source to achieve a cell production of 50 g / L and a polyester content of 50% (see Non-Patent Document 7). However, since Aeromonas hydrophila is pathogenic to humans (see Non-Patent Document 8), it cannot be said that it is a suitable species for industrial production. In addition, since an expensive carbon source is used in these culture productions, utilization of an inexpensive carbon source is also required from the viewpoint of manufacturing cost.
このため、安全な宿主での生産及び生産性の向上を目指した取組みが行なわれた。アエロモナス・キャビエよりポリヒドロキシアルカン酸（ＰＨＡ）合成酵素遺伝子がクローニングされた（特許文献５、非特許文献９参照）。本遺伝子をワーテルシア・ユートロファ（Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ、旧ラルストニア・ユートロファ）に導入した形質転換体を用いてＰ（３ＨＢ−ｃｏ−３ＨＨ）の生産を行った結果、菌体生産性は４ｇ／Ｌ、ポリエステル含量は３０％であった。更に本形質転換体を炭素源として植物油脂を用いて培養した結果、菌体生産量４ｇ／Ｌ、ポリエステル含量８０％が達成された（非特許文献１０参照）。更に培養条件の改善により、菌体生産量４５ｇ／Ｌ、ポリエステル含量６２．５％、３ＨＨ組成８．１％にまで向上したように、培養方法に関する研究もなされている（特許文献６参照）。
また、フラクトースを炭素源としてＰ（３ＨＢ−ｃｏ−３ＨＨ）を生産できるワーテルシア・ユートロファも構築されたが、本菌株のポリエステル生産性は低く、実生産に適しているとはいえなかった（非特許文献１１参照）。
For this reason, efforts were made to improve production and productivity in a safe host. A polyhydroxyalkanoic acid (PHA) synthase gene was cloned from Aeromonas caviae (see Patent Document 5 and Non-Patent Document 9). As a result of producing P (3HB-co-3HH) using a transformant in which this gene was introduced into Waterusia eutropha (formerly Ralstonia eutropha), the cell productivity was 4 g / L, and the polyester content Was 30%. Furthermore, as a result of culturing the transformant using vegetable oils and fats as a carbon source, a bacterial cell production amount of 4 g / L and a polyester content of 80% were achieved (see Non-Patent Document 10). Furthermore, research on the culture method has been conducted so that the bacterial cell production amount is improved to 45 g / L, the polyester content 62.5%, and the 3HH composition 8.1% by improving the culture conditions (see Patent Document 6).
In addition, Watersia eutropha, which can produce P (3HB-co-3HH) using fructose as a carbon source, was constructed, but the polyester productivity of this strain was low and could not be said to be suitable for actual production (non-patented) Reference 11).
大腸菌を宿主としたＰ（３ＨＢ−ｃｏ−３ＨＨ）生産株も構築された。アエロモナス属のＰＨＡ合成酵素遺伝子及びワーテルシア・ユートロファのＮＡＤＰ−アセトアセチルＣｏ―Ａ還元酵素遺伝子等を大腸菌に導入した株が構築された。ドデカンを炭素源として同大腸菌を培養した結果、４０．８時間の培養で菌体量７９ｇ／Ｌ、ポリエステル含量２７．２％、３ＨＨ組成１０．８％という生産性であった（非特許文献１２参照）。 A P (3HB-co-3HH) producing strain using E. coli as a host was also constructed. A strain was constructed in which the PHA synthase gene of Aeromonas and the NADP-acetoacetyl Co-A reductase gene of Watercia eutropha were introduced into E. coli. As a result of culturing the Escherichia coli using dodecane as a carbon source, the cell mass was 79 g / L, the polyester content was 27.2%, and the 3HH composition was 10.8% after 40.8 hours of cultivation (Non-patent Document 12). reference).
Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の生産性向上並びに３ＨＨ組成制御を目指して、ＰＨＡ合成酵素の人為的な改変が行なわれた。アエロモナス・キャビエ由来のＰＨＡ合成酵素変異体のなかで、１４９番目のアミノ酸アスパラギンがセリンに置換された変異体酵素や、１７１番目のアスパラギン酸がグリシンに置換された変異体酵素は、大腸菌内でのＰＨＡ合成酵素活性や３ＨＨ組成が向上していることが示された（非特許文献１３参照）。また、同酵素の５１８番目のフェニルアラニンがイソロイシンに置換された変異体酵素や２１４番目のバリンがグリシンに置換された変異体酵素は大腸菌でのＰＨＡ合成酵素活性やポリエステル含量が向上したことが報告されている（非特許文献１４参照）。しかし、これらは宿主として特殊な大腸菌を用いており未だポリエステル含量は低い。 In order to improve the productivity of P (3HB-co-3HH) and control the 3HH composition, artificial modification of PHA synthase was performed. 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 was shown that PHA synthase activity and 3HH composition are improved (see Non-Patent Document 13). It was also reported that the mutant enzyme in which the 518th phenylalanine of the enzyme was replaced with isoleucine and the mutant enzyme in which the 214th valine was replaced with glycine had improved PHA synthase activity and polyester content in Escherichia coli. (See Non-Patent Document 14). However, these use special E. coli as a host and the polyester content is still low.
実用的物性に優れたＰ（３ＨＢ−ｃｏ−３ＨＨ）を大腸菌に産生させるためには基質モノマー供給のための遺伝子をさらに共存させる必要や、または高価な脂肪酸などを培地へ供給させる必要があり、なお、高い生産効率を達成するための妨げになっている。
Ｐ（３ＨＢ−ｃｏ−３ＨＨ）を高生産性で商業生産するためには、安全性、モノマー基質供給量や原材料の観点から宿主としてワーテルシア・ユートロファのＰ（３ＨＢ）非生産株、すなわちＰＨＢ−４株を宿主として使用することがより合理的であると考えられてきたが、更なる生産性向上が求められていた。
In order to commercially produce P (3HB-co-3HH) with high productivity, from the viewpoint of safety, monomer substrate supply amount and raw materials, as a host, a non-P (3HB) producing strain of Watercia Eutropha, ie, PHB-4 Although it has been considered more reasonable to use strains as hosts, further productivity improvements have been sought.
以上のように、ＰＨＡの効率的商業生産には未だ多くの課題が存在した。そこで本発明では、工業的規模の培養プロセスにおいて、ＰＨＡ、特に広範な用途が期待できるＰ（３ＨＢ−ｃｏ−３ＨＨ）を高レベルで生産する組換え微生物株を提供すること、およびそれを利用したＰＨＡの製造方法を提供することを目的とする。 As described above, there are still many problems in the efficient commercial production of PHA. Therefore, the present invention provides a recombinant microbial strain that produces PHA, particularly P (3HB-co-3HH), which can be expected to have a wide range of uses, in an industrial-scale culture process, and uses the same. It aims at providing the manufacturing method of PHA.
本発明者らはＰ（３ＨＢ−ｃｏ−３ＨＨ）を商業的に高生産する方法を鋭意研究した結果、従来用いられてきたＰＨＢ−４株の代わりに、部位特異的変異によってＰＨＡ非生産性とした微生物を宿主として使用することにより、ＰＨＡの生産性を向上させることが可能であることを見出した。
これまでの遺伝子組換えを利用したＰＨＡ生産では、代表的なＰ（３ＨＢ）非生産株であるワーテルシア・ユートロファＰＨＢ−４株（ＤＳＭ Ｎｏ．５４１，以下ＰＨＢ−４株）がその宿主として多く用いられてきた。ＰＨＢ−４株は、１９７０年にワーテルシア・ユートロファ（当時名ハイドロゲノモナス）の野生株であるＨ１６株（ＤＳＭ Ｎｏ．４２８、以下Ｈ１６株）から変異剤を用いた突然変異処理によって作製された菌株である（Ｈ．Ｇ．Ｓｃｈｌｅｇｅｌ等、Ａｒｃｈ．Ｍｉｋｒｏｂｉｏｌ．７１：２８３−２９４，（１９７０））。非特異的な変異剤によって処理された株であるため、目的としたＰ（３ＨＢ）非生産という点だけでなく、その他の遺伝子にも変異が導入されている可能性を否定できない。事実、Ｈ．Ｇ．Ｓｃｈｌｅｇｅｌ等によると、ＰＨＢ−４株は親株Ｈ１６株と比べて増殖能力やある種の生物活性が劣っており、ＰＨＡ生産用宿主株として最高の生産能力を発揮できているかどうか不明であった。
As a result of intensive research on a method for commercially producing P (3HB-co-3HH), the present inventors have determined that PHA non-productivity can be obtained by site-directed mutagenesis instead of the conventionally used PHB-4 strain. It was found that the productivity of PHA can be improved by using the obtained microorganism as a host.
In PHA production using genetic recombination so far, Watersia Eutropha PHB-4 strain (DSM No. 541, hereinafter referred to as PHB-4 strain), which is a typical P (3HB) non-producing strain, is often used as its host. Has been. The PHB-4 strain was produced in 1970 by mutation treatment using a mutagen from the H16 strain (DSM No. 428, hereinafter referred to as H16 strain), which is a wild strain of Watersia Eutropha (then known as Hydrogenomonas). (HG Schlegel et al., Arch. Mikubiol. 71: 283-294, (1970)). Since it is a strain treated with a non-specific mutagen, it cannot be denied that not only the target P (3HB) is not produced, but also that other genes have mutations introduced. In fact, H.C. G. According to Schlegel et al., The PHB-4 strain was inferior in growth ability and certain biological activities as compared to the parent strain H16, and it was unclear whether or not it was able to demonstrate the highest production capacity as a host strain for PHA production.
ワーテルシア・ユートロファにおける染色体遺伝子の部位特異的変異は、Ｙｏｒｋ等、Ｐｏｔｔｅｒ等（Ｙｏｒｋ等、Ｊｏｕｒｎａｌ ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ、１８０（１）：５９−６６（２００２）、Ｐｏｔｔｅｒ等、Ｍｉｃｒｏｂｉｏｌｏｇｙ、１５１：８２５（２００５））によっても開示されている。しかし、この報告では、ワーテルシア・ユートロファｐｈｂＣ遺伝子破壊株や不活性株をＰＨＡ合成酵素遺伝子発現ベクターの宿主株として用いてはおらず、その生産性等は、ＰＨＢ−４株を宿主とした場合との優劣が不明であった。 Site-specific mutations of chromosomal genes in Watercia Eutropha are described in York et al., Potter et al. (York et al., Journal of Bacteriology, 180 (1): 59-66 (2002), Potter et al., Microbiology, 151: 825 (2005)). Is also disclosed. However, this report does not use a Watersia Eutropha phbC gene disruption strain or an inactive strain as a host strain for the PHA synthase gene expression vector, and its productivity is the same as that when the PHB-4 strain is used as a host. The superiority or inferiority was unknown.
そこで本発明者等は、ワーテルシア・ユートロファ Ｈ１６株の染色体上に存在するＰ（３ＨＢ）合成酵素遺伝子ｐｈｂＣを塩基配列の相同組換えを用いて部位特異的に破壊し、Ｐ（３ＨＢ）非生産という性質のみが野生株と異なる宿主用株を種々作製した。
これらのｐｈｂＣ遺伝子破壊株を宿主とし、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）合成酵素遺伝子発現プラスミドを導入することにより、従来よりも飛躍的に生産性が向上することを見出し、本発明を完成するに至った。
Therefore, the inventors of the present invention called the P (3HB) non-production by destroying the P (3HB) synthase gene phbC present on the chromosome of the Watercia Eutropha H16 strain in a site-specific manner using homologous recombination of the base sequence. Various host strains were produced which differed only in nature from the wild strain.
In order to complete the present invention, it has been found that by using these phbC gene-disrupted strains as hosts and introducing a P (3HB-co-3HH) synthase gene expression plasmid, the productivity can be dramatically improved as compared with the prior art. It came.
即ち、第一の本発明は、
（１）部位特異的変異により微生物が本来有しているポリヒドロキシアルカン酸合成酵素遺伝子のみを不活性化した微生物を宿主として用い、外因性のポリヒドロキシアルカン酸合成酵素遺伝子を導入して得られた形質転換体を培養することを特徴とする生分解性ポリエステルの製造方法、
（２）生分解性ポリエステルが３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸とのモノマー単位で構成されることを特徴とする上記記載の製造方法、
（３）微生物がワーテルシア・ユートロファである上記記載の製造方法、
（４）微生物がワーテルシア・ユートロファ Ｈ１６株である上記記載の製造方法、
（５）宿主が配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子内部の塩基を欠失、置換又は挿入した変異株である上記記載の製造方法、
（６）宿主が配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子内部のＢｇｌＩＩからＢｃｌＩ間を欠失させた株である上記記載の製造方法、
（７）宿主が配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子内部のＳａｃＩからＳａｃＩ間を欠失させた株である上記記載の製造方法、
（８）宿主が配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子の３１９番目のアミノ酸であるシステインをアラニンに変異させた株である上記記載の製造方法、
（９）宿主が配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子の１０７番目のアミノ酸であるトリプトファンをコードするコドンをストップコドンに変異させた株である上記記載の製造方法、
（１０）宿主が配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子を完全に欠失させた株である上記記載の製造方法、
に関する。
第二の本発明は、
（１１）配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子のＢｇｌＩＩからＢｃｌＩ間を欠失させることによってポリヒドロキシアルカン酸非生産性となった変異株、
（１２）配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子のＳａｃＩからＳａｃＩ間を欠失させることによってポリヒドロキシアルカン酸非生産性となった変異株、
（１３）配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子の１０７番目のアミノ酸であるトリプトファンをコードするコドンをストップコドンに変異させることによってポリヒドロキシアルカン酸非生産性となった変異株、
（１４）上記記載の変異株を用いて得られた形質転換体、
に関する。
第三の本発明は、
（１５）上記記載の変異株を宿主として用い、外因性のポリヒドロキシアルカン酸合成酵素遺伝子を導入して得られた形質転換体を培養することを特徴とする生分解性ポリエステルの製造方法、
（１６）生分解性ポリエステルが３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸をモノマー単位で構成されることを特徴とする上記記載の生分解性ポリエステルの製造方法、
に関する。
That is, the first invention is
(1) Obtained by introducing an exogenous polyhydroxyalkanoate synthase gene using as a host a microorganism in which only the polyhydroxyalkanoate synthase gene originally possessed by the microorganism is inactivated by site-specific mutation. A method for producing a biodegradable polyester, characterized by culturing the transformed product,
(2) The production method as described above, wherein the biodegradable polyester is composed of monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid,
(3) The production method as described above, wherein the microorganism is Watercia eutropha,
(4) The production method as described above, wherein the microorganism is Watersia eutropha H16 strain,
(5) The production method according to the above, wherein the host is a mutant strain in which the base in the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24 is deleted, substituted or inserted
(6) The production method according to the above, wherein the host is a strain in which a portion between BglII and BclI in the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24 is deleted.
(7) The production method according to the above, wherein the host is a strain in which a portion between SacI and SacI in the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24 is deleted.
(8) The production method according to the above, wherein the host is a strain obtained by mutating cysteine, which is the 319th amino acid of the polyhydroxyalkanoic acid synthase gene shown in SEQ ID NO: 24, to alanine,
(9) The production method according to the above, wherein the host is a strain in which a codon encoding tryptophan which is the 107th amino acid of the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24 is mutated to a stop codon,
(10) The production method according to the above, wherein the host is a strain in which the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24 is completely deleted,
About.
The second aspect of the present invention is
(11) A mutant strain that has become non-productive in polyhydroxyalkanoate by deleting between BglII and BclI of the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24,
(12) a mutant strain that has become non-productive in polyhydroxyalkanoate by deleting between SacI and SacI of the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24,
(13) A mutant strain that has become non-productive in polyhydroxyalkanoate by mutating a codon encoding tryptophan, which is the 107th amino acid of the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24, to a stop codon,
(14) A transformant obtained using the mutant strain described above,
About.
The third aspect of the present invention is
(15) A method for producing a biodegradable polyester, characterized by culturing a transformant obtained by introducing an exogenous polyhydroxyalkanoate synthase gene using the mutant strain described above as a host,
(16) The method for producing a biodegradable polyester as described above, wherein the biodegradable polyester is composed of monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid,
About.
以下に本発明を詳細に説明する。
本発明における生分解性ポリエステルとは、以下の一般式：
The present invention is described in detail below.
The biodegradable polyester in the present invention has the following general formula:
（式中、Ｒ１及びＲ２は炭素数１以上であるアルキル基を表し、ポリマー中同一でも異なっていてもよい。ｍおよびｎは該ポリマーを構成するモノマー単位数を表し、１以上の整数である）で表される３−ヒドロキシアルカン酸をモノマー単位とする重合体であり、３−ヒドロキシ酪酸、３−ヒドロキシヘキサン酸、３−ヒドロキシヘプタン酸、３−ヒドロキシオクタン酸、３−ヒドロキシノナン酸、３−ヒドロキシデカン酸、３−ヒドロキシウンデカン酸、３−ヒドロキシドデカン酸、３−ヒドロキシトリデカン酸、３−ヒドロキシテトラデカン酸、３−ヒドロキシペンタデカン酸、３−ヒドロキシヘキサデカン酸からなる群より選択されるモノマー単位の２種以上から構成される共重合ポリマーが好ましい。より好ましくは、３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸とのモノマー単位で構成される共重合ポリマーであるＰ（３ＨＢ−ｃｏ−３ＨＨ）である。 (In the formula, R 1 and R 2 represent an alkyl group having 1 or more carbon atoms, and may be the same or different in the polymer. M and n represent the number of monomer units constituting the polymer and are integers of 1 or more. In which 3-hydroxyalkanoic acid is a monomer unit, 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid , 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytridecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, 3-hydroxyhexadecanoic acid Copolymers composed of two or more monomer units are preferred. More preferred is P (3HB-co-3HH), which is a copolymer polymer composed of monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.
本発明で用いる宿主用微生物は、ＰＨＡ合成酵素遺伝子を生物種として原初的にその染色体ＤＮＡ等に有する微生物であれば特に制限なく使用することができ、別の炭素源が利用できるように改変された微生物、基質モノマーの生成や取り込み能が改変された微生物、または生産を増大するように改変された微生物を含む。例としては、Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ（分類学上、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ、Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒと同一）、Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ、Ａｌｃａｌｉｇｅｎｅｓ ｌａｔｕｓ、Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ、Ｐｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ等の細菌類が含まれる。安全性及び生産性の観点から好ましくはＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａであり、より好ましくはＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株である。 The host microorganism used in the present invention can be used without particular limitation as long as it is a microorganism originally having a PHA synthase gene in its chromosomal DNA as a biological species, and has been modified so that another carbon source can be used. Microorganisms that have been modified in their ability to produce and incorporate substrate monomers, or microorganisms that have been modified to increase production. Examples include Waterusia eutropha (taxonomically identical to Ralstonia eutropha, Cupriavidus necator), Aeromonas cavaeae, Alcaligenes latus, Pseudomonas aeruginosa eusoidosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, and Pseudomonas aeruginosa. From the viewpoint of safety and productivity, Waterseutraeupha is preferable, and Watersia eutropha H16 strain is more preferable.
染色体上の遺伝子を部位特異的に変異する方法は当業者に周知である。代表的な方法としてはトランスポゾンと相同組換えの機構を利用した方法（Ｏｈｍａｎ等、Ｊ．Ｂａｃｔｅｒｉｏｌ．，１６２：１０６８−１０７４（１９８５））や相同組換えの機構によって起こる部位特異的な組み込みと第二段階の相同組換えによる脱落を原理とした方法（Ｎｏｔｉ等、Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ．，１５４：１９７−２１７（１９８７））などがあり、また、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ由来のｓａｃＢ遺伝子を共存させて、第二段階の相同組換えによって遺伝子が脱落した微生物株をシュークロース耐性株として容易に単離する方法（Ｓｃｈｗｅｉｚｅｒ、Ｍｏｌ．Ｍｉｃｒｏｂｉｏｌ．，６：１１９５−１２０４（１９９２）、Ｌｅｎｚ等、Ｊ．Ｂａｃｔｅｒｉｏｌ．，１７６：４３８５−４３９３（１９９４））も利用することができるが、染色体上のＰＨＡ合成酵素遺伝子を不活性化できればその方法は特に制限されない。 Methods for site-specific mutation of genes on chromosomes are well known to those skilled in the art. Typical methods include transposon and homologous recombination mechanisms (Ohman et al., J. Bacteriol., 162: 1068-1074 (1985)) and site-specific integration and homologous recombination mechanisms. There are methods based on the principle of elimination by two-stage homologous recombination (Noti et al., Methods Enzymol., 154: 197-217 (1987)), and the second stage in which a sacB gene derived from Bacillus subtilis coexists. A method of easily isolating a microbial strain from which a gene has been dropped by homologous recombination as a sucrose resistant strain (Schweizer, Mol. Microbiol., 6: 1955-1204 (1992), Lenz et al., J. Bacteriol., 176: 4385-4393 (1994)) can also be used, but the method is not particularly limited as long as the PHA synthase gene on the chromosome can be inactivated.
以下に、ＰＨＡ合成酵素遺伝子（ｐｈｂＣ）を特異的に不活性化する方法を、Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株を例に挙げてより具体的に例示する。
まず、変異断片を作製する。Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株のＰＨＡ合成酵素遺伝子はこれまで多数の研究者により解析され、その３１９番目のアミノ酸であるシステイン残基（以下Ｃ３１９）が活性中心であることが明らかになっている（Ｙｏｒｋ等、Ｊｏｕｒｎａｌ ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ、１８０（１）：５９−６６（２００２））。従って、このＰＨＡ合成酵素遺伝子を不活性化するには、配列番号２４に示すポリヒドロキシアルカン酸合成酵素遺伝子内部の塩基を欠失、置換又は挿入すること、より具体的には、Ｃ３１９を含む適当な遺伝子断片を欠失させること、塩基の挿入や欠失によって以後のアミノ酸配列を変化させるフレームシフトを起こさせること、又は３１９番目のアミノ酸であるシステインを別のアミノ酸に変異させること（例えば、システインをアラニンに変異させるＣ３１９Ａ変異）等、ＰＨＡ合成酵素活性発現に重要なアミノ酸残基を置換させること等が挙げられる。すなわち、例えばＰＨＡ合成酵素活性中心部分の塩基配列を含まないＤＮＡ断片との置換や、配列番号２４で示すＤＮＡの制限酵素ＢｇｌＩＩ部位−ＢｃｌＩ部位間の欠失、制限酵素ＳａｃＩ部位間の欠失、或いはストップコドンの導入や配列番号２４の完全な欠失等によりＰＨＡ合成酵素遺伝子を不活性化することができる。また、染色体上に存在する配列番号２４の約１０塩基上流のリボソーム結合配列（ＳＤ配列）を改変することによってもＰＨＡ合成酵素遺伝子の不活性化は可能である。
配列番号２４で示すＤＮＡにおいて、制限酵素ＢｇｌＩＩ部位は１９５番目のアミノ酸辺りに、ＢｃｌＩ部位は５１３番目のアミノ酸辺りに存在する。また、ＳａｃＩ部位は２６０番目のアミノ酸辺りと４５０番目のアミノ酸辺りに存在する。
上記ストップコドンの導入は、例えば、配列番号２４で示すアミノ酸配列の１０７番目のアミノ酸であるトリプトファンをコードするコドンをストップコドンに変異させること（Ｗ１０７＊変異）等が挙げられる。
Hereinafter, a method for specifically inactivating the PHA synthase gene (phbC) will be more specifically exemplified by taking the Watersia eutropha H16 strain as an example.
First, a mutant fragment is prepared. A number of researchers have analyzed the PHA synthase gene of the Watersia eutropha H16 strain, and it has been revealed that the 319th amino acid cysteine residue (hereinafter C319) is the active center (York et al., Journal of Bacteriology, 180 (1): 59-66 (2002)). Therefore, in order to inactivate this PHA synthase gene, the base in the polyhydroxyalkanoate synthase gene shown in SEQ ID NO: 24 is deleted, substituted or inserted, more specifically, an appropriate one containing C319. Deleting a gene fragment, causing a frame shift that changes the subsequent amino acid sequence by insertion or deletion of a base, or mutating cysteine, which is the 319th amino acid, to another amino acid (for example, cysteine Substitution of amino acid residues important for expression of PHA synthase activity, etc. That is, for example, replacement with a DNA fragment not containing the base sequence of the PHA synthase active center portion, deletion between the restriction enzyme BglII site and BclI site of the DNA shown in SEQ ID NO: 24, deletion between the restriction enzyme SacI sites, Alternatively, the PHA synthase gene can be inactivated by introduction of a stop codon, complete deletion of SEQ ID NO: 24, or the like. In addition, the PHA synthase gene can be inactivated by modifying a ribosome binding sequence (SD sequence) about 10 bases upstream of SEQ ID NO: 24 present on the chromosome.
In the DNA represented by SEQ ID NO: 24, the restriction enzyme BglII site exists around the 195th amino acid, and the BclI site exists around the 513th amino acid. The SacI site is present around the 260th amino acid and around the 450th amino acid.
Examples of the introduction of the stop codon include mutating a codon encoding tryptophan, which is the 107th amino acid of the amino acid sequence shown in SEQ ID NO: 24, to a stop codon (W107 * mutation).
変異断片の上流配列と下流配列は、染色体上の遺伝子と相同組換えを起こすために必要な相同配列であって、一般的にはその長さが長いほど組換え頻度は高くなるが、相同組換えさえ起こればよく、その長さは任意に設定できる。上記相同配列は、通常５０ｂｐ以上、好ましくは５００ｂｐ以上あればよい。 The upstream and downstream sequences of the mutant fragment are homologous sequences necessary for homologous recombination with the gene on the chromosome. Generally, the longer the length, the higher the frequency of recombination. All that needs to be changed is its length, which can be set arbitrarily. The homologous sequence is usually 50 bp or more, preferably 500 bp or more.
変異断片には遺伝子置換の際に選択マーカーとなる遺伝子を付加することができる。選択マーカーとなる遺伝子は例えばカナマイシン、クロラムフェニコール、ストレプトマイシン、アンピシリン等の抗生物質の耐性遺伝子や各種の栄養要求性を相補する遺伝子等が使用できる。Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株で行う場合にはカナマイシンの耐性遺伝子が好適である。
さらにそれらに加えて、第二段階の相同組み換えによってマーカー遺伝子が脱落した微生物株の選択を容易にするための遺伝子が付加できる。そのような遺伝子としてはＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ由来のｓａｃＢ遺伝子が例示できる（Ｓｃｈｗｅｉｚｅｒ、Ｍｏｌ．Ｍｉｃｒｏｂｉｏｌ．，６：１１９５−１２０４（１９９２））。この遺伝子が発現している微生物株はシュークロースを含む培地で生育できないことが知られており、シュークロースを含む培地での生育により、第二段階の相同的組換えによってこの遺伝子を失った株を選択することが容易となる。
A gene serving as a selection marker at the time of gene replacement can be added to the mutant fragment. As a gene serving as a selection marker, for example, an antibiotic resistance gene such as kanamycin, chloramphenicol, streptomycin, ampicillin, a gene that complements various auxotrophy, and the like can be used. A kanamycin resistance gene is preferred when the assay is performed using the Watersia eutropha H16 strain.
In addition to these, a gene for facilitating the selection of a microbial strain from which the marker gene has been eliminated by the second-stage homologous recombination can be added. An example of such a gene is a sacB gene derived from Bacillus subtilis (Schweizer, Mol. Microbiol., 6: 1195-1204 (1992)). Microbial strains expressing this gene are known to be unable to grow on medium containing sucrose, and have lost this gene due to second-stage homologous recombination due to growth on medium containing sucrose. It becomes easy to select.
これらで構成された変異断片は、宿主微生物株中で自律複製不可能なベクターに接続することによって遺伝子変異用のプラスミドが作製される。ワーテルシア属やシュードモナス属等で利用できるこのようなベクターには、例えば、ｐＵＣベクター、ｐＢｌｕｅｓｃｒｉｐｔベクター、ｐＢＲ３２２ベクターあるいはそれらと同じ複製起点を持つベクター等が挙げられる。さらには、接合伝達を可能にするｍｏｂ、ｏｒｉＴなどのＤＮＡ配列を共存させることも可能である。 The mutant fragment composed of these is connected to a vector that cannot autonomously replicate in the host microorganism strain to produce a plasmid for gene mutation. Examples of such vectors that can be used in the genus Watersia and Pseudomonas include, for example, pUC vectors, pBluescript vectors, pBR322 vectors, and vectors having the same origin of replication. Furthermore, it is possible to coexist DNA sequences such as mob and oriT that enable junction transfer.
このような構成で作製された遺伝子変異用のプラスミドは、エレクトロポレーション法や接合伝達法などの公知の方法によりＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株に導入することができる。
次に、相同組換えによって染色体上にプラスミドが挿入された株の選択を行う。選択は遺伝子変異用プラスミドに共存させた選択用の遺伝子に基づいた方法によって行うことができる。カナマイシン耐性遺伝子を用いた場合にはカナマイシンを含む培地で生育してきた株から選ぶことができる。
The gene mutation plasmid produced in such a configuration can be introduced into the Watersuteria europa strain H16 by known methods such as electroporation and conjugation transfer.
Next, a strain having a plasmid inserted on the chromosome by homologous recombination is selected. The selection can be performed by a method based on the gene for selection coexisting with the gene mutation plasmid. When a kanamycin resistance gene is used, it can be selected from strains grown in a medium containing kanamycin.
次の段階で第二の相同組換えによって染色体上からマーカー遺伝子を含む領域が脱落した株を選択する。挿入時に利用した選択用の遺伝子に基づいて、例えばカナマイシンを含む培地で生育できなくなった株を選択してもよいが、ｓａｃＢ遺伝子を遺伝子変異用プラスミドに共存させている場合は、シュークロースを含む培地で生育してくる株から容易に選択できる。遺伝子が変異していることの確認には、ＰＨＡ生産能の有無を簡易的に調べるＮｉｌｅｒｅｄ染色法、ＰＣＲ法、サザンハイブリダイゼーション法、ＤＮＡ塩基配列の決定など、公知の方法が使用できる。以上のようにしてＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株のＰＨＡ合成酵素遺伝子（ｐｈｂＣ）が不活性化された株を取得することができる。 In the next step, a strain in which the region containing the marker gene has been removed from the chromosome by the second homologous recombination is selected. For example, a strain that can no longer grow on a medium containing kanamycin may be selected based on the selection gene used at the time of insertion, but if the sacB gene coexists in the gene mutation plasmid, sucrose is included. It can be easily selected from strains that grow on the medium. For confirming that the gene has been mutated, known methods such as the Nilered staining method, PCR method, Southern hybridization method, and determination of the DNA base sequence for simply examining the presence or absence of PHA production ability can be used. As described above, a strain in which the PHA synthase gene (phbC) of the Watersia eutropha H16 strain is inactivated can be obtained.
次に、上記の方法等により作製した宿主用株にＰＨＡ合成酵素発現プラスミドを導入する方法を説明する。
本発明で用いる外因性のＰＨＡ合成酵素遺伝子は各種のＰＨＡ蓄積生物に由来する遺伝子のうち生分解性ポリエステルを蓄積させるものであればどのようなものでもよい。そのような遺伝子としては例えば、Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ（非特許文献９）、Ｎｏｃａｒｄｉａ ｃｏｒａｌｌｉｎａ（ＧｅｎＢａｎｋアクセッション番号ＡＦ０１９９６４）、Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ（Ｔｉｍｍ 等、Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．、２０９：１５−３０（１９９２））、Ｐｓｅｕｄｏｍｏｎａｓ ｏｌｅｏｖｏｒａｎｓ（Ｈｕｉｓｍａｎ等、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６６：２１９１−２１９８（１９９１））、Ｔｈｉｏｃｙｓｔｉｓ ｖｉｏｌａｃｅａｅ（Ｌｉｅｂｅｒｇｅｓｅｌｌ等、Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．Ｂｉｏｔｅｃｈｎｏｌ．，３８：４９３−５０１（１９９３））などから単離されているＰＨＡ合成酵素遺伝子がある。
Next, a method for introducing a PHA synthase expression plasmid into a host strain prepared by the above method will be described.
The exogenous PHA synthase gene used in the present invention may be any gene that accumulates biodegradable polyester among genes derived from various PHA accumulating organisms. Examples of such genes include Aeromonas caviae (Non-Patent Document 9), Nocardia corallina (GenBank accession number AF01964), Pseudomonas aeruginosa (Timm et al., Eur. J. Biochem., 209: 15-30 (1992), Pseudomonas oleovarans (Huisman et al., J. Biol. Chem., 266: 2191-2198 (1991)), Thiocystis violaceae (Liebergesell et al., Appl. Microbiol. Biotechnol., 19: 93). There are PHA synthase genes.
また、目的とする酵素活性が失われない範囲内でアミノ酸配列が改変するように、ＰＨＡ合成酵素遺伝子の塩基配列の一部を改変したものも使用することができる。例えば、非特許文献１３に記載されている、１４９番目のアミノ酸のアスパラギンがセリンに置換されたアエロモナス・キャビエ由来であるＰＨＡ合成酵素遺伝子（Ｎ１４９Ｓ変異体遺伝子）、１７１番目のアミノ酸のアスパラギン酸がグリシンに置換されたアエロモナス・キャビエ由来であるＰＨＡ合成酵素遺伝子（Ｄ１７１Ｇ変異体遺伝子）、または、上記の２つのアミノ酸置換が組み合わされたアエロモナス・キャビエ由来であるＰＨＡ合成酵素遺伝子等を好ましく用いることができる。上記の遺伝子はプロモーターおよび／またはリボソーム結合部位を有し得るが、必ずしも必要ではない。 Moreover, what modified a part of base sequence of a PHA synthetase gene so that an amino acid sequence may modify | change within the range in which the target enzyme activity is not lost can also be used. For example, as described in Non-Patent Document 13, the PHA synthase gene (N149S mutant gene) derived from Aeromonas caviae in which the asparagine of the 149th amino acid is substituted with serine, the aspartic acid of the 171st amino acid is glycine PHA synthase gene (D171G mutant gene) derived from Aeromonas caviae substituted with the above, or the PHA synthase gene derived from Aeromonas caviae combined with the above two amino acid substitutions can be preferably used. . The above genes may have promoters and / or ribosome binding sites, but are not necessary.
ＰＨＡ合成酵素遺伝子を発現させるベクターはそれぞれ宿主内で自律的複製が可能なベクターであれば、どのようなベクターであっても使用することができる。例えば広宿主域ベクターであるｐＪＲＤ２１５やｐＢＢＲ１やそれらの誘導体を用いることができる。
本発明において、ＰＨＡ合成酵素遺伝子を発現させるベクターが宿主内で安定して保持されるように、ｐａｒ領域を含有させてもよい。
また、ＰＨＡ合成酵素遺伝子を発現ベクターで導入する代わりに、ｐｈｂＣ遺伝子を不活性化した株の染色体、メガプラスミド等に前述の相同組換えの機構を利用して外因性のＰＨＡ合成酵素遺伝子を組み込むことも可能である。
Any vector can be used as the vector for expressing the PHA synthase gene as long as it can replicate autonomously in the host. For example, pJRD215, pBBR1, and their derivatives, which are broad host range vectors, can be used.
In the present invention, a par region may be included so that the vector for expressing the PHA synthase gene is stably maintained in the host.
Also, instead of introducing the PHA synthase gene with an expression vector, the exogenous PHA synthase gene is incorporated into the chromosome, megaplasmid, etc. of the strain in which the phbC gene is inactivated using the above-mentioned homologous recombination mechanism. It is also possible.
発現ベクターは、エレクトロポレーション法や接合伝達法などの公知の方法により宿主用株に導入することができる。
選択マーカーとしては例えばカナマイシン、クロラムフェニコール、ストレプトマイシン、アンピシリン等の抗生物質の耐性遺伝子や各種の栄養要求性を相補する遺伝子等が使用できる。ｐＪＲＤ２１５やｐＢＢＲ１を用いた発現ベクターをＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａに導入する場合には、カナマイシンの耐性遺伝子が好適である。
ポリエステル生産確認の簡易法としては、Ｎｉｌｅｒｅｄを用いた染色法を利用できる。すなわち、組換え菌が生育する寒天培地にＮｉｌｅｒｅｄを加え、組換え菌を１〜７日間培養し、組換え菌が赤変するか否かを観察することにより、ポリエステル生産の有無を確認できる。
The expression vector can be introduced into the host strain by a known method such as electroporation or conjugation transfer.
As the selection marker, for example, resistance genes of antibiotics such as kanamycin, chloramphenicol, streptomycin, ampicillin, genes complementary to various auxotrophy, and the like can be used. When an expression vector using pJRD215 or pBBR1 is introduced into Watersia eutropha, a kanamycin resistance gene is preferred.
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.
本発明において、本発明の微生物を炭素源存在下で増殖させることにより、微生物体内に生分解性ポリエステルを蓄積させることができる。炭素源としては、糖、油脂または脂肪酸等を用いることができる。炭素源以外の栄養源として、窒素源、無機塩類、そのほかの有機栄養源を任意に用いることができる。
糖としては、例えばシュークロース、フラクトース等の炭水化物が挙げられる。油脂としては、炭素数が１０以上である飽和・不飽和脂肪酸を多く含む油脂、例えばヤシ油、パーム油、パーム核油等が挙げられる。脂肪酸としては、ヘキサン酸、オクタン酸、デカン酸、ラウリン酸、オレイン酸、パルミチン酸、リノール酸、リノレン酸、ミリスチン酸等の飽和・不飽和脂肪酸、あるいはこれら脂肪酸のエステルや塩等の脂肪酸誘導体が挙げられる。
窒素源としては、例えばアンモニア、塩化アンモニウム、硫酸アンモニウム、リン酸アンモニウム等のアンモニウム塩の他、ペプトン、肉エキス、酵母エキス等が挙げられる。
無機塩類としては、例えばリン酸水素二カリウム、リン酸二水素カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム等が挙げられる。
そのほかの有機栄養源としては、例えばグリシン、アラニン、セリン、スレオニン、プロリン等のアミノ酸；ビタミンＢ１、ビタミンＢ１２、ビタミンＣ等のビタミン等が挙げられる。
In the present invention, the biodegradable polyester can be accumulated in the microorganism by growing the microorganism of the present invention in the presence of a carbon source. As the carbon source, sugar, fat or fatty acid, or the like can be used. Nitrogen sources, inorganic salts, and other organic nutrient sources can be arbitrarily used as nutrient sources other than the carbon source.
Examples of the sugar include carbohydrates such as sucrose 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 dipotassium hydrogen phosphate, potassium dihydrogen 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.
培養温度は、その菌が生育可能な温度であればよいが、２０℃から４０℃が好ましい。培養時間は、特に制限はないが、１〜１０日間程度で良い。
得られた該培養菌体に蓄積されたポリエステルは公知の方法により回収することができる。例えば次のような方法により行うことができる。培養終了後、培養液から遠心分離器等で菌体を分離し、その菌体を蒸留水およびメタノール等により洗浄し、乾燥させる。この乾燥菌体から、クロロホルム等の有機溶剤を用いてＰＨＡを抽出する。このＰＨＡを含んだ有機溶剤溶液から、濾過等によって菌体成分を除去し、そのろ液にメタノールやヘキサン等の貧溶媒を加えてＰＨＡを沈殿させる。さらに、濾過や遠心分離によって上澄み液を除去し、乾燥させてＰＨＡを回収することができる。
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.
The polyester accumulated in the cultured cells thus obtained can be recovered by a known method. For example, it can be performed by 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. PHA is extracted from the dried cells using an organic solvent such as chloroform. Cellular components are removed from the organic solvent solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. Furthermore, the supernatant can be removed by filtration or centrifugation and dried to recover PHA.
本発明によれば、産業用発酵課程においてポリヒドロキシアルカン酸（ＰＨＡ）を高生産できる組換え微生物株が提供され、高純度のポリヒドロキシアルカン酸（ＰＨＡ）が簡便かつ大量に、しかも安価に製造することが可能となる。 According to the present invention, a recombinant microbial strain capable of producing polyhydroxyalkanoic acid (PHA) at a high rate in an industrial fermentation process is provided, and high-purity polyhydroxyalkanoic acid (PHA) can be produced easily, in large quantities, and at low cost. It becomes possible to do.
以下に実施例で本発明を詳細に説明するが、本発明はこれら実施例によって何ら制限されるものではない。なお全体的な遺伝子操作は、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ（Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ、（１９８９））に記載されているように行うことができる。また、遺伝子操作に使用する酵素、クローニング宿主等は、市場の供給者から購入し、その説明に従い使用することができる。なお、酵素としては、遺伝子操作に使用できるものであれば特に限定されない。 EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. The 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.
（実施例１）遺伝子置換用プラスミドの作製
ｐＵＣ１９に新たに制限酵素部位を付与するため、配列番号１の合成ＤＮＡを用いてＨｉｎｄＩＩＩ部位をＮｏｔＩ部位に、更に配列番号２と配列番号３の合成ＤＮＡを用いてＳａｃＩ部位をＳｗａＩに変換したベクターｐＮＳ２Ｘを作製した。続いて、プラスミドｐＭＴ５０７１（Ｔｓｕｄａ、ＧＥＮＥ，２０７：３３−４１（１９９８））を制限酵素ＢａｍＨＩで処理してクロラムフェニコール耐性遺伝子を除去し、代わりにｐＪＲＤ２１５由来のカナマイシン耐性遺伝子を配列番号４と配列番号５を用いてＢａｍＨＩ断片としてＰＣＲ増幅させた断片を挿入してｐＭＴ５０７１−Ｋｍを得た。このプラスミドをＮｏｔＩで切断し、ｏｒｉＴ＋Ｋｍ＋ｓａｃＢ遺伝子を含む約５．７ｋｂのＤＮＡ断片を単離し、ｐＮＳ２ＸのＮｏｔＩ部位に挿入してｐＮＳ２Ｘ−ｓａｃＢを作製した。
(Example 1) Preparation of a gene replacement plasmid In order to newly give a restriction enzyme site to pUC19, a synthetic DNA of SEQ ID NO: 1 was used, a HindIII site was changed to a NotI site, and further, synthetic DNAs of SEQ ID NO: 2 and SEQ ID NO: 3 Was used to prepare a vector pNS2X in which the SacI site was converted to SwaI. Subsequently, the plasmid pMT5071 (Tsuda, GENE, 207: 33-41 (1998)) was treated with the restriction enzyme BamHI to remove the chloramphenicol resistance gene. Instead, the kanamycin resistance gene derived from pJRD215 was replaced with SEQ ID NO: 4 A fragment amplified by PCR as a BamHI fragment using SEQ ID NO: 5 was inserted to obtain pMT5071-Km. This plasmid was cleaved with NotI, and a DNA fragment of about 5.7 kb containing the oriT + Km + sacB gene was isolated and inserted into the NotI site of pNS2X to prepare pNS2X-sacB.
（実施例２）ｐｈｂＣ遺伝子を含む断片のクローニング
Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株の染色体上のｐｈｂＣ遺伝子を不活性化させるため、まずその上流（プロモーター側）及び下流（ｐｈｂＡ側）遺伝子を含むｐｈｂＣＡ（Ｓｗａ）断片３．１ｋｂをクローニングした。染色体ＤＮＡを鋳型とし、配列番号６と配列番号７で示されるプライマーを用いてＰＣＲ反応を行った。ＰＣＲ条件は（１）９５℃で２分、（２）９６℃で１０秒、（３）５５℃で１０秒、（４）６８℃で３分、（２）から（４）を２５サイクル、（５）６８℃で５分であり、ポリメラーゼとしてはＴａＫａＲａ ＬＡ Ｔａｑ（タカラバイオ製）を用いた。得られた断片をｐＮＳ２ＸのＳｗａＩ部位にサブクローニングしｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｓｗａ）を作製した。
Example 2 Cloning of Fragment Containing phbC Gene In order to inactivate the phbC gene on the chromosome of Watersuteria europa strain H16, first, a phbCA (Swa) fragment containing upstream (promoter side) and downstream (phbA side) genes 3.1 kb was cloned. PCR reaction was performed using the chromosomal DNA as a template and the primers shown in SEQ ID NO: 6 and SEQ ID NO: 7. PCR conditions were (1) 95 ° C for 2 minutes, (2) 96 ° C for 10 seconds, (3) 55 ° C for 10 seconds, (4) 68 ° C for 3 minutes, (2) to (4) 25 cycles, (5) 5 minutes at 68 ° C., and TaKaRa LA Taq (manufactured by Takara Bio Inc.) was used as the polymerase. The obtained fragment was subcloned into the SwaI site of pNS2X to prepare pNS2X-phbCA (Swa).
（実施例３）ｐｈｂＣ不活性化断片の調製
前述したようにｐｈｂＣ酵素の活性中心は３１９番目のアミノ酸であるシステインとされている。従って不活性化は、１）ＢｇｌＩＩからＢｃｌＩ間の欠失、２）ＳａｃＩ間の欠失、３）Ｃ３１９Ａ変異導入、４）Ｗ１０７＊変異導入、５）ｐｈｂＣ遺伝子の完全欠失、の５種類で行った。以下にそれぞれの作製方法を示す。
１）ＢｇｌＩＩ及びＢｃｌＩ間の欠失
ｐｈｂＣ遺伝子の中で、ＢｇｌＩＩ部位は１９５番目のアミノ酸辺りに、またＢｃｌＩ部位は５１３番目のアミノ酸辺りに存在する。従って実施例２のｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｓｗａ）をＢｇｌＩＩ及びＢｃｌＩで切断し、そのまま連結反応を行って中間部分が欠失したクローンｐＮＳ２Ｘ−ｐｈｂＣＡ（ΔＢ）を得た。
２）ＳａｃＩ間の欠失
ｐｈｂＣ遺伝子の中でＳａｃＩ部位は２６０番目のアミノ酸辺りと４５０番目のアミノ酸辺りに存在する。従って実施例２のｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｓｗａ）をＳａｃＩで切断し、そのまま連結反応を行って中間部分が欠失したクローンｐＮＳ２Ｘ−ｐｈｂＣＡ（ΔＳ）を得た。
３）Ｃ３１９Ａ変異導入
Ｙｏｒｋ等によれば、３１９番目のアミノ酸であるシステインをアラニンに変換するとｐｈｂＣ酵素活性が消失する。従って、配列番号６と配列番号８を用いてｐｈｂＣＡ（Ｓｗａ）の前半部分を増幅させ、配列番号９と配列番号７を用いてｐｈｂＣＡ（Ｓｗａ）の後半部分を増幅させた。その後、２つの増幅断片を混合し、オーバーラップＰＣＲを行った。増幅した断片をＳｗａＩで切断し、ｐＮＳ２ＸのＳｗａＩ部位に挿入してｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｃ３１９Ａ）を得た。
４）Ｗ１０７＊変異導入
Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａのＰ（３ＨＢ）非生産変異株であるＰＨＢ−４株のｐｈｂＣ遺伝子の塩基配列を決定したところ、１０７番目のアミノ酸であるトリプトファン（ＴＧＧ）がストップコドン（ＴＡＧ）に置換されており、その後の蛋白合成が行われていない可能性が高いことが分かった。従ってこの変異と同じ変異を導入することによってｐｈｂＣ遺伝子の不活性化を行った。すなわち、ＰＨＢ−４株の染色体ＤＮＡを鋳型とし、配列番号６と配列番号７で示したプライマーを用いてＰＣＲを行った。鋳型が異なる点以外は実施例２と同条件である。その後、ＳｗａＩで切断し、ｐＮＳ２ＸのＳｗａＩ部位に挿入してｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｗ１０７＊）を得た。
５）ｐｈｂＣ遺伝子の完全欠失
ｐｈｂＣ遺伝子のＡＴＧコドンから下流側ｐｈｂＡ遺伝子のＡＴＧコドン直前までを正確に欠失させた変異体断片は以下のようにして作製した。配列番号６と配列番号１０でプロモーターを含む断片をＰＣＲで増幅させた。また配列番号１１と配列番号７でｐｈｂＡ遺伝子のＡＴＧコドンからの一部をＰＣＲで増幅させた。それぞれの断片をＳｗａＩ及びＢｓｐＨＩで切断後、ｐＮＳ２ＸのＳｗａＩ部位に挿入してｐＮＳ２Ｘ−ｐｈｂＣＡ（ｄ−ｐｈｂＣ）を得た。
(Example 3) Preparation of inactivated phbC fragment As described above, the active center of the phbC enzyme is cysteine, which is the 319th amino acid. Therefore, there are 5 types of inactivation: 1) deletion between BglII and BclI, 2) deletion between SacI, 3) C319A mutation, 4) W107 * mutation, and 5) complete deletion of phbC gene. went. Each manufacturing method is shown below.
1) In the deleted phbC gene between BglII and BclI, the BglII site exists around the 195th amino acid, and the BclI site exists around the 513th amino acid. Therefore, pNS2X-phbCA (Swa) of Example 2 was cleaved with BglII and BclI, and a ligation reaction was carried out as it was to obtain a clone pNS2X-phbCA (ΔB) from which the intermediate portion was deleted.
2) Deletion between SacI In the phbC gene, a SacI site exists around the 260th amino acid and around the 450th amino acid. Therefore, pNS2X-phbCA (Swa) of Example 2 was cleaved with SacI, and a ligation reaction was carried out as it was to obtain clone pNS2X-phbCA (ΔS) from which the intermediate portion was deleted.
3) According to C319A mutation-introduced York et al., Cysteine, the 319th amino acid, is converted to alanine, and the phbC enzyme activity disappears. Therefore, SEQ ID NO: 6 and SEQ ID NO: 8 were used to amplify the first half of phbCA (Swa), and SEQ ID NO: 9 and SEQ ID NO: 7 were used to amplify the second half of phbCA (Swa). Thereafter, the two amplified fragments were mixed and overlap PCR was performed. The amplified fragment was cut with SwaI and inserted into the SwaI site of pNS2X to obtain pNS2X-phbCA (C319A).
4) When the nucleotide sequence of the phbC gene of PHB-4, which is a P (3HB) non-producing mutant of W107 * mutagenesis Waterteria eutropha, was determined, the 107th amino acid tryptophan (TGG) was the stop codon (TAG). It was found that there is a high possibility that subsequent protein synthesis is not performed. Therefore, the phbC gene was inactivated by introducing the same mutation. That is, PCR was carried out using the chromosomal DNA of the PHB-4 strain as a template and the primers shown in SEQ ID NO: 6 and SEQ ID NO: 7. The conditions are the same as in Example 2 except that the mold is different. Then, it cut | disconnected with SwaI and inserted in the SwaI site | part of pNS2X, and pNS2X-phbCA (W107 *) was obtained.
5) Complete deletion of the phbC gene A mutant fragment in which the portion from the ATG codon of the phbC gene to the portion immediately before the ATG codon of the downstream phbA gene was deleted was prepared as follows. Fragments containing the promoters of SEQ ID NO: 6 and SEQ ID NO: 10 were amplified by PCR. Further, in SEQ ID NO: 11 and SEQ ID NO: 7, a part from the ATG codon of the phbA gene was amplified by PCR. Each fragment was cleaved with SwaI and BspHI and then inserted into the SwaI site of pNS2X to obtain pNS2X-phbCA (d-phbC).
（実施例４）変異ｐｈｂＣ遺伝子導入用プラスミドの構築
実施例３で構築したｐＮＳ２Ｘ−ｐｈｂＣＡ（ΔＢ）、ｐＮＳ２Ｘ−ｐｈｂＣＡ（ΔＳ）、ｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｃ３１９Ａ）、ｐＮＳ２Ｘ−ｐｈｂＣＡ（Ｗ１０７＊）、ｐＮＳ２Ｘ−ｐｈｂＣＡ（ｄ−ｐｈｂＣ）をそれぞれＳｗａＩで処理し、それぞれから変異導入ＳｗａＩ断片を調製した。その断片を実施例１で作製したｐＮＳ２Ｘ−ｓａｃＢのＳｗａＩに挿入し、ｐＮＳ２Ｘ−ｓａｃＢ−ｐｈｂＣＡ（ΔＢ）、ｐＮＳ２Ｘ−ｓａｃＢ−ｐｈｂＣＡ（ΔＳ）、ｐＮＳ２Ｘ−ｓａｃＢ−ｐｈｂＣＡ（Ｃ３１９Ａ）、ｐＮＳ２Ｘ−ｓａｃＢ−ｐｈｂＣＡ（Ｗ１０７＊）、ｐＮＳ２Ｘ−ｓａｃＢ−ｐｈｂＣＡ（ｄ−ｐｈｂＣ）をそれぞれ構築した。
(Example 4) Construction of plasmid for introducing mutant phbC gene pNS2X-phbCA (ΔB), pNS2X-phbCA (ΔS), pNS2X-phbCA (C319A), pNS2X-phbCA (W107 *), pNS2X- constructed in Example 3 Each phbCA (d-phbC) was treated with SwaI, and a mutated SwaI fragment was prepared from each. The fragment was inserted into SwaI of pNS2X-sacB prepared in Example 1, and pNS2X-sacB-phbCA (ΔB), pNS2X-sacB-phbCA (ΔS), pNS2X-sacB-phbCA (C319A), pNS2X-sacB-phbCA. (W107 *) and pNS2X-sacB-phbCA (d-phbC) were constructed.
（実施例５）変異ｐｈｂＣ遺伝子導入株の作製
実施例４で構築した５種類の変異ｐｈｂＣ遺伝子導入用プラスミドで大腸菌Ｓ１７−１株（ＡＴＣＣ４７００５）を形質転換し、Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株とＮｕｔｒｉｅｎｔ Ａｇａｒ培地（Ｄｉｆｃｏ社製）上で混合培養して接合伝達を行った。
２５０ｍｇ／Ｌのカナマイシンを含むシモンズ寒天培地（クエン酸ナトリウム２ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ、硫酸マグネシウム・７水塩０．２ｇ／Ｌ、リン酸二水素アンモニウム１ｇ／Ｌ、リン酸水素二カリウム１ｇ／Ｌ、寒天１５ｇ／Ｌ、ｐＨ６．８）上で生育してきた菌株を選択して、プラスミドがＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株の染色体上に組み込まれた株を取得した。この株をカナマイシンを含まないＮｕｔｒｉｅｎｔ Ｂｒｏｔｈ培地（Ｄｉｆｃｏ社製）で２世代培養した後、１５％のシュークロースを含むＮｕｔｒｉｅｎｔ Ａｇａｒ培地上に希釈して塗布し、生育してきた菌株を選択してカナマイシン感受性且つＰ（３ＨＢ）非生産株を取得した。さらにＰＣＲ或いは塩基配列の解析を行い、ｐｈｂＣ遺伝子が目的通り変異導入されていることを確認した。これにより、ＢｇｌＩＩ及びＢｃｌＩ間の欠失変異株としてΔＢ１１３３株、ＳａｃＩ間の欠失変異株としてΔＳ１３１２３株、Ｃ３１９Ａ変異株としてＣ７株、Ｗ１０７＊変異株としてＰ３株、ｐｈｂＣ遺伝子の完全欠失変異株としてｄ１株をそれぞれ作製した。
(Example 5) Preparation of mutant phbC gene introduction strain E. coli S17-1 strain (ATCC 47005) was transformed with the five types of mutant phbC gene introduction plasmids constructed in Example 4, and the Watersia eutropha H16 strain and the Nutrient Agar medium ( Conjugate transmission was performed by mixed culture on Difco).
Simmons agar medium containing 250 mg / L kanamycin (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8) was selected to obtain a strain in which the plasmid was integrated on the chromosome of the Watersia eutropha H16 strain. This strain was cultured for 2 generations in Nutrient Broth medium (Difco) without kanamycin, then diluted and applied to Nutrient Agar medium containing 15% sucrose, and the grown strain was selected to be sensitive to kanamycin. And the P (3HB) non-production strain was acquired. Furthermore, PCR or nucleotide sequence analysis was performed to confirm that the phbC gene was mutated as intended. As a result, a ΔB1133 strain as a deletion mutant between BglII and BclI, a ΔS13123 strain as a deletion mutant between SacI, a C7 strain as a C319A mutant, a P3 strain as a W107 * mutant, and a complete deletion mutant of the phbC gene D1 strains were prepared respectively.
（実施例６）プラスミドベクターｐＣＵＰ２の作製
本実施例において、ＰＨＡ合成酵素遺伝子を導入するプラスミドベクターとしては、Ｗａｕｔｅｒｓｉａ属細菌にて使用可能なものであれば特に制限はない。本実施例にて作製したプラスミドベクターは、Ｃｕｐｒｉａｖｉｄｕｓ ｍｅｔａｌｌｉｄｕｒａｎｓ ＣＨ３４株が保有するメガプラスミド（ｐＭＯＬ２８）の複製開始領域（配列番号１２）及び配列番号１３に記載のｐａｒ領域を含んでいる。
具体的な作製手順としては、まず、Ｃｕｐｒｉａｖｉｄｕｓ ｍｅｔａｌｌｉｄｕｒａｎｓ ＣＨ３４株からＤＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ｐｒｏｍｅｇａ社製）を使用し、メガプラスミドを含むＤＮＡを調製、このＤＮＡを鋳型に、配列番号１４及び１５に記載のプライマーを用いてＰＣＲ法によって約４ｋｂｐの配列番号１２及び１３の配列を含むＤＮＡ領域を増幅した。ＰＣＲ条件は（１）９８℃で２分、（２）９８℃で３０秒、（３）５５℃で３０秒、（４）７２℃で５分、（２）から（４）を３０サイクル、（５）７２℃で５分であり、ポリメラーゼとしてはＴａＫａＲａ Ｐｙｒｏｂｅｓｔ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）を用いた。増幅断片を大腸菌用のクローニングベクターｐＣＲ−Ｂｌｕｎｔ２−ＴＯＰＯ（Ｉｎｖｉｔｒｏｇｅｎ社製）にクローニングした。
(Example 6) Preparation of plasmid vector pCUP2 In this example, the plasmid vector into which the PHA synthase gene is introduced is not particularly limited as long as it can be used in bacteria belonging to the genus Watersia. The plasmid vector prepared in this example contains the replication initiation region (SEQ ID NO: 12) of the megaplasmid (pMOL28) possessed by Cupriavidus metallidurans strain CH34 and the par region described in SEQ ID NO: 13.
As a specific production procedure, first, DNA Purification Kit (manufactured by Promega) was prepared from Cupriavidus metallidrans CH34 strain, and DNA containing a megaplasmid was prepared. Using this DNA as a template, primers described in SEQ ID NOs: 14 and 15 The DNA region containing the sequences of SEQ ID NOS: 12 and 13 of about 4 kbp was amplified by PCR. PCR conditions are (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 30 seconds, (3) 55 ° C. for 30 seconds, (4) 72 ° C. for 5 minutes, (2) to (4) for 30 cycles, (5) 5 minutes at 72 ° C., TaKaRa Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) was used as the polymerase. The amplified fragment was cloned into a cloning vector pCR-Blunt2-TOPO (manufactured by Invitrogen) for E. coli.
次に、配列番号１６及び１７に記載のプライマーを用いてＰＣＲ法によってｐＣＲ−Ｂｌｕｎｔ２−ＴＯＰＯベクターの２０６１ｂｐ−２７０２ｂｐ領域の両端より外側に向かって増幅反応を行い、ＤＮＡリガーゼキット（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ（東洋紡社製））で連結することにより６４１ｂｐを欠失させたベクターｐＣＵＰを作製した（図１）。ＰＣＲ条件は（１）９８℃で２分、（２）９８℃で３０秒、（３）５５℃で３０秒、（４）７２℃で７分、（２）から（４）を３０サイクル、（５）７２℃で７分であり、ポリメラーゼとしてはＴａＫａＲａ Ｐｙｒｏｂｅｓｔ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）を用いた。
更にプラスミドベクターへの遺伝子挿入を容易にするため、得られたｐＣＵＰに制限酵素ＭｕｎＩサイトを導入した。具体的な作製手順としては、まず配列番号１８及び１９に記載のプライマーを用いてｐＣＵＰを鋳型にしてＰＣＲを行い、増幅断片をＤＮＡリガーゼキット（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ（東洋紡社製））で連結し、図２に示されるｐＣＵＰ２を作製した。ＰＣＲ条件は（１）９８℃で２分、（２）９８℃で３０秒、（３）５５℃で３０秒、（４）７２℃で５分、（２）から（４）を３０サイクル、である。ポリメラーゼとしてはＴａＫａＲａ Ｐｙｒｏｂｅｓｔ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）を用いた。このようにして、配列番号１２で示されるＤＮＡ領域及び配列番号１３で示されるＤＮＡ領域を含有し、ｍｏｂ遺伝子群及びｏｒｉＴ配列など接合伝達に関与する遺伝子を含有しないプラスミドベクターｐＣＵＰ２を作製した。
Next, an amplification reaction was performed from the both ends of the 2061 bp-2702 bp region of the pCR-Blunt2-TOPO vector by PCR using the primers shown in SEQ ID NOs: 16 and 17, and a DNA ligase kit (Ligation High (Toyobo) The vector pCUP from which 641 bp was deleted was produced by ligation in (Fig. 1). PCR conditions were (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 30 seconds, (3) 55 ° C. for 30 seconds, (4) 72 ° C. for 7 minutes, (2) to (4) for 30 cycles, (5) 7 minutes at 72 ° C. TaKaRa Pyrobest DNA Polymerase (Takara Bio Inc.) was used as the polymerase.
Furthermore, in order to facilitate gene insertion into the plasmid vector, a restriction enzyme MunI site was introduced into the obtained pCUP. As a specific production procedure, first, PCR was performed using pCUP as a template using the primers shown in SEQ ID NOs: 18 and 19, and the amplified fragments were ligated with a DNA ligase kit (Ligation High (manufactured by Toyobo)). The pCUP2 shown in 2 was prepared. PCR conditions are (1) 98 ° C. for 2 minutes, (2) 98 ° C. for 30 seconds, (3) 55 ° C. for 30 seconds, (4) 72 ° C. for 5 minutes, (2) to (4) for 30 cycles, It is. As the polymerase, TaKaRa Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) was used. In this manner, a plasmid vector pCUP2 containing the DNA region represented by SEQ ID NO: 12 and the DNA region represented by SEQ ID NO: 13 and not containing genes involved in conjugative transfer such as the mob gene group and the oriT sequence was prepared.
（実施例７）Ｐ（３ＨＢ−ｃｏ−３ＨＨ）合成酵素発現ベクターの構築
アエロモナス・キャビエ由来のＮ１４９Ｓ／Ｄ１７１Ｇ変異体遺伝子断片は、国際公開第２００５／０９８００１号パンフレットに記載されているようにＰＣＲ法により作製した。次にプロモーター領域の変換を行った。すなわち、Ｈ１６株の染色体を鋳型とし、配列番号２０と配列番号２１とでＰＣＲを行い、ｐｈｂオペロンプロモーター断片（約４６０ｂｐ）を得た。また、Ｎ１４９Ｓ／Ｄ１７１Ｇ断片を鋳型に配列番号２２と配列番号２３とでＰＣＲを行い、増幅断片を得た。それぞれの断片をＥｃｏＲＩ及びＢｓｐＴ１０４Ｉで切断し、ｐＵＣ１９のＥｃｏＲＩ部位に挿入してＥＥＲＥＰ１４９ＮＳ／１７１ＤＧ断片を得た。この断片を実施例６で作製したｐＣＵＰ２をＭｕｎＩで切断した部位に挿入して図３に示す発現ベクターｐＣＵＰ２ＥＥＲＥＰ１４９ＮＳ／１７１ＤＧを構築した。
(Example 7) Construction of P (3HB-co-3HH) synthase expression vector The N149S / D171G mutant gene fragment derived from Aeromonas caviae was subjected to the PCR method as described in WO 2005/098001. It was produced by. Next, the promoter region was converted. That is, using the chromosome of H16 strain as a template, PCR was performed with SEQ ID NO: 20 and SEQ ID NO: 21 to obtain a phb operon promoter fragment (about 460 bp). In addition, PCR was performed with SEQ ID NO: 22 and SEQ ID NO: 23 using the N149S / D171G fragment as a template to obtain an amplified fragment. Each fragment was cleaved with EcoRI and BspT104I and inserted into the EcoRI site of pUC19 to obtain the EEREP149NS / 171DG fragment. This fragment was inserted into the site cleaved with MunI from pCUP2 prepared in Example 6 to construct the expression vector pCUP2EEREP149NS / 171DG shown in FIG.
（実施例８）形質転換体の作製
実施例７で得られた発現ベクターを導入した各種ｐｈｂＣ遺伝子変異株（５種）の形質転換体を電気パルス法により作製した。つまり、遺伝子導入装置はＢｉｏｒａｄ社製のジーンパルサーを用い、キュベットは同じくＢｉｏｒａｄ社製のｇａｐ０．２ｃｍのものを用いた。キュベットに、コンピテント細胞４００μｌと発現ベクター２０μｌを注入してパルス装置にセットし、静電容量２５μＦ、電圧１．５ｋＶ、抵抗値８００Ωの条件で電気パルスをかけた。パルス後、キュベット内の菌液をＮｕｔｒｉｅｎｔ Ｂｒｏｔｈ培地（ＤＩＦＣＯ社製）で３０℃、３時間振とう培養し、選択プレート（Ｎｕｔｒｉｅｎｔ Ａｇａｒ培地（ＤＩＦＣＯ社製）、カナマイシン１００ｍｇ／Ｌ）で、３０℃にて２日間培養して、形質転換体を取得した。
(Example 8) Production of transformants Transformants of various phbC gene mutants (5 types) into which the expression vector obtained in Example 7 was introduced were produced by an electric pulse method. That is, a gene pulsar manufactured by Biorad was used as the gene transfer device, and a cuvette having a gap of 0.2 cm manufactured by Biorad was used. 400 μl of competent cells and 20 μl of expression vector 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 (DIFCO) at 30 ° C. for 3 hours, and then selected plate (Nutient Agar medium (DIFCO), kanamycin 100 mg / L) at 30 ° C. And cultured for 2 days to obtain transformants.
（実施例９）Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の生産と精製
種母培地の組成は１ｗ／ｖ％ Ｍｅａｔ−ｅｘｔｒａｃｔ、１ｗ／ｖ％ Ｂａｃｔｏ−Ｔｒｙｐｔｏｎ、０．２ｗ／ｖ％ Ｙｅａｓｔ−ｅｘｔｒａｃｔ、０．９ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１５ｗ／ｖ％ ＫＨ２ＰＯ４、ｐＨ６．８とした。
前培養培地の組成は１．１ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１９ｗ／ｖ％ ＫＨ２ＰＯ４、１．２９ｗ／ｖ％（ＮＨ４）２ＳＯ４、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、２．５ｗ／ｖ％パームＷオレイン油、０．５ｖ／ｖ％微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの。）、５×１０−６ｗ／ｖ％ カナマイシンとした。
(Example 9) Production of P (3HB-co-3HH) and composition of purified seed medium were 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0 0.9 w / v% Na 2 HPO 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 HPO 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, 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 5 × 10 −6 w / v% kanamycin.
ポリエステル生産培地の組成は０．３８５ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．０６７ｗ／ｖ％ ＫＨ２ＰＯ４、０・２９１ｗ／ｖ％（ＮＨ４）２ＳＯ４、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、０．５ｖ／ｖ％ 微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの。）、０．０５ｗ／ｖ％ＢＩＯＳＰＵＲＥＸ２００Ｋ（消泡剤：コグニスジャパン製）、５×１０−６ｗ／ｖ％ カナマイシンとした。炭素源はパーム核油を分別した低融点画分であるパーム核油オレインを単一炭素源として用い、培養全般を通じ、比基質供給速度が０．０８〜０．１（ｇ油脂）×（ｇ正味乾燥菌体重量）−１×（ｈ）−１となるように流加した。 The composition of the polyester production medium is 0.385 w / v% Na 2 HPO 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 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% BIOSPUREX 200K (antifoaming agent: manufactured by Cognis Japan), 5 × 10 −6 w / v% kanamycin was used. As a carbon source, palm kernel oil olein, which is a low melting point fraction obtained by separating 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) x (g Net dry cell weight) -1 x (h) -1 was fed.
実施例８で作製した各種形質転換株のグリセロールストック（５０μｌ）を種母培地（１０ｍｌ）に接種して２４時間培養し、１．８Ｌの前培養培地を入れた３Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＬ−３００型）に１．０ｖ／ｖ％接種した。運転条件は、培養温度３０℃、攪拌速度５００ｒｐｍ、通気量１．８Ｌ／ｍｉｎとし、ｐＨは６．７〜６．８の間でコントロールしながら２８時間培養した。ｐＨコントロールには７％水酸化アンモニウム水溶液を使用した。
ポリエステル生産培養は６Ｌの生産培地を入れた１０Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＬ−１０００型）に前培養種母を５．０ｖ／ｖ％接種した。運転条件は、培養温度２８℃、攪拌速度４００ｒｐｍ、通気量３．６Ｌ／ｍｉｎとし、ｐＨは６．７から６．８の間でコントロールした。ｐＨコントロールには７％水酸化アンモニウム水溶液を使用した。培養は約６５時間行い、培養終了後、遠心分離によって菌体を回収、メタノールで洗浄、凍結乾燥し、乾燥菌体重量を測定した。
得られた乾燥菌体約１ｇに１００ｍｌのクロロホルムを加え、室温で一昼夜攪拌して、菌体内のポリエステルを抽出した。菌体残渣をろ別後、エバポレーターで総容量が約３０ｍｌになるまで濃縮後、約９０ｍｌのヘキサンを徐々に加え、ゆっくり攪拌しながら、１時間放置した。析出したポリエステルをろ別後、５０℃で３時間真空乾燥した。乾燥ポリエステルの重量を測定し、ポリエステル生産量を算出した。その結果を表１に示す。
Glycerol stocks (50 μl) of various transformants prepared in Example 8 were inoculated into seed medium (10 ml), cultured for 24 hours, and 3 L jar fermenter (Maruhishi Bio Inc.) containing 1.8 L of preculture medium. Enzyme 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 a 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.
In the polyester production culture, a 10 L jar fermenter (MDL-1000, manufactured by Maruhishi Bioengine) 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. Culture was performed for about 65 hours, and after completion of the culture, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the dry cell weight was measured.
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 production was calculated. The results are shown in Table 1.
（比較例１）
従来の菌株であるＰＨＢ−４株を宿主とし、発現ベクターｐＪＲＤ１４９ＮＳ／１７１ＤＧ（国際公開第２００５／０９８００１号パンフレット）を導入した形質転換体を同様の方法にて培養した。また、同発現ベクターをΔＢ１１３３株及びΔＳ１３１２３株に導入した形質転換体も同様の方法にて培養した。結果を表２に示す。
(Comparative Example 1)
A transformant into which the expression vector pJRD149NS / 171DG (International Publication No. 2005/098001) was introduced was cultured in the same manner using the PHB-4 strain, which is a conventional strain, as a host. Moreover, the transformant which introduce | transduced the same expression vector into (DELTA) B1133 strain | stump | stock and (DELTA) S13123 strain | stump | stock was cultured by the same method. The results are shown in Table 2.
本発明は、生分解性ポリエステルであるポリヒドロキシアルカン酸（ＰＨＡ）の商業的生産に有用な、改良された微生物株と、それを利用するＰＨＡの製造方法を提供するものである。 The present invention provides an improved microbial strain useful for the commercial production of polyhydroxyalkanoic acid (PHA), a biodegradable polyester, and a process for producing PHA using the same.
Claims (16)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006085314A JP2007259708A (en) | 2006-03-27 | 2006-03-27 | New method for producing biodegradable polyester |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006085314A JP2007259708A (en) | 2006-03-27 | 2006-03-27 | New method for producing biodegradable polyester |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2007259708A true JP2007259708A (en) | 2007-10-11 |
Family
ID=38633336
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2006085314A Pending JP2007259708A (en) | 2006-03-27 | 2006-03-27 | New method for producing biodegradable polyester |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2007259708A (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012102371A1 (en) | 2011-01-27 | 2012-08-02 | 株式会社カネカ | Microorganism producing high-molecular-weight pha |
| WO2014042076A1 (en) | 2012-09-14 | 2014-03-20 | 国立大学法人東京工業大学 | Method for producing polyhydroxyalkanoate using modified fat or oil composition |
| WO2014065253A1 (en) | 2012-10-22 | 2014-05-01 | 株式会社カネカ | High molecular weight pha-producing microbe and method of producing high molecular weight pha using same |
| WO2015020104A1 (en) | 2013-08-06 | 2015-02-12 | 株式会社カネカ | Soft thermoplastic resin composition |
| WO2015020105A1 (en) | 2013-08-06 | 2015-02-12 | 株式会社カネカ | Soft thermoplastic resin composition |
| WO2015115619A1 (en) | 2014-01-31 | 2015-08-06 | 株式会社カネカ | MICROORGANISM HAVING ADJUSTED EXPRESSION OF R-SPECIFIC ENOYL-CoA HYDRATASE GENE, AND METHOD FOR MANUFACTURING POLYHYDROXYALKANOATE COPOLYMER USING SAME |
| WO2018117168A1 (en) | 2016-12-20 | 2018-06-28 | 株式会社カネカ | Method for producing polyhydroxyalkanoic acid, and microbes |
| WO2018216726A1 (en) | 2017-05-25 | 2018-11-29 | 株式会社カネカ | Pha-producing micro-organism in which glycerol kinase activity is enhanced, and pha production method using said same |
| WO2019142845A1 (en) | 2018-01-17 | 2019-07-25 | 株式会社カネカ | Transformed microorganism for producing pha copolymer comprising 3hh monomer unit at high composition rate and method for producing pha using same |
| WO2019142717A1 (en) | 2018-01-16 | 2019-07-25 | 株式会社カネカ | Mutant polyhydroxyalkanoic acid synthase, gene and transformant thereof, and method for producing polyhydroxyalkanoic acid |
| WO2020174988A1 (en) | 2019-02-28 | 2020-09-03 | 株式会社カネカ | Transformed microorganism and production method of polyhydroxyalkanoic acid |
| WO2021049207A1 (en) | 2019-09-09 | 2021-03-18 | 株式会社カネカ | Transformed microorganism, and method for producing polyhydroxyalkanoic acid using said microorganism |
| WO2021059762A1 (en) | 2019-09-25 | 2021-04-01 | 株式会社カネカ | Method for producing polyhydroxyalkanoate |
| WO2021206155A1 (en) | 2020-04-10 | 2021-10-14 | 株式会社カネカ | Method for manufacturing copolymer polyhydroxyalkanoic acid mixture, and transformed microorganism |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005098001A1 (en) * | 2004-04-09 | 2005-10-20 | Kaneka Corporation | Novel transformant |
-
2006
- 2006-03-27 JP JP2006085314A patent/JP2007259708A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005098001A1 (en) * | 2004-04-09 | 2005-10-20 | Kaneka Corporation | Novel transformant |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5969395B2 (en) * | 2011-01-27 | 2016-08-17 | 株式会社カネカ | High molecular weight PHA producing microorganism |
| WO2012102371A1 (en) | 2011-01-27 | 2012-08-02 | 株式会社カネカ | Microorganism producing high-molecular-weight pha |
| WO2014042076A1 (en) | 2012-09-14 | 2014-03-20 | 国立大学法人東京工業大学 | Method for producing polyhydroxyalkanoate using modified fat or oil composition |
| WO2014065253A1 (en) | 2012-10-22 | 2014-05-01 | 株式会社カネカ | High molecular weight pha-producing microbe and method of producing high molecular weight pha using same |
| WO2015020104A1 (en) | 2013-08-06 | 2015-02-12 | 株式会社カネカ | Soft thermoplastic resin composition |
| WO2015020105A1 (en) | 2013-08-06 | 2015-02-12 | 株式会社カネカ | Soft thermoplastic resin composition |
| WO2015115619A1 (en) | 2014-01-31 | 2015-08-06 | 株式会社カネカ | MICROORGANISM HAVING ADJUSTED EXPRESSION OF R-SPECIFIC ENOYL-CoA HYDRATASE GENE, AND METHOD FOR MANUFACTURING POLYHYDROXYALKANOATE COPOLYMER USING SAME |
| WO2018117168A1 (en) | 2016-12-20 | 2018-06-28 | 株式会社カネカ | Method for producing polyhydroxyalkanoic acid, and microbes |
| WO2018216726A1 (en) | 2017-05-25 | 2018-11-29 | 株式会社カネカ | Pha-producing micro-organism in which glycerol kinase activity is enhanced, and pha production method using said same |
| WO2019142717A1 (en) | 2018-01-16 | 2019-07-25 | 株式会社カネカ | Mutant polyhydroxyalkanoic acid synthase, gene and transformant thereof, and method for producing polyhydroxyalkanoic acid |
| WO2019142845A1 (en) | 2018-01-17 | 2019-07-25 | 株式会社カネカ | Transformed microorganism for producing pha copolymer comprising 3hh monomer unit at high composition rate and method for producing pha using same |
| WO2020174988A1 (en) | 2019-02-28 | 2020-09-03 | 株式会社カネカ | Transformed microorganism and production method of polyhydroxyalkanoic acid |
| WO2021049207A1 (en) | 2019-09-09 | 2021-03-18 | 株式会社カネカ | Transformed microorganism, and method for producing polyhydroxyalkanoic acid using said microorganism |
| WO2021059762A1 (en) | 2019-09-25 | 2021-04-01 | 株式会社カネカ | Method for producing polyhydroxyalkanoate |
| WO2021206155A1 (en) | 2020-04-10 | 2021-10-14 | 株式会社カネカ | Method for manufacturing copolymer polyhydroxyalkanoic acid mixture, and transformed microorganism |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2007259708A (en) | New method for producing biodegradable polyester | |
| US7384766B2 (en) | Gene-substituted microorganisms, and production method of polyesters using the same | |
| JP5670728B2 (en) | Improved polyhydroxyalkanoate-producing microorganism and method for producing polyhydroxyalkanoate using the same | |
| JP5468779B2 (en) | Gene replacement microorganism and method for producing polyester using the same | |
| JP2006204255A (en) | ACETYL-CoA ACYLTRANSFERASE GENE-BROKEN POLYHYDROXYALKANOATE-PRODUCING MICROORGANISM, AND METHOD FOR PRODUCING POLYHYDROXYALKANOATE THEREWITH | |
| JP2006204257A (en) | Isogenic strain of polyhydroxyalkanoate synthetase gene-disrupted polyhydroxyalkanoate-producing microorganism, and method for producing polyhydroxyalkanoate therewith | |
| WO2015115619A1 (en) | MICROORGANISM HAVING ADJUSTED EXPRESSION OF R-SPECIFIC ENOYL-CoA HYDRATASE GENE, AND METHOD FOR MANUFACTURING POLYHYDROXYALKANOATE COPOLYMER USING SAME | |
| JPWO2005098001A1 (en) | New transformant | |
| JP6313708B2 (en) | High molecular weight PHA producing microorganism and method for producing high molecular weight PHA using the same | |
| JP4960033B2 (en) | Method for producing copolymerized polyester using microorganisms having reduced enzyme activity | |
| Chen et al. | A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates | |
| JP4995469B2 (en) | Genetically modified microorganism and method for producing polyester using the same | |
| JPWO2008090873A1 (en) | Process for producing polyhydroxyalkanoate | |
| US20090130731A1 (en) | Microorganism capable of accumulating ultra high molecular weight polyester | |
| JP2008086238A (en) | Method for producing polyhydroxyalkanoate | |
| US20060160195A1 (en) | Novel vector | |
| WO2014133088A1 (en) | Production method for copolymer polyhydroxyalkanoate using genetically modified strain of fatty acid β-oxidation pathway | |
| Thakor et al. | Microbiological and biotechnological aspects of biodegradable plastics: Poly (hydroxyalkanoates) | |
| JP5839854B2 (en) | Microbial culture method | |
| US10876140B2 (en) | Method for producing polyhydroxyalkanoic acid, and microbes | |
| WO2015133468A1 (en) | Production method for copolymer polyester using recombinant strain having modified protein locus for polyester granule binding | |
| JP2005333933A (en) | New expression plasmid | |
| JP2009225775A (en) | Method of producing polyhydroxyalkanoic acid | |
| CN104845927A (en) | Gene substituted microbe and polyester preparation method using gene substituted microbe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20090121 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20110913 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20120417 |