JPS62186786A - L-tryptophan-producing microbial strain produced by transformation and production of l-tryptophan - Google Patents
L-tryptophan-producing microbial strain produced by transformation and production of l-tryptophanInfo
- Publication number
- JPS62186786A JPS62186786A JP2802886A JP2802886A JPS62186786A JP S62186786 A JPS62186786 A JP S62186786A JP 2802886 A JP2802886 A JP 2802886A JP 2802886 A JP2802886 A JP 2802886A JP S62186786 A JPS62186786 A JP S62186786A
- Authority
- JP
- Japan
- Prior art keywords
- tryptophan
- transformed
- dna
- microbial strain
- bacillus
- 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
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 57
- 229960004799 tryptophan Drugs 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 230000000813 microbial effect Effects 0.000 title abstract description 4
- 230000009466 transformation Effects 0.000 title description 3
- 108020004414 DNA Proteins 0.000 claims abstract description 27
- 244000005700 microbiome Species 0.000 claims abstract description 24
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 15
- 239000012634 fragment Substances 0.000 claims abstract description 10
- 230000002068 genetic effect Effects 0.000 claims abstract description 10
- 102000053602 DNA Human genes 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims description 28
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 claims description 18
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 11
- 239000013611 chromosomal DNA Substances 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 239000013612 plasmid Substances 0.000 abstract description 12
- 108091008146 restriction endonucleases Proteins 0.000 abstract description 11
- 238000007796 conventional method Methods 0.000 abstract description 6
- 241000588724 Escherichia coli Species 0.000 abstract description 5
- 102000003960 Ligases Human genes 0.000 abstract description 5
- 108090000364 Ligases Proteins 0.000 abstract description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 abstract description 3
- 229960000723 ampicillin Drugs 0.000 abstract description 3
- 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 abstract description 3
- 239000001110 calcium chloride Substances 0.000 abstract description 3
- 229910001628 calcium chloride Inorganic materials 0.000 abstract description 3
- 239000004098 Tetracycline Substances 0.000 abstract description 2
- 229960002180 tetracycline Drugs 0.000 abstract description 2
- 229930101283 tetracycline Natural products 0.000 abstract description 2
- 235000019364 tetracycline Nutrition 0.000 abstract description 2
- 150000003522 tetracyclines Chemical class 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 22
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000002609 medium Substances 0.000 description 7
- 229960005091 chloramphenicol Drugs 0.000 description 5
- 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 5
- 239000000126 substance Substances 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 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 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 101150035767 trp gene Proteins 0.000 description 3
- 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
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 101100261636 Methanothermobacter marburgensis (strain ATCC BAA-927 / DSM 2133 / JCM 14651 / NBRC 100331 / OCM 82 / Marburg) trpB2 gene Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 101100408135 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) phnA gene Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 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 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 101150081616 trpB gene Proteins 0.000 description 2
- 101150111232 trpB-1 gene Proteins 0.000 description 2
- 101150044170 trpE gene Proteins 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- YYGJQPKPUWCRIM-JTQLQIEISA-N (2s)-2-(fluoroamino)-3-(1h-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(C[C@@H](C(=O)O)NF)=CNC2=C1 YYGJQPKPUWCRIM-JTQLQIEISA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- -1 2H 2o1g Chemical compound 0.000 description 1
- INPQIVHQSQUEAJ-UHFFFAOYSA-N 5-fluorotryptophan Chemical compound C1=C(F)C=C2C(CC(N)C(O)=O)=CNC2=C1 INPQIVHQSQUEAJ-UHFFFAOYSA-N 0.000 description 1
- HUNCSWANZMJLPM-UHFFFAOYSA-N 5-methyltryptophan Chemical compound CC1=CC=C2NC=C(CC(N)C(O)=O)C2=C1 HUNCSWANZMJLPM-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 241000193411 Paenibacillus amylolyticus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101100370749 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) trpC1 gene Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QLULGSLAHXLKSR-UHFFFAOYSA-N azane;phosphane Chemical compound N.P QLULGSLAHXLKSR-UHFFFAOYSA-N 0.000 description 1
- 229940054340 bacillus coagulans Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 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
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000003317 industrial substance Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 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 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 description 1
- 229960005287 lincomycin Drugs 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 235000011118 potassium hydroxide Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000007017 scission Effects 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
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 101150019416 trpA gene Proteins 0.000 description 1
- 101150016309 trpC gene Proteins 0.000 description 1
- 101150079396 trpC2 gene Proteins 0.000 description 1
- 101150100816 trpD gene Proteins 0.000 description 1
- 101150079930 trpGD gene Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔技術分野〕
本発明は形質転換によシ、Ｌ −） ＩＪブトファン生
産能が高められた微生物及びこれを用いたし−トリプト
ファンの製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a microorganism with enhanced ability to produce L-)IJ butophane through transformation, and a method for producing sti-tryptophan using the microorganism.
〔従来技術゛及び発明刈解決しようとする問題点〕Ｌ−
トリプトファンは必須アミノ酸の一種として重要な化合
物であり特に栄養学的に不足し易い物質であるためその
経済的な製造法の開発が強く望まれており、そのため従
来より種々の方法が提案されているが、就中発酵法によ
る方法が注目を集め、その際特にその基礎となるＬ −
） ＩＪブトファン生産性微生物の改良は重要な課題と
なっている。[Prior art and problems to be solved by invention] L-
Tryptophan is an important compound as a type of essential amino acid, and is a substance that is particularly susceptible to nutritional deficiencies, so there is a strong desire to develop an economical method for producing it, and for this reason various methods have been proposed. However, the fermentation method is attracting attention, especially when the L −
) Improvement of IJ butophane-producing microorganisms has become an important issue.
有効な改良法として、Ｔｒｐ遺伝子を宿主菌の染色体Ｄ
ＮＡに挿入（インテグレーション）することによシ遺伝
子増幅する方法があるが、この方法では遺伝子増幅の効
率が低い。そこで発明者らは、広い範囲にわたる宿主菌
を用いることができ、宿主菌内で導入遺伝子が安定に存
在し得るというインテグレーション法の長所を生かしさ
らに遺伝子増幅の効率を上げるべく研究を重ねた結果、
本発明をなすに至った。As an effective improvement method, the Trp gene is transferred to chromosome D of the host bacterium.
There is a method of amplifying genes by insertion (integration) into NA, but this method has low gene amplification efficiency. Therefore, the inventors conducted repeated research to further improve the efficiency of gene amplification by taking advantage of the advantages of the integration method, in that a wide range of host bacteria can be used and the introduced gene can stably exist within the host bacteria.
The present invention has been accomplished.
本発明によれば、Ｌ−トリプトファンの生合成に関する
遺伝情報を有するＤＮＡ断片を複数個連結させた部分を
有するＤＮＡ分子を、バチルス属に属する微生物から選
ばれる宿主菌の染色体ＤＮＡに挿入することによシ、Ｌ
−）リットファン高生産性微生物が提供される。According to the present invention, a DNA molecule having a portion in which a plurality of DNA fragments having genetic information related to the biosynthesis of L-tryptophan are linked is inserted into the chromosomal DNA of a host bacterium selected from microorganisms belonging to the genus Bacillus. Yoshi, L
-) Litfan high productivity microorganisms are provided.
本発明によれば、更にこれらのＬ−１リプトフアン高生
産性微生物を培地に培養して培養物中にＬ−トリプトフ
ァンを生成せしめ、これを採取すること全特徴とするＬ
−トリプトファンが経済的に得られる製造法が提供され
る。According to the present invention, the L-1-lyptophan-producing microorganisms are further cultured in a medium to produce L-tryptophan in the culture, and the L-tryptophan is collected.
- A process for producing tryptophan economically is provided.
以下に本発明の微生物とその取得方法及びこれ全周いた
Ｌ −トＩＪブトファンの製造法について更に詳細に説
明する。The microorganism of the present invention, the method for obtaining the microorganism, and the method for producing L-IJ butophane using the microorganism will be explained in more detail below.
本発明におけるＬ −トＩＪブトファンの生合成に関す
る遺伝情報を有するＤＮＡ断片は、適当な微生物の染色
体ＤＮＡから適当な制限酵素によって切シ出されたもの
が用いられるが、原則としてその由来については制限は
ない。一般には、Ｌ −） ＩＪブトファン生産能を有
する微生物を用いるが、Ｌ−トリプトファン生合成系の
一部酵素の欠損等の理由によ、９．Ｌ−トリプトファン
生産能を有しない微生物を用いることも何らさしつかえ
ない。例えば、土壌や他の天然物から分離されるＬ −
トリプトファン生産能を有する野性法は勿論のこと、そ
れらを紫外線照射や化学物質による処理をして得られる
突然変異株或いは遺伝子組換え技術を用いて得られる組
み換えＤＮＡ等いずれでもよい。尚、この場合、ＤＮＡ
断片は、Ｌ −トリプトファンの生合成に関する遺伝情
報以外のＤＮＡ部分を含むこともあり得るが、その部分
が、Ｌ−トリプトファンの生合成に悪影響を及ぼさない
限り用いることができる。また、ＤＮＡ断片は、Ｌ−ト
リプトファンの生合成に関する遺伝情報のすべてを有す
る必要はなく、その一部分のみを有しているものでもか
まわない。In the present invention, the DNA fragment having genetic information related to the biosynthesis of L-toIJbutophane is excised from the chromosomal DNA of an appropriate microorganism with an appropriate restriction enzyme, but as a general rule, there are no restrictions regarding its origin. There isn't. Generally, microorganisms having the ability to produce L-)IJbutophane are used; however, due to defects in some enzymes in the L-tryptophan biosynthesis system, etc. There is nothing wrong with using microorganisms that do not have the ability to produce L-tryptophan. For example, L − isolated from soil or other natural products
Any of the wild methods capable of producing tryptophan, mutant strains obtained by irradiating them with ultraviolet rays or treatment with chemical substances, or recombinant DNA obtained using genetic recombination techniques may be used. In this case, DNA
The fragment may contain DNA parts other than genetic information related to L-tryptophan biosynthesis, but can be used as long as such parts do not adversely affect L-tryptophan biosynthesis. Further, the DNA fragment does not need to have all of the genetic information regarding the biosynthesis of L-tryptophan, and may have only a part of it.
また、ＤＮＡ断片は宿主菌の染色体ＤＮＡと相同性が高
いほうが望ましいが、ＤＮＡ分子の他の部分と宿主菌の
染色体ＤＮＡの相同性が高い場合は、特に相同性が高い
必要はない。Furthermore, it is desirable that the DNA fragment has high homology with the chromosomal DNA of the host bacterium, but if the other parts of the DNA molecule have high homology with the chromosomal DNA of the host bacterium, the homology does not need to be particularly high.
このようなＬ−トリプトファンの生合成に関する遺伝情
報を有する染色体ＤＮＡ断片中有する微生物としては、
例えば、バチルス・アミロリクイファシェンス、バチル
ス・アミロリティカス、バチルス・アルカロフィラス、
バチルス・コアギユランス、バチルス・ライケニホルミ
ス、バチルス・ナラトウ、バチルス・スフチルス、バチ
ルス・ステアロサーモフィラス等のバチルス属に属する
微生物や、それらの変異株など、およびそれらを親株と
して遺伝子組み換えによって育種した株等が掲げられる
。Microorganisms that have genetic information related to the biosynthesis of L-tryptophan in their chromosomal DNA fragments include:
For example, Bacillus amyloliquefacens, Bacillus amylolyticus, Bacillus arcalophilus,
Microorganisms belonging to the genus Bacillus such as Bacillus coagulans, Bacillus licheniformis, Bacillus naratou, Bacillus sphutilus, and Bacillus stearothermophilus, their mutant strains, and strains bred by genetic recombination using these as parent strains, etc. is listed.
また、これらＤＮＡよりＤＮＡ断片を切出すのに用いら
れる制限酵素としては特に制限はないが、目的のＤＮＡ
断片中に切断部位が少ないほうが望ましく、例えば、Ｅ
ｃｏＲｌ 、 ＢａｍＨｉ 、 ５ａｌＩ、５ａｃｌ
。There are no particular restrictions on the restriction enzymes used to cut out DNA fragments from these DNAs, but
It is desirable to have fewer cleavage sites in the fragment; for example, E
coRl, BamHi, 5alI, 5acl
.
ｐｖｕｌｌ、ＸｈｏＩ １ＸｂａＩ、Ｍｂｏｌ 、 Ｍ
ｌｕＩ等があげられる。pbull, XhoI 1XbaI, Mbol, M
Examples include luI.
本発明において、宿主菌内で自己複製しないベクターと
しては所謂宿主−村りター系として成り立つものであれ
ばいずれでもよく、例えば、Ｃｏ ＩＥＩ、ｐｓｃｌｏ
ｌ、ｐＢＲ３２５及びｐＢＲ３２８等の大腸菌由来のプ
ラスミドがあげられる。形質転換菌の取得を容易にする
ため、宿主菌としてＴｒｐ遺伝子の一部が変異してトリ
プトファン要求性になった株、例えばバチルス５Ｄ−５
３（特開昭５９−１７００４７）。In the present invention, vectors that do not self-replicate within host bacteria may be of any type as long as they can form a so-called host-return system, such as CoIEI, psclo
Examples include plasmids derived from Escherichia coli, such as pBR325 and pBR328. In order to facilitate the acquisition of transformed bacteria, strains in which part of the Trp gene has mutated to become tryptophan auxotrophic, such as Bacillus 5D-5, are used as host bacteria.
3 (Japanese Unexamined Patent Publication No. 59-170047).
ＶＯＴＯ５３１（東京大学応用微生物研究所）等を用い
ることができる。あるいは、導入するＤＮＡ分子に選択
可能な遺伝子マーカーを組み込むことによっても形質転
換菌を容易に選択できる。ただし、これらはあくまで選
択を容易にする方法であり、本発明においては必ずしも
必須の要件ではない。VOTO531 (Institute of Applied Microbiology, University of Tokyo), etc. can be used. Alternatively, transformed bacteria can be easily selected by incorporating a selectable genetic marker into the introduced DNA molecule. However, these are merely methods to facilitate selection, and are not necessarily essential requirements in the present invention.
本発明の方法において用いる選択可能な遺伝子マーカー
とは、宿主菌内で発現するものであればいずれもよく、
−例をあげると次のようなものがある。薬剤耐性遺伝子
、アミノ酸生合成遺伝子、核酸生合成遺伝子、ビタミン
生合成遺伝子、その他の生体内物質の生合成遺伝子など
、薬剤耐性遺伝子の例としては、クロラムフェニコール
耐性、テトラサイクリン耐性、エリスロマイシン耐性、
カナマイシン耐性、ネオマイシン耐性、ストレプトマイ
シン耐性、ペニシリン耐性、リンコマイシン耐性などが
ある。The selectable genetic marker used in the method of the present invention may be any gene marker as long as it is expressed within the host bacterium.
- Examples include: Examples of drug resistance genes include drug resistance genes, amino acid biosynthesis genes, nucleic acid biosynthesis genes, vitamin biosynthesis genes, and biosynthesis genes for other biological substances.
These include kanamycin resistance, neomycin resistance, streptomycin resistance, penicillin resistance, and lincomycin resistance.
ＤＮＡ分子を導入すべき宿主菌としては、その染色体Ｄ
ＮＡが導入するＤＮＡ分子と相同性を有するものであれ
ば原則として如何なるものでも良いが、実際上は本発明
の本来の目的である醗酵法によるＬ −トＩＪブトファ
ンの製造に適した微生物であることが必要であり、かか
る観点から病原菌及び工業的使用の際管理が繁雑なもの
や使用不能なものは除外される。実用的には培養が容易
で、工業的に実際に使用の実績もあるバチルス属に属す
る微生物が用いられる。好ましくは、トリプトファンの
生成蓄積により微生物固有のトリプトファン生合成のフ
ィードバック抑制機構の解除された菌、例工ば、トリプ
トファンアナログ（５−フルオロトリプトファン、５−
メチルトリプトファン等）耐性菌であり、代表的なもの
を例示すれば、例えば、特開昭４８−１８８２８、同４
９−２’０３９１、同４９−８５２８９、同５１−６４
９２１、同５３−１３５８、同５３−３９５１７、同５
６−９２７９６、同５８−９４３９１、同５８−１０７
１９０、同５８−１０７１９３、同５８−１０７１９４
、同５８−１０７１９５、同５８−１３８３８９、同５
８−２２０６９３、同５９−１２００９１、同５９−１
３０１８１等に記載の菌が掲げられる。The host bacterium into which the DNA molecule should be introduced should have its chromosome D.
In principle, any microorganism may be used as long as the NA has homology to the DNA molecule to be introduced, but in practice it is a microorganism suitable for the production of L-toIJ butophane by the fermentation method, which is the original purpose of the present invention. From this perspective, pathogenic bacteria and those that are difficult to manage or cannot be used for industrial use are excluded. Practically, microorganisms belonging to the genus Bacillus are used, which are easy to culture and have a track record of actual industrial use. Preferably, microorganisms in which the feedback inhibition mechanism of tryptophan biosynthesis inherent in microorganisms is released by producing and accumulating tryptophan, such as tryptophan analogs (5-fluorotryptophan, 5-
Methyltryptophan, etc.) resistant bacteria.
9-2'0391, 49-85289, 51-64
921, 53-1358, 53-39517, 5
6-92796, 58-94391, 58-107
190, 58-107193, 58-107194
, 58-107195, 58-138389, 5
8-220693, 59-120091, 59-1
The bacteria described in 30181 and the like are listed.
上記宿主菌の形質転換は、公知の方法、例えば、コンピ
ーテントセル法［Ｊ、Ｂａｃｔｅｒｉａ１８１，７４１
（１９６１）］或いはプロトプラスト法１： Ｍｏ１ｅ
ｃ、ｇｅｎ ＊ Ｇｅｎｅｔ 。The transformation of the host bacteria can be carried out using known methods, such as the competent cell method [J, Bacteria 181, 741
(1961)] or protoplast method 1: Mo1e
c, gen*Genet.
１６８．１１１（１９７９）Ｅ等によって行なわれる。168.111 (1979) E et al.
以下に本発明のＬ −） ＩＪブトファン高生産性微生
物について、代表的な例を示し、更に具体的に説明する
。但しこれらは単なる例示であシ、本発明はこれらのみ
に限られない。Representative examples of the L-)IJ butophane highly productive microorganism of the present invention will be shown below and explained in more detail. However, these are merely examples, and the present invention is not limited to these.
プラスミドｐｓＤ２９６１ （特願昭５９−２０７０４
５号参照）を制限酵素Ｈｉｎｄｌｌｒで部分切断し、Ｔ
４ファージリガーゼを用いて再結合させる。このＤＮＡ
を用い、塩化カルシウム処理した大腸菌Ｃ６００を常法
により形質転換し、トリプトファン非要求性でアンピシ
リン耐性、テトラサイクリン感受性の形質転換株を選択
した。これらの形質転換株よりプラスミドを常法により
分離精製し、第１図に示すようなプラスミドｐｓＤ３１
７１ ’に得た。このプラスミドを用いて各種トリプト
ファン要求株（ｔｒｐＡ、ｔｒｐＢ 、 ｔｒｐＣ、ｔ
ｒｐＤまたはｔｒｐＥ等の突然変異株）を形質転換した
ところ、全てにトリプトファン非要求性の形質転換菌が
高頻度に出現することからこの短小化されたＤＮＡもト
リプトファンの生合成を調整する遺伝情報を有すると考
えられる。Plasmid psD2961 (Patent application 1986-20704)
(see No. 5) was partially cut with the restriction enzyme Hindllr, and T
4. Religate using phage ligase. this DNA
Escherichia coli C600 treated with calcium chloride was transformed using a conventional method, and a transformant strain that did not require tryptophan, was resistant to ampicillin, and was sensitive to tetracycline was selected. Plasmids were isolated and purified from these transformed strains by conventional methods, and plasmid psD31 as shown in Figure 1 was obtained.
Got it at 71'. Using this plasmid, various tryptophan auxotrophs (trpA, trpB, trpC, t
When mutant strains such as rpD or trpE) were transformed, non-tryptophan auxotrophic transformed bacteria frequently appeared in all of them, indicating that this shortened DNA also contains genetic information that regulates tryptophan biosynthesis. It is considered to have.
一方、ｐｓＤ３０５１ （特願昭６０−３１５８号参照
）をｇｃｏＲＩで部分切断、５ａｌｌで完全切断し、Ｅ
ｃｏＲＩＳａＩＩで切断したｐｓＤ３１７１と混合し、
Ｔ４７フージリガーゼを用いて結合させる。このＤＮＡ
を用いてバチルスズプチルスＢＤ２２４ （ｔｒｐＣ２
ｔｈｒ−５ｒｅｃＥ４）をコンピテントセル法により形
質転換し、クロラムフェニコール耐性でかつＴｒｐ非要
求性を示す形質転換菌を取得する。該形質転換菌から組
換えプラスミドを常法により分離精製し、制限酵素地図
を作成したところ、第２図のような制限酵素地図を持つ
プラスミドが得られた。このプラスミド（ｐｓＤ３６５
１と称する）にはｐｓＤ３０５１の５ａｌ（切断点とＥ
ｃｏＲＩ切断点のうちの一方の間に約４．６メガダレト
ンのＤＮＡが挿入されていた。この挿入ＤＮＡは各種、
トリプトファン要求株（ｔ′ｒｐＡ 、 ｔｒｐＢ ５
ｔｒｐｃ 、 ｔｒｐＤ 、またばｔｒｐＥ等の突然変
異株）を受容菌としてｐｓＤ３６５１を供与体ＤＮＡと
した時、全てにＴｒｐ非要求性の形質転換菌が高頻度に
出現せしめる。ことがらＴｒｐ遺伝子を含むと考えられ
る。次に、ｐｓＤ３１７１をＥｃｏＲＩで切断し、同じ
（ＥｅｏＲＩで切断したｐｓＤ３６５１と混合し、Ｔ４
ファージリガーゼを用いて結合させる。このＤＮＡ ｉ
用い、塩化カルシウム処理した大腸菌Ｃ６００株を常法
により形質転換し、クロラムフェニコール耐性、アンピ
シリン耐性でかつＴｒｐ非要求性を示す株を取得した。On the other hand, psD3051 (see Japanese Patent Application No. 60-3158) was partially cleaved with gcoRI, completely cleaved with 5all, and E
mixed with psD3171 cut with coRISaII,
Ligation is performed using T47 fusiligase. this DNA
using Bacillus subtilis BD224 (trpC2
thr-5recE4) is transformed by the competent cell method to obtain transformed bacteria that are resistant to chloramphenicol and exhibit no requirement for Trp. When the recombinant plasmid was isolated and purified from the transformed bacteria by a conventional method and a restriction enzyme map was prepared, a plasmid having a restriction enzyme map as shown in FIG. 2 was obtained. This plasmid (psD365
1) is the 5al (cutting point and E) of psD3051.
Approximately 4.6 megadaletons of DNA had been inserted between one of the coRI breakpoints. This inserted DNA is of various types.
Tryptophan auxotrophs (t'rpA, trpB 5
When psD3651 is used as a donor DNA and psD3651 is used as a recipient strain (mutant strains of trpc, trpD, or trpE, etc.), transformants that do not require Trp appear frequently in all of them. It is thought that the Trp gene is included in some cases. Next, psD3171 was cut with EcoRI, mixed with psD3651 cut with the same (EeoRI), and T4
Ligate using phage ligase. This DNA i
Escherichia coli strain C600 treated with calcium chloride was transformed using the above-described method to obtain a strain that was resistant to chloramphenicol, ampicillin, and non-requiring for Trp.
該形質転換菌からｉ換えプラスミドを常法により分離精
製し、制限酵素地図を作成したところ、第３図のような
制限酵素地図を持つプラスミドｐｓＤＴ３９１が得られ
た。When the i-transformed plasmid was isolated and purified from the transformed bacteria by a conventional method and a restriction enzyme map was prepared, plasmid psDT391 having a restriction enzyme map as shown in FIG. 3 was obtained.
次にｐｓＤＴ３９１を用いて、ＩＡＭ１５２１株由来の
５〜フルオロトリプトフアン耐性株バチルスアミロリク
イファシェンス５Ｄ３０ （特開昭５９−１３０１８１
号参照）を形質転換し、クロラムフェニコール耐性の形
質転換株を選択する。これらのクロラムフェニコール耐
性の形質転換株をアントラニル酸を含む液体培地中で培
養し、Ｌ −トＩＪブトファン生産性の向上した菌株を
選択し、バチルス５Ｄ１０１２を得た。この菌は工業技
術院微生物工業技術研究所に微工研菌寄第８６２２号と
して寄託されている。Next, using psDT391, a 5- to fluorotryptophan-resistant strain Bacillus amyloliquefacens 5D30 derived from the IAM1521 strain (Japanese Patent Application Laid-Open No. 130181-1982)
) and select chloramphenicol-resistant transformants. These chloramphenicol-resistant transformants were cultured in a liquid medium containing anthranilic acid, and a strain with improved L-toIJ butophane productivity was selected to obtain Bacillus 5D1012. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Microtechnology Research Institute No. 8622.
この菌株の菌学的性質は原株バチルス・アミロリクイフ
ァシェンス５Ｄ３０とクロラムフェニコール耐性である
こと及びＬ−トリプトファンの生産能力が高いことを除
いて実質的には同じである。The mycological properties of this strain are substantially the same as the original strain Bacillus amyloliquifacens 5D30, except that it is chloramphenicol resistant and has a high L-tryptophan production capacity.
得られた形質転換菌バチルス５Ｄ１０１２のＬ−）＋７
プトフアンシンセターゼ活性の測定結果を示すと次のと
うりである。L−)+7 of the obtained transformed bacterium Bacillus 5D1012
The measurement results of putophane synthetase activity are as follows.
菌 Ｌ−トリプトファンシンセター
ゼ活性５Ｄ３０ １００Ｓ
Ｄ１０１２ ２１０これらの
菌に関してアントラニル酸（８０ｐｐｍ ）存在下にお
けるスビディゼン最小培地で培養（３７℃、１．５時間
）した時のＬ−トリプトファン蓄積結果を示す。Fungus L-tryptophan synthetase activity 5D30 100S
D1012 210 The results of L-tryptophan accumulation when these bacteria were cultured (37°C, 1.5 hours) in subvidizen minimal medium in the presence of anthranilic acid (80 ppm) are shown.
菌 株 Ｌ−トリプトファン蓄積
（μｇ／−）
バチルス嗜
アミロリクイ
ファシェンス
５Ｄ−３０３４
バチルス
５Ｄ１０１２ ７３本発明方法に従
えば、バチルス５Ｄ１０１２ ’にアントラニル酸又は
その塩を含む培地で培養することにａすＬ−トリプトフ
ァンを生成せしめることができる。栄養培地中のアント
ラニル酸又はその塩の濃度には特に限定はないが目的Ｌ
−トＩＪブトファンの収量、培養条件及び経済的観点
から一般には０．１〜３０００ｍｇ／ｌ、好ましくは１
００〜１００１００Ｏ／ Ｌの濃度とする。Bacterial strain L-tryptophan accumulation (μg/-) Bacillus amyloliquifacens 5D-3034 Bacillus 5D1012 73 According to the method of the present invention, Bacillus 5D1012' can be cultured in a medium containing anthranilic acid or its salt. L-tryptophan can be produced. There is no particular limitation on the concentration of anthranilic acid or its salt in the nutrient medium;
- Generally 0.1 to 3000 mg/l, preferably 1
The concentration should be between 00 and 100100 O/L.
本発明方法において使用することのできる培地としては
、前記微生物が培養によシ増殖し得るものであれば任意
のものでよく、例えば、炭素源としては、ブドウ糖、糖
蜜、蔗糖、澱粉、澱粉糖化液、セルロース分解物等の糖
類、酢酸、エチルアルコール、グリセリンなど、窒素源
としては、アンモニア、硫安、塩安、硝安、燐安なとの
アンモニウム塩や尿素、硝酸塩等が適宜使用される。無
機塩としては燐酸、カリウム、マグネシウム、マンガン
等の塩類、例えば燐酸アンモニウム、燐酸カリウム、燐
酸ナトリウム、硫酸マグネシウム、硫酸第一鉄、硫酸マ
ンガン、苛性カリ等の工業的薬品で良く、他に微量元素
としてカルシウム、亜鉛、硼素、銅、コバルト、モリブ
デン等の塩類を添加してもよく、また微量有機栄養素と
してビタミン、アミノ酸、核酸関連物質等は菌の成育上
特に必要ではないが、これらを添加したり、肉エキス、
酵母エキス、コーンステイープリカー、ヘフトン等の有
機物を添加してもよい。アントラニル酸はナトリウム塩
、カリウム塩、アンモニウム塩等の水溶液や遊離酸のエ
タノール又はメタノール溶液として添加すれば良い。The medium that can be used in the method of the present invention may be any medium as long as the microorganisms can be cultured and grown. For example, the carbon source may be glucose, molasses, sucrose, starch, or starch saccharification. As the nitrogen source, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphorus, urea, nitrates, etc. are used as appropriate. Examples of inorganic salts include salts such as phosphoric acid, potassium, magnesium, and manganese, as well as industrial chemicals such as ammonium phosphate, potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, and caustic potash, as well as other trace elements. Salts such as calcium, zinc, boron, copper, cobalt, and molybdenum may be added, and trace organic nutrients such as vitamins, amino acids, and nucleic acid-related substances are not particularly necessary for the growth of bacteria, but these may be added. , meat extract,
Organic substances such as yeast extract, cornstarch liquor, and hefton may also be added. Anthranilic acid may be added as an aqueous solution of sodium salt, potassium salt, ammonium salt, etc., or an ethanol or methanol solution of the free acid.
本発明方法における培養は好気的条件下に、例えば通気
攪拌や往復振盪方法によって培養することができる。あ
るいは固定化菌体法なども用いることができるのはいう
までもない。培養条件は、特に限定はないが、一般的に
言えば、温度３０〜４５℃、ＰＩ（６，０〜８．０及び
１５〜６０時間程度の条件で実施する。Culture in the method of the present invention can be carried out under aerobic conditions, for example, by aerated stirring or reciprocating shaking. It goes without saying that an immobilized bacterial cell method can also be used. Culture conditions are not particularly limited, but generally speaking, the culture is carried out at a temperature of 30 to 45°C, a PI of 6.0 to 8.0, and a period of 15 to 60 hours.
培養液又は培養物からの目的のＬ −） ＩＪブトファ
ンの採取方法は慣用方法に従って行うことができる。例
えば、培養液を遠心分離し、その上清からイオン交換樹
脂処理法、活性炭処理法などの操作を適宜組み合せてＬ
−ト１７プトフアンを単離することができる。The target L-)IJ butophane can be collected from the culture medium or culture according to a conventional method. For example, the culture solution is centrifuged, and the supernatant is treated with an ion exchange resin, activated carbon, etc. in an appropriate combination.
-17ptophan can be isolated.
実施例
以下に本発明の詳細な説明するが、本発明の範囲をこれ
らの実施例に限定するものでないことはいうまでもない
。EXAMPLES The present invention will be described in detail below, but it goes without saying that the scope of the present invention is not limited to these Examples.
例 １
グルコース５％、硫安０．２％、Ｋ２ＨＰＯ４１゜４％
、ＫＩ（２ＰＯ４０，６％、クエン酸ナトリウム・２Ｈ
２ｏ１ｇ、ＭｇＳＯ４・７Ｈ２ｏ０．０２％、ＦｅＳＯ
４・７７Ｈ２Ｏ１ｐｐ及びＭｎＳＯ４１Ｐｐｍ’含む液
体借地（、Ｈ７，０）２Ａにアントラニル酸８００ ｐ
ｐｍ ｆ添加し、これに上で得たバチルス５Ｄ１０１２
を植菌し、３５℃で５ｔのジャーファーメンタ−で通気
攪拌培養した。Example 1 Glucose 5%, Ammonium sulfate 0.2%, K2HPO41°4%
, KI (2PO40.6%, sodium citrate, 2H
2o1g, MgSO4・7H2o0.02%, FeSO
800 p of anthranilic acid in 2A of liquid (H7,0) containing 1 ppp of 4.77 H2O and 1 Ppm' of MnSO4.
Bacillus 5D1012 obtained above was added with pm f.
was inoculated and cultured with aeration and agitation at 35°C in a 5 t jar fermentor.
培養中、アントラニル酸濃度が５０ ｐｐｍまで減少し
た時点でアントラニル酸濃度が約１０００ ｐｐｍにな
るように適宜追加添加し、また培養途中グルコースを１
００ｇ追加し、更にアンモニア水の添加により、借地の
、Ｈを７．０±０．４に保ちながら１５時間培養した。During the culture, when the anthranilic acid concentration decreased to 50 ppm, additional addition was made as appropriate to bring the anthranilic acid concentration to about 1000 ppm, and during the culture, glucose was added at 1 liter.
00g was added, and the culture was continued for 15 hours while maintaining the H of the borrowed area at 7.0±0.4 by adding ammonia water.
Ｌ −ト！Ｊブトファンの蓄積は９．９９／ｌで平行運
転したバチルスアミロリクイファシェンス５Ｄ３０株の
２．１１倍を示した。L-to! The accumulation of J-butophane was 2.11 times that of Bacillus amyloliquefaciens 5D30 strain operated in parallel at 9.99/l.
第１図はｐｓＤ３１７１ 、第２図はｐｓＤ３６５１及
び第３図はｐｓＤＴ３９１のそれぞれ制限酵素地図を示
す。FIG. 1 shows the restriction enzyme maps of psD3171, FIG. 2 shows the restriction enzyme maps of psD3651, and FIG. 3 shows the restriction enzyme maps of psDT391.
Claims (1)
するＤＮＡ断片を複数個連結させた部分を有するＤＮＡ
分子を用い、該ＤＮＡ分子をバチルス属に属する微生物
から選ばれる宿主菌の染色体ＤＮＡに挿入してなるＬ−
トリプトファン生産性形質転換菌。 ２）宿主菌がトリプトファンアナログ耐性を有すること
を特徴とする特許請求の範囲第１項に記載のＬ−トリプ
トファン生産性形質転換菌。 ３）特許請求の範囲第１項に記載の形質転換菌を培地に
培養して培養物中にＬ−トリプトファンを生成せしめこ
れを採取することを特徴とするＬ−トリプトファンの製
造法。 ４）培地がアントラニル酸またはその塩を含むことを特
徴とする特許請求の範囲第３項に記載のＬ−トリプトフ
ァンの製造法。[Scope of Claims] 1) DNA having a portion in which multiple DNA fragments having genetic information related to L-tryptophan biosynthesis are linked together
molecule and inserting the DNA molecule into the chromosomal DNA of a host bacterium selected from microorganisms belonging to the genus Bacillus.
Tryptophan-producing transformed bacteria. 2) The L-tryptophan-producing transformed bacterium according to claim 1, wherein the host bacterium has tryptophan analog resistance. 3) A method for producing L-tryptophan, which comprises culturing the transformed bacterium according to claim 1 in a medium to produce L-tryptophan in the culture and collecting the L-tryptophan. 4) The method for producing L-tryptophan according to claim 3, wherein the medium contains anthranilic acid or a salt thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2802886A JPS62186786A (en) | 1986-02-13 | 1986-02-13 | L-tryptophan-producing microbial strain produced by transformation and production of l-tryptophan |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2802886A JPS62186786A (en) | 1986-02-13 | 1986-02-13 | L-tryptophan-producing microbial strain produced by transformation and production of l-tryptophan |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62186786A true JPS62186786A (en) | 1987-08-15 |
Family
ID=12237289
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2802886A Pending JPS62186786A (en) | 1986-02-13 | 1986-02-13 | L-tryptophan-producing microbial strain produced by transformation and production of l-tryptophan |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62186786A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003010334A1 (en) * | 2001-07-10 | 2003-02-06 | Apogene Biotechnologie Gmbh & Co. Kg | Method for identifying reading frame mutations |
-
1986
- 1986-02-13 JP JP2802886A patent/JPS62186786A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003010334A1 (en) * | 2001-07-10 | 2003-02-06 | Apogene Biotechnologie Gmbh & Co. Kg | Method for identifying reading frame mutations |
| US7368235B2 (en) | 2001-07-10 | 2008-05-06 | Apogene Biotechnologie Gmbh & Co. Kg | Method for identifying reading frame mutations |
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