CN102093997B - Method for producing doramectin by modifying streptomyces avermitilis - Google Patents
Method for producing doramectin by modifying streptomyces avermitilis Download PDFInfo
- Publication number
- CN102093997B CN102093997B CN 201010568764 CN201010568764A CN102093997B CN 102093997 B CN102093997 B CN 102093997B CN 201010568764 CN201010568764 CN 201010568764 CN 201010568764 A CN201010568764 A CN 201010568764A CN 102093997 B CN102093997 B CN 102093997B
- Authority
- CN
- China
- Prior art keywords
- gene
- sequence
- starting module
- pnp4
- pnp1
- 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.)
- Expired - Fee Related
Links
- QLFZZSKTJWDQOS-YDBLARSUSA-N doramectin Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O3)C=C[C@H](C)[C@@H](C3CCCCC3)O4)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C QLFZZSKTJWDQOS-YDBLARSUSA-N 0.000 title abstract description 7
- 229960003997 doramectin Drugs 0.000 title abstract description 7
- 238000004519 manufacturing process Methods 0.000 title abstract description 5
- 241001468227 Streptomyces avermitilis Species 0.000 title abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 112
- 238000000034 method Methods 0.000 claims abstract description 33
- -1 pnAT1ACP1 and pnP1 Chemical compound 0.000 claims abstract description 20
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108091008053 gene clusters Proteins 0.000 claims abstract description 8
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 6
- 241001655322 Streptomycetales Species 0.000 claims description 37
- 239000011574 phosphorus Substances 0.000 claims description 31
- 229910052698 phosphorus Inorganic materials 0.000 claims description 31
- YZEUHQHUFTYLPH-UHFFFAOYSA-N 2-nitroimidazole Chemical compound [O-][N+](=O)C1=NC=CN1 YZEUHQHUFTYLPH-UHFFFAOYSA-N 0.000 claims description 30
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 30
- 239000013612 plasmid Substances 0.000 claims description 24
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims description 22
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 claims description 19
- 239000005516 coenzyme A Substances 0.000 claims description 19
- 229940093530 coenzyme a Drugs 0.000 claims description 19
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 claims description 19
- 108010030975 Polyketide Synthases Proteins 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 16
- 229960003276 erythromycin Drugs 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 10
- 238000012546 transfer Methods 0.000 claims description 9
- 239000004927 clay Substances 0.000 claims description 8
- 230000003570 biosynthesizing effect Effects 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 3
- 238000006073 displacement reaction Methods 0.000 claims 1
- 238000011144 upstream manufacturing Methods 0.000 claims 1
- 230000001851 biosynthetic effect Effects 0.000 abstract description 14
- 239000005660 Abamectin Substances 0.000 abstract description 8
- IBSREHMXUMOFBB-JFUDTMANSA-N 5u8924t11h Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O3)C=C[C@H](C)[C@@H](C(C)C)O4)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 IBSREHMXUMOFBB-JFUDTMANSA-N 0.000 abstract description 6
- 229950008167 abamectin Drugs 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 5
- 230000004151 fermentation Effects 0.000 abstract description 5
- QRSKGVRHSLILFG-TYHXJLICSA-N cyclohexane-1-carbonyl-CoA Chemical compound O=C([C@H](O)C(C)(COP(O)(=O)OP(O)(=O)OC[C@@H]1[C@H]([C@@H](O)[C@@H](O1)N1C2=NC=NC(N)=C2N=C1)OP(O)(O)=O)C)NCCC(=O)NCCSC(=O)C1CCCCC1 QRSKGVRHSLILFG-TYHXJLICSA-N 0.000 abstract 2
- 229930187140 phoslactomycin Natural products 0.000 abstract 2
- 102000003960 Ligases Human genes 0.000 abstract 1
- 108090000364 Ligases Proteins 0.000 abstract 1
- 229930001119 polyketide Natural products 0.000 abstract 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 21
- 241000894006 Bacteria Species 0.000 description 17
- 230000000968 intestinal effect Effects 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 238000006555 catalytic reaction Methods 0.000 description 8
- 230000008676 import Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 101100244439 Alkaliphilus metalliredigens (strain QYMF) pnp4 gene Proteins 0.000 description 5
- 101100244438 Alkaliphilus metalliredigens (strain QYMF) pnp3 gene Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 230000001131 transforming effect Effects 0.000 description 4
- 102000057234 Acyl transferases Human genes 0.000 description 3
- 108700016155 Acyl transferases Proteins 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 3
- 102000013142 Amylases Human genes 0.000 description 3
- 108010065511 Amylases Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 235000019418 amylase Nutrition 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 239000013605 shuttle vector Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- CWGATOJEFAKFBK-UHFFFAOYSA-N Ac-(E)-8-Tridecen-1-ol Natural products C1C(O)C(C)C(C(C)CC)OC11OC(CC=C(C)C(OC2OC(C)C(OC3OC(C)C(O)C(OC)C3)C(OC)C2)C(C)C=CC=C2C3(C(C(=O)O4)C=C(C)C(O)C3OC2)O)CC4C1 CWGATOJEFAKFBK-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 2
- 108010047524 Deoxyribonuclease HindIII Proteins 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000000749 insecticidal effect Effects 0.000 description 2
- AEWHYWSPVRZHCT-NDZSKPAWSA-N isobutyryl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C(C)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 AEWHYWSPVRZHCT-NDZSKPAWSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 229920001470 polyketone Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 125000004493 2-methylbut-1-yl group Chemical group CC(C*)CC 0.000 description 1
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- CWGATOJEFAKFBK-PDVFGPFMSA-N 5-o-demethyl-22,23-dihydro-23-hydroxy-(13r,23s)-avermectin a1a Chemical compound C1[C@H](O)[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 CWGATOJEFAKFBK-PDVFGPFMSA-N 0.000 description 1
- 101710146995 Acyl carrier protein Proteins 0.000 description 1
- 108700037654 Acyl carrier protein (ACP) Proteins 0.000 description 1
- 102000048456 Acyl carrier protein (ACP) Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 235000003276 Apios tuberosa Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000010744 Arachis villosulicarpa Nutrition 0.000 description 1
- 101000596279 Bacillus subtilis Type II restriction enzyme BglII Proteins 0.000 description 1
- 108010089921 CTCGAG-specific type II deoxyribonucleases Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 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
- 241000238631 Hexapoda Species 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- GAIPQMSJLNWRGC-MZAVDHTQSA-N Phoslactomycin B Chemical compound CC[C@H]1C=CC(=O)O[C@H]1\C=C\[C@](O)(CCN)[C@H](OP(O)(O)=O)C[C@@H](O)\C=C/C=C\C1CCCCC1 GAIPQMSJLNWRGC-MZAVDHTQSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 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
- 230000002141 anti-parasite Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 1
- SHURRSUZDBDBMX-JLSLGBNPSA-N avermectin B2a Natural products CC[C@H](C)[C@H]1O[C@@]2(C[C@@H]3C[C@@H](CC=C(/C)[C@@H](O[C@H]4C[C@H](OC)[C@@H](O[C@H]5C[C@H](OC)[C@@H](O)[C@H](C)O5)[C@H](C)O4)[C@@H](C)C=CC=C6/OC[C@@H]7[C@H](O)C(=C[C@@H](C(=O)O3)[C@]67O)C)O2)C[C@@H](O)[C@@H]1C SHURRSUZDBDBMX-JLSLGBNPSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- VZFUCHSFHOYXIS-UHFFFAOYSA-N cycloheptane carboxylic acid Natural products OC(=O)C1CCCCCC1 VZFUCHSFHOYXIS-UHFFFAOYSA-N 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 230000024835 cytogamy Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- RXUATCUKICAIOA-ZMHDXICWSA-N pentanoyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCCC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RXUATCUKICAIOA-ZMHDXICWSA-N 0.000 description 1
- GAIPQMSJLNWRGC-UHFFFAOYSA-N phoslactomycin B Natural products CCC1C=CC(=O)OC1C=CC(O)(CCN)C(OP(O)(O)=O)CC(O)C=CC=CC1CCCCC1 GAIPQMSJLNWRGC-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 101150002083 pnp4 gene Proteins 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a technique for producing doramectin by modifying an abamectin biosynthesis method by utilizing phoslactomycin biosynthetic gene, in particular a technique for producing the doramectin by modifying an abamectin producing strain. A polyketide synthetase starting module in the abamectin biosynthesis route is replaced, and a biosynthesis route for synthesizing cyclohexyl formyl-CoA is introduced, so that the streptomyces avermitilis has the capacity of producing the doramectin through fermentation. Genes used for replacing and coding the starting module, and coding biosynthetic enzyme of cyclohexyl formyl-CoA, namely pnAT1ACP1 and pnP1, pnP2, pnP3 and pnP4 come from a biosynthetic gene cluster of phoslactomycins. The invention also discloses method for producing doramectin mutant strains by utilizing the gene structures, and application thereof. The yield and purity of the doramectin obtained through corresponding mutant strains are greatly improved compared with those of unmodified strains.
Description
Technical field
The present invention relates to genetically engineered field and microbiology field, the method that relates to the class gene relevant with doractin production and operate such genes produce doractin.
Technical background
Doractin is described as the most outstanding insecticidal materials of Avrmectin family, is a kind of novel Macrolide anti-parasite medicine, nematode, insect and mite is all had efficiently kill effect.Have efficiently, consumption is little, the characteristics that safety range is high, and the relative Avrmectin of its biological activity, insecticidal activity is higher, and lower to the toxicity of human body and livestock, begins for subcutaneous injection at present.It only is that from the difference of Avrmectin the 25th bit substituent is different from chemical structure, abamectin Bla is a 2-methyl butyl at the 25th, and doractin is a cyclohexyl at the 25th, and causing the different major cause of this part-structure from biosynthetic angle is the substrate difference that catalysis forms the polyketide synthases starting module selection of this compound.
From the Avrmectin biosynthetic process, the starting module of Avrmectin comprises an acyltransferase (AT) and an acyl carrier protein (ACP), and their identification methylbutyryl-CoAs or isobutyryl coenzyme A are as start element.[Science (1998) 279 in existing work, 199-202] show that the biosynthetic starting module of Avrmectin has the wide in range property of selecting substrate, the at present production of doractin has also utilized this point just, namely by interrupting the biosynthetic pathway of start element, then add during the fermentation hexahydrobenzoic acid and reach the purpose that produces doractin as start element.But because the suitableeest substrate of starting module remains methylbutyryl-CoA or isobutyryl coenzyme A, so often be mixed with Avrmectin in the product.
The present invention is directed to the limitation of aforesaid method, by being replaced, Avrmectin biosynthesizing starting module becomes the starting module take hexahydrobenzoic acid salt as specific substrate [Chinese patent 201010547279.7] that derives from the biosynthetic polyketide synthases of phosphorus azomycin, and then the gene of coding collar hexyl formyl coenzyme A biosynthetic enzyme carried out heterogenous expression in the mutant strain of replacing, successfully having obtained producing doractin is main mutant strain.If in the process of fermentation, add again hexahydrobenzoic acid, by adding the supply of larger precursor, meeting is so that produce the amount of doractin and further improve, directly feed hexahydrobenzoic acid generation doractin with respect to original strain significant raising has been arranged, reduced the complicacy of the component of producing, and the component of producing mainly is doractin.
Summary of the invention
The purpose of this invention is to provide a kind of gene and method that Abamectin producing bacterium is produced doractin of transforming.
In one aspect of the invention, provide two kinds be positioned at phosphorus azomycin biosynthesis gene sequence with gene order and uses thereof.Become the gene of coding catalysis phosphorus azomycin biosynthesizing polyketone enzyme starting module by the Gene Replacement of the catalysis Avrmectin biosynthesizing polyketone enzyme starting module of in deinsectization streptomycete, will encoding, import again the gene that coding catalysis forms the enzyme of cyclohexyl formyl coenzyme A required in the phosphorus azomycin biosynthetic process, improved the output of doractin, described gene is selected from: encoding gene pnAT1ACP1 gene and the pnP1 of phosphorus azomycin polyketide synthases starting module, pnP2, pnP3, pnP4, and the gene of telling is from phosphorus azomycin biological synthesis gene cluster [Chinese patent 201010547279.7].
A second aspect of the present invention provides a kind of method that improves doractin expression amount in the deinsectization streptomycete by the supply that increases precursor.May further comprise the steps: in the deinsectization streptomycete genome, introduce the gene that coding catalysis forms the enzyme of cyclohexyl formyl coenzyme A, and the gene of telling is selected from: pnP1, pnP2, pnP3, the pnP4 gene adds hexahydrobenzoic acid in fermention medium.
In a preference of the present invention, described goal gene is replaced at the deinsectization streptomycete genome may further comprise the steps:
(a) structure contains the replacement carrier that needs are replaced the encoding gene pnAT1ACP1 gene of target gene 5 and 3 ' flanking sequence and phosphorus azomycin polyketide synthases starting module.
The host strain that (b) will contain the gene replacement vector is as F+strain, and the method by F+strain-streptomycete conjugal transfer imports in the deinsectization streptomycete, obtains containing the deinsectization streptomycete transformant of gene replacement vector.
(c) transformant obtains single cross through high temperature induction and changes mutant strain, single cross is changed the restructuring deinsectization streptomycete mutant strain that the homology double exchange has occured in the rear acquisition of going down to posterity again.
(d) the encoding gene pnAT1ACP1 gene 5 '-3 ' sequence of 5 ' of gene end flanking sequence, phosphorus azomycin polyketide synthases starting module is connected with gene in the described replacement carrier ' hold flanking sequence to connect in turn by said sequence and by above-mentioned direction.
Being used for making up the carrier that sets out of replacing carrier is any one intestinal bacteria-streptomycete shuttle vectors, such as pKC1139, and pKC505, pIJ653, pIJ8154 is preferably pKC1139.Gene replacement vector take pKC1139 as the vector construction that sets out is as pWJb7381.
In another preference of the present invention, described cyclohexyl formyl coenzyme A biosynthetic enzyme heterogenous expression in the strain of deinsectization streptomycete Substitution be may further comprise the steps:
(a) make up the heterogenous expression carrier that contains coding collar hexyl formyl coenzyme A complete genome sequence and erythromycin promotor.
The host strain that (b) will contain the gene replacement vector is as F+strain, and the method by F+strain-streptomycete conjugal transfer imports in the deinsectization streptomycete, obtains containing the deinsectization streptomycete transformant of gene replacement vector.
(c) transformant obtains mono-clonal with resistance through resistance screening, through its stability of test that goes down to posterity, obtains stable mutant strain again
(d) required heterogenous expression gene connects the erythromycin promoter sequence at its promoter region.According to being linked in sequence of PermE (erythromycin promotor)-pnP1-pnP4.
Be used for making up any one intestinal bacteria of the carrier-streptomycete shuttle vectors that sets out of heterogenous expression carrier, such as pKC1139, pKC505, pIJ653, pIJ8154, pSET152 is preferably pSET152.Gene replacement vector take pSET152 as the vector construction that sets out is as pWJb835.
Host cell is Bacillus coli cells in another preference of the present invention.
The 4th aspect of the present invention provides a kind of method of producing doractin, comprises following step:
(1) the above-mentioned deinsectization streptomycete through replacement and heterogenous expression cyclohexyl formyl coenzyme A synthetic enzyme of fermentation the present invention;
(2) from tunning, separate doractin.
In a preference of the present invention, the described gene that is replaced is arranged in the deinsectization streptomycete genome, and only has a copy.
In the preference of the present invention, be used for the gene of replacement and the gene of wish expression and all come from phosphorus azomycin biosynthesis gene sequence.
Aspect bright the 6th of we, the purposes of described deinsectization streptomycete through replacing and introduce cyclohexyl formyl coenzyme A biosynthetic pathway is provided, it is characterized in that for the production of doractin.
Other side of the present invention is because disclosure of the present invention is apparent for a person skilled in the art.
Description of drawings:
Fig. 1: Avrmectin biosynthesis gene starting module is replaced design.
Phoslactomycin B: phosphorus azomycin, Avermectin: Avrmectin, Doramectin: doractin, Cyclohexanecarboxylic acid: hexahydrobenzoic acid
Fig. 2: the building process of aveAT1ACP1 gene substitution plasmid pWJb7381 in the Avrmectin biosynthesis gene sequence in the replacement deinsectization streptomycete.
PWJb6-24-4, pWJb6-24-3, pWJb7-62, pWJb6-24-1, pWJb7381: certainly name recombinant plasmid; Derived from pANT841: setting out with carrier pANT841 makes up derived frompGEM-5zf: with the carrier pGEM-5zf structure that sets out; Derived from pKC1139: with the carrier pKC1139 structure that sets out, derived from pAGe-1: with the carrier pAGe-1 structure that sets out.Restriction enzyme site: HindIII, BglII, XbaI, XhoI.
Fig. 3: the building process of the plasmid pWJb835 of heterogenous expression cyclohexyl formyl coenzyme A.
PnP1, pnP2, pnP3, pnP4: the gene of coding collar hexyl formyl coenzyme A synthetic enzyme; Primer 7, primer8, and primer 9, and primer 10: primer 7, primer 8, primer 9, primer 10; PWJb7981, pWJb7982, pWJb7942, pWJb7100, pWJb806, pWJb835: certainly name recombinant plasmid; Derived from pANT841: with the carrier pANT841 structure that sets out; Derived from pGEM-5zf: with the carrier pGEM-5zf structure that sets out; Derived from pSP72: with the carrier pSP72 structure that sets out, derived from pSET152: with the carrier pSET152 structure that sets out.Restriction enzyme site: HindIII, SpeI, BamHI, XbaI, EcoRI.PermE: erythromycin promoter gene.
Fig. 4: the PCR checking of aveAT1ACP1 gene substitution mutant strain.Amplifying the sequence of about 1.2Kb with primer pnLDF:5 '-TTTCTCGAGCGGGTCGAGGTGATCCAG-3 ' and primer pnLDR:5 '-TTTAGATCTCAGTTCCTCCGCCAGCGC-3 ', is the primer sequence verification with pnLDF and pnLDR after cutting glue and reclaiming. Swimming lane 1,3,7,9,10,11,12,15,18:1.2kb zone display, swimming lane 13:5000bp marker (being followed successively by from top to bottom 5KB, 3Kb, 2Kb, 1Kb, 750bp, 500bp)
Fig. 5: the analysis contrast of final mutant strain and wild type strain tunning.(A) HPLC collection of illustrative plates; (B) column comparison diagram, 1 represents wild-type, and the final mutant strain of 2 representatives all adds hexahydrobenzoic acid (200mg/L) in fermention medium.Ave B2a: avermectin B2a component, Ave Bla: abamectin Bla component, Dor B2: doractin B2 component, Dor: doractin component.
Embodiment
The inventor has found that through extensive and deep research five can be used for transforming the gene that deinsectization streptomycete (Avrmectin) is produced doractin, and described gene is the encoding gene pnAT1ACP1 of phosphorus azomycin polyketide synthases starting module, pnp1, pnp2, pnp3, pnp4 gene.The encoding gene pnAT1ACP1 gene of described phosphorus azomycin polyketide synthases starting module is replaced the aveAT1ACP1 that catalysis forms the synthetic enzyme of Avrmectin in the deinsectization streptomycete genome, in fermention medium, add hexahydrobenzoic acid, can greatly improve the output of doractin.Finished the present invention based on above.Described gene is from the biosynthetic gene cluster of phosphorus azomycin.As used herein, the albumen of used genes encoding is for participating in skeleton and the synthetic secondary metabolism process of precursor in the phosphorus azomycin biosynthetic process, and the new gene of introducing can not affect the growth conditions of deinsectization streptomycete.
In the present invention, used " pnAT1ACP1 " refers in phosphorus azomycin biosynthetic process, the acyltransferase of the selection of responsible selection start element and the gene of acyl carrier protein in the coding catalysis phosphorus azomycin skeleton building-up process.It has controlled the start element that uses in the phosphorus azomycin biosynthetic process, and the present invention falls the module gene that the responsible Avrmectin start element of coding is selected in the Avrmectin biosynthetic process in the deinsectization streptomycete with this Gene Replacement, after replacing, do not affect the growth conditions of deinsectization streptomycete.
In the present invention, the biosynthetic gene of synthesizing cyclohexyl formyl coenzyme A " pn1; pn2; pn3; pn4 " refers in phosphorus azomycin biosynthetic process in the used phosphorus azomycin gene cluster, coding catalysis forms the gene of the enzyme of start element cyclohexyl formyl coenzyme A, the present invention with these four genes according to the order from pn1-pn4, after the initiation region adds an erythromycin promotor, after being building up to integrating vector pSET152, import in the mutant strain that the deinsectization streptomycete replaced replaced, form final mutant strain, the introducing of this approach can not affect the growth conditions of deinsectization streptomycete.
In the present invention, " deinsectization streptomycete " (Streptomyces avermitilis) is a kind of bacterium that can produce Avrmectin (avermectins AVM), and only produces Avrmectin B component through transforming.
In the present invention, intestinal bacteria-streptomycete shuttle vectors refers to that a class can forward plasmid in the streptomycete to by intestinal bacteria, comprises (but being not limited to) pKC1139, pSET152.
In the present invention, " transgenosis " refers to the genetic material of external source is imported to process in the recipient bacterium by the donor bacterium, and it can be realized by means such as conversion, conjugal transfer, transduction, cytogamy." conjugal transfer " refers to that bacterium trafficability characteristic pili interconnects communication, and genetic material (mainly being plasmid DNA) is transferred to recipient bacterium from the donor bacterium.
The phosphorus azomycin biosynthetic enzyme starting module gene pnAT1ACP1 that the present invention is used for replacing is shown in nucleotides sequence tabulation sequence 1;
The present invention is used for the biosynthetic gene pn1-pn4 of phosphorus azomycin gene cluster synthesizing cyclohexyl formyl coenzyme A of heterogenous expression shown in nucleotides sequence tabulation sequence 2.
The construction process of replacement plasmid provided by the invention, system adds by carrier with in multiple clone site needs the goal gene flanking sequence of replacing and the gene order that is used for replacing to form, carrier is intestinal bacteria and streptomycete shuttle plasmid, can be pKC1139, p0J260, pWHM3 etc. and similar carrier thereof.
The construction process of heterogenous expression plasmid provided by the invention, system is added in multiple clone site by carrier needs the goal gene of heterogenous expression and erythromycin promoter gene sequence to form, being entitled as intestinal bacteria and streptomycete shuttle plasmid, can be its similar carriers such as pSET152.
Further be described below:
Gene of the present invention can strengthen the biosynthesizing of doractin, after being incorporated into this genoid in the deinsectization streptomycete, (replacing Avrmectin biosynthesizing starting module and directly import coding collar hexyl formyl coenzyme A biosynthetic enzyme genes), can improve the output of doractin.And the inventor has selected suitable conjugal transfer carrier, namely with the plasmid pKC1139 of temperature sensitive type replicon, utilize homologous recombination technique, obtained stable Gene Replacement mutant strain, by utilizing stable integrating vector pSET152 to import the biosynthetic pathway of cyclohexyl formyl coenzyme A, obtain stable final mutant strain again.
When detecting Avrmectin output, can be standard by the content of measuring the active principle Bla (avemectin Bla) that starting strain and improved strain produce, whether generation and the output of detection doractin improve.
Be used for the numerals such as the shown composition consumption of embodiment or other guide, reaction conditions among the present invention and be about numerical value.Unless therefore dated especially in the literary composition, the above-mentioned digital parameters of this specification sheets is approximation, it can be changed according to the required result of the present invention who obtains.
Except specifying.The same meaning that used technical term and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the method for the present invention.Not marked experimental technique in the experiment, usually according to people such as normal condition such as Sambrook, molecular cloning: condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) or the condition of advising according to manufacturer.
Principal feature of the present invention is:
1. found that two kinds are applied to transform the gene that the deinsectization strepto-produces doractin.
2. find that with Avrmectin biosynthesizing starting module Gene Replacement be the output that the encoding gene pnAT1ACP1 gene of phosphorus azomycin polyketide synthases starting module can improve the doractin of deinsectization streptomycete greatly.
3. find cyclohexyl formyl coenzyme A biosynthetic enzyme genes is imported to the output that can further improve doractin in the Substitution strain
4. find when mutant strain ferments, in fermention medium, to add hexahydrobenzoic acid, can suppress the generation of Avrmectin component, improve the quantity of component.
5. the present invention adopts the means of conjugal transfer will replace plasmid and the heterogenous expression plasmid imports in the recipient bacterium, induces the generation double exchange, has obtained stable genotype mutant strain.
Further specify the present invention below in conjunction with specific embodiment.It should be understood that used embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.
Used medium among the embodiment, except specified otherwise, liquid is all used the YEME substratum, and solid is all used the MS substratum.
Embodiment one: replace the aveAT1ACP1 gene
1: about the exchange homology arm acquisition
Take the genome of Streptomyces avermitilis as template, adopt primer 5 '-TTTAAGCTTACGCCCGGCGCGGGCTGAG-3 ' and primer 5 '-TTTCTCGAGCCCGGGAGCGTCCGGCGAG-3 ' amplification to obtain the PCR product of the about 2kb of left arm, flush end is connected in the EcoRV otch in the multiple clone site of pGEM-5zf, use primer 5 '-TTTAGATCTGCGGCGCTCGGCCACCTC-3 ' and primer 5 '-TTTTCTAGAATCCCCCGCGTCCCGGCG-3 ' to obtain the PCR product of the about 2kb of right arm, flush end is connected in the multiple clone site StuI otch of pANT841 again.
2: the acquisition of the encoding gene pnAT1ACP1 fragment of phosphorus azomycin polyketide synthases starting module
Take the clay that includes this fragment gene as template, adopt primer 5 '-TTTCTCGAGCGGGTCGAGGTGATCCAG-3 ' and primer 5 '-TTTAGATCTCAGTTCCTCCGCCAGCGC-3 ' amplification obtain the PCR product of about 1.15Kb, flush end is connected in the EcoRV otch in the polyclone position of pGEM-5zf.
3: the acquisition of recombinant plasmid pWJb7381
Left arm is cut out with endonuclease HindIII and XhoI from carrier, right arm is cut out from the plasmid that makes up with endonuclease BglII and XbaI, the encoding gene pnAT1ACP1 of phosphorus azomycin polyketide synthases starting module is cut out with BglII and XhoI from carrier, resulting fragment is connected among the pKC1139 that cuts with HindIII and XbalI together, specifically makes up visible accompanying drawing 1.
4: the acquisition of replacing aveAT1ACP1 transgenation strain
To be transferred to intestinal bacteria ET12567 for the recombinant plasmid pWJb7381 that replaces, by engaging between the genus between intestinal bacteria and the streptomycete to shift recombinant plasmid is imported in the streptomycete, 37 ℃ of integration relax afterwards and induce the generation double exchange, the double exchange mutant strain utilizes the PCR checking, the genotype the result is seen Fig. 2, because the gene fragment itself of replacing is more or less the same with original gene, sequence verification after therefore the object tape that amplifies being reclaimed obtains mutant strain 2.
Embodiment two: the biosynthetic pathway that imports cyclohexyl formyl coenzyme A
1: the acquisition of gene fragment
Take the clay that comprises this gene fragment as template, obtain the fragment of the about 1Kb of 5 ' end of pnp1 with primer 5 '-AAGCTTACCGACGCCATCGAATCG-3 ' and primer 5 '-CCTGCTGGATCCGGACAC-3 ' amplification, flush end is connected in the StuI otch in the pANT841 multiple clone site.Cut above-mentioned template clay with endonuclease BamHI and EcoRI, obtain a 3 ' end part and a complete pnp2 who has comprised pnp1,5 ' the end part of pnp3 and pnp4 is the fragment of 5.482Kb altogether, and it is connected among the BamHI and EcoRI double digestion otch in the pSP72 multiple clone site.With primer 5 '-GGCGGAATTCGCCGAGAC-3 ' and primer 5 '-TCTAGACGCACGTTCTCACCCCAC-3 ', take above-mentioned clay as template, amplification obtains the about 800bp sequence of part of pnp4 remainder, and flush end is connected in the EcoRV otch in the pGEM-5zf multiple clone site.
2: the acquisition of erythromycin promoter gene sequence
Cut the plasmid pLL6214 that contains the erythromycin promotor with endonuclease HindIII and EcoRI, obtain the erythromycin promoter sequence of about 500bp
3: the structure of heterogenous expression plasmid pWJb835
5 ' the end of pnp1 is cut out with HindIII and BamHI, include the pnp1 remaining part, pnp2,5 of pnp3 and pnp4 ' holds the fragment of a part to cut out with BamHI and EcoRI, three fragments are connected among the HindIII and EcoRI double digestion otch in the multiple clone site of carrier pGEM-3zf, again will be even this good fragment (comprise complete pnp1, pnp2, the fragment of the 5 ' end of pnp3 and pnp4) cuts out with HindIII and EcoRI and to obtain about 6.5Kb fragment, 3 ' the end of pnp4 is cut out with XbaI and EcoRI from cloning vector, these two fragments are connected among the HindIII and XbaI double digestion otch in the multiple clone site of pGEM-3zf.Again this plasmid is cut out with HindIII and XbaI and obtain about 7.3Kb fragment of (comprising complete pnp1-pnp4), together with erythromycin promoter gene sequence (cutting out with HindIII and EcoRI), be connected among the XbaI and EcoRI double digestion otch in the pSET152 multiple clone site, obtain final recombinant plasmid pWJb835.
4: the acquisition of final mutant strain
To be transferred to intestinal bacteria ET12567 for the recombinant plasmid pWJb835 of heterogenous expression, by engaging between the genus between intestinal bacteria and the streptomycete to shift recombinant plasmid is imported in the streptomycete mutant strain 2, obtain the mutant strain with the A Bula resistance, Here it is final mutant strain.
3: mutant strain and original starting strain produce the doractin potency ratio and measure
The component analysis that bacterial strain after contrast starting strain under the identical fermentation condition and sudden change produces doractin Bla.
After the spore of keeping thawed; be applied on the plate culture medium, the plate culture medium prescription is: glucose (Chemical Reagent Co., Ltd., Sinopharm Group) 1.5%, aspargine (Huamei Bio-Engrg Co.) 0.05%, imported beef medicinal extract (China Medicine (Group) Shanghai Chemical Reagent Co.) 0.3%, potassium primary phosphate (Tianjin chemical reagent six factories) 0.05%, agar 1.8%.Cultivate after five days, get a fritter with punch tool and be put in kind of the bottle for 30 ℃.
The seed culture based formulas is: W-Gum 3%, bean cake powder 0.8%, groundnut meal 1%, yeast extractive substance 0.4%, cobalt chloride (China Medicine (Group) Shanghai Chemical Reagent Co.,, 1% solution) 0.3%, amylase 4 μ/g starch.
During preparation first with corn starch pasting (a small amount of water dissolution of W-Gum, add amylase, stir, pour in the boiling water of about 1/2 total amount, boil), add other raw materials (also mixing well with cold water), adjust pH 6.8～7 behind the constant volume, packing 40ml/250ml triangular flask, 121 ℃ of sterilization 25min.28 ℃, 200rpm cultivates two days later, draws 1.5ml in shaking flask.
Fermentative medium formula is: W-Gum 14%, bean cake powder 2.8%, yeast powder 1%, Sodium orthomolybdate (1%) 2.2 ‰, manganous sulfate (0.1%) 2.3 ‰, ammonium sulfate (5%) 5 ‰, cobalt chloride (1%) 2 ‰.
First W-Gum is mixed well with cold-water solution during preparation, add amylase (1.5～2 units do not restrain starch), heating in water bath, thinning rear timing insulation 12min is (in temperature-rise period, feed liquid can become translucent starchiness by oyster white, begin timing this moment), take out, add other raw material (mixing well with cold water first), add hexahydrobenzoic acid (200mg/L), constant volume, transfer pH to 7.5, then add calcium carbonate 0.8 ‰, stir, packing 30ml/250ml triangular flask while stirring, 121 ℃ of sterilization 25min.
Shaking flask was cultivated ten days under the condition of 200rpm at 28 ℃, got 1ml bacterium liquid, added 2ml methyl alcohol, ultrasonic 10min, and the centrifuging and taking supernatant, HPLC detects.
Testing conditions is: detect wavelength: UV=245nm; Pillar: VARIAN 250/4.6SN 282566MICROSORB-MV 100-5C18R008620005; Moving phase condition: v=1mL/min; A solution=H
2O; B solution=CH
3OH; Elution requirement is 85%B, 15%A, and elution time is 20min.
Analyze HPLC result and can find out that the output of original starting strain doractin is about 15ug/ml, the output of mutant strain is 60ug/ml, and volume increase is 300%.Concrete HPLC collection of illustrative plates and yield comparison column diagram are seen accompanying drawing 5.
Claims (3)
1. transform the method that deinsectization streptomycete produces doractin for one kind, it is characterized in that utilizing the encoding gene aveAT1ACP1 of the encoding gene pnAT1ACP1 displacement Avrmectin polyketide synthases starting module of phosphorus azomycin polyketide synthases starting module, simultaneously with biosynthesis gene pnP1-pnP4 heterogenous expression in replacement mutant of synthesizing cyclohexyl formyl coenzyme A in the phosphorus azomycin gene cluster;
Wherein, the nucleotide sequence of the encoding gene pnAT1ACP1 of described phosphorus azomycin polyketide synthases starting module is as follows:
cgggtcgagg tgatccagcc ggccctgttc gccgtcatgg tgtcgctcgc ggagctgtgg 60
cggtcgaacg gcgtcacgcc cgccgcggtc gtgggccaca gtttcggtga gatcgcggcg 120
gtgaccgccg ccggagcgct caccctggcg gacggcgcaa ggctggtcgc cgccgtcagc 180
aaggcactgg cacagctcca gggcaacgga gagatggtcg ccgtggcgct ccccgacgcg 240
caggtcaccg aactggtcgc cgagtgggat ctggacctgg acatcgcggt cgtcaacggc 300
cctcgttcga cggtcgtcgc gggccccacc gaggccgcga cagccttggt ggagcggctg 360
cgggaccggg acgtgcgggc cacactgctg cccatcggga tcgccgggca ctcgcggcgc 420
atggagcccg cccacgcata cctggtacag gaggcctcgg cggtacggcc gcgtgccacc 480
gggatacccg tgtacacgtc cacgacgacc gatcctctcg acaccggcga cctggacgcc 540
gagcactggt tccacagcct tcgggagccc gcccggttcc agcaggtcat cgaggaactg 600
ctcggccagg gccaccgggt gttcgtggag atgagcccgc acccggtgct cgccctgtcg 660
atcgaggaga ccgccgcgca cctcggccgg gacgtcgtcg tcctcgaaac gatgcgtcgt 720
gacgacgcgg gccacgaccg ctacctacgc gccctggccg aggcacacct gcacggcgtc 780
gcccccgact ggagcaccgt cctccccgac gcacgccgcg tcacgctgcc tccgtaccgg 840
ctcgacctgg acacggcgga cgccgccggc agcggcggca ccgagccggg cgccggactc 900
cgcgagcggc tgctgggggt cgccccggaa cgacgcatcg aagaggccgt acgcctcgtg 960
gtggacgccg tggccgcgca catcgagccg tcggcgacgg ccatcggcgc ggaccaggcg 1020
ttccggtcgc tgggggtgga ctccgcgggc gccctccggg tccgcaaccg gctggtcgag 1080
acgaccgggc tgcggctgcc cgcgacgatc ctgttcgacc accccactcc gcgcgcgctg 1140
gcggaggaac tg 1152
The biosynthesis gene of synthesizing cyclohexyl formyl coenzyme A in the described phosphorus azomycin gene cluster
The nucleotide sequence of pnP1-pnP4 gene is as follows:
gacaccgacg ccatcgaatc ggcgaccgcc gaagaacttc tcgcattcgt cgagaagcat 60
ttcgactagt tccccggtcc cgcgcaggag cgggaccgcg ccgcagcatt aggggccact 120
aggggttagt gaatcgatcc aataccggag agcattcatc aagaggatct gcgaaatgcc 180
taggagtgaa catatcccat gcaccttctc gtcaagggag ttcagcaccg gatttccagc 240
gaaatcctcg taccgaactc caaatatcat gcacaccgtg cgctgatcct cgcttcgctc 300
gcggacggag agagccgcat ccacggcctg tccgacgccc ggcacgtcga gtacaccgtt 360
cggctgctgc gcgacctcgg cgtacagatc gtccgcgagg gcgacacctt tgtggtgcgg 420
gggctcggcg gccactaccg gccggtccgg gacaccgtct cggccggcag ctccggcacc 480
accctctatt tcatggccgg gctcgcgtcc ctgtcgaacc gcgcggtcac gatcaccggg 540
cagaaatact ttcggaaacg ccccgtgggt ccgctgctgc gcgcgctgga acagctgggc 600
gtacgcctgg agtcggtaaa tgactgcccg cccattcagg tccgaagcca gcggcccacg 660
ggcggcgagg tgaccattcc gggcaccttg tcccagtgga tatccggcct gctgctgctc 720
gcgccgttcg ccaccggccc caccgtcatc accgtggcgg gcacgctgaa cgaacgcacc 780
tatctggagc tgaccgtcgc gatgatgcgg cagttcggcc tggaagtgac cgtggccccc 840
gactggcggc gcttcgacgt ggcgccgcac cagagcgtgc ggcccaccga gctgacgttg 900
ccccccgaca tcggttccgc ggccttcgcc gtcgccaccg ccgcgctgca cccctccgac 960
gtactcctgc gcggtatgcc gagcctgacc ggtggccccg ccgaccaccc cgagttccac 1020
ttcatcgaca tagcacgctc gatgggtgtc cccatggaac tggacccggt ggccggcggt 1080
gtccggatcc agcaggacgc cccgctgctg aaggccgtcg acgtcgactg ccgtgacgtg 1140
ccggacatgc ttcccgtcct gtccaccctc gccaccttcg cgcacggcga gtcggtgttc 1200
cacaatgtcg cgcacacccg gctgaaggag tccgaccggg ccggcgcgat gctccagctc 1260
aactccatgg gcgccgcact ggagttggcc gacgacaccc tgcgggtgac cggggtggac 1320
ggtctggtcg gcgcggagct gtcgtcgtac aacgaccacc gggtgctgat gtcgctggcc 1380
gtcgccgcct cacgcgcccg gggccactcc acgctgacct acccccacgc ctaccgcatc 1440
tcgtatccgg gcttcctcga catgatgaat gccgtcgggg tgccgacgtc cgtggagaag 1500
ggccccggac gctccgccag gagccgtgcc cgcacacccg tccggcccgt cgcggacccg 1560
gaccacgcct gccaggtcac cttgccggag tggctggccc ggcacgcggc gtcccgcccc 1620
ggggacaccg ccctcgtgga cgtacgcccg gacggcgact ccgtcgtcac ctggagcgag 1680
ctggccggcc aggtggacaa ggccgccgcg ctgctgctcg aactcggcgt gcagccgggc 1740
gagaacgtgg cgtaccagtt gcccaaccgc ctggagtgcg tggtgctgtc gctggcggcc 1800
ctgcggatcg gcgccgtctg ctgtccgatc attccgttct tccgggaacg cgaactcggc 1860
ttcgttctgc gccgctccgg ggcccgcgtc ctcgtcgtca tggacgagta ccggtcccgg 1920
cgccccgcgg aggaggcact cgccctcgcc gcgaccggtg gcccggacac ggcacacctg 1980
gagcatgtcg tggtgctggc ccggtcggcc ggcggcagcc gactgccgca gccgcctccg 2040
aacggcgtcc gcctgtacga ctgggggacg gccctgaaga cgacgacggt cgaccgggcg 2100
gccctcgacg cgatccaccc gacggccgag acgacggcgc agctgctgtt cacctcgggc 2160
accaccggcg agcccaaggg ggtcatgcag ccgtcctcgc acctggtccg ggccgtctcg 2220
atggagatcc ggcatctcgg cctcggtccc gaggacgccg tctgggtgcc gtctccgctc 2280
tcccaccaga ccgggttcct gtacgggatg acgctggcga ccgtgctggg cgtgccccag 2340
atcctgcagt ccgagtggga cgcgcggcgc gccctcgtct cgctgaacac gcaccgggcg 2400
acgttcgtcc aggccgccac gccgttcctg gccgacctgg tgaaagcggt cgaggagatc 2460
ggcgaggcgc cccggcatct gcggatcttc gtggccaccg gcgccaccgt gccccgctcg 2520
ctggccgagc acgcgagcga ggtgctgggc acgaaggtgt gcggcgcctt cggcaccacc 2580
gaaacctgcc tcggcgcgat gtccacgccg accgacgcac cggagcggcg ctggggcacc 2640
gacgggcggg tcctcgacgg cgtacggcta agggtcaccg acgaccaggg cgaggtgctg 2700
gcaccgggcg ccgagggcaa cttcgagatc ctctcgccga cgacgttcga ggggtatctc 2760
gaccgccccg acctgaccgc ggaggccttc accccggacg gctggttccg caccggcgac 2820
ctcgcggtga tcgacgagtc gggctacgtc cgtatcaccg gccggatcaa ggacgtgatc 2880
aaccgcggcg gcgagaagat ccccgtcgcc gagatggagc agctgctgtt cggtcacccc 2940
gccgtcgacg acgtggccat cgtggccatg cccgacgagc ggctcggcga gcgtgcctgt 3000
gcgttcgtcg ttctcaagga cggcgcgctg ctcggcttcg aggagatgtg ccactacctg 3060
gacgggcacc aggcagccaa gcagtactgg ccggaacggc tccagctcgt tgcggacctc 3120
ccgcgtaacc cgatcggcaa ggtccagaag ttcgaactgc gggccagggc acgccacctg 3180
cgcccccata ccgtgcagtg acaggagaag acggagtgcg acagatgatg gacgggcagc 3240
agttcaacaa ggtccgtgac gcggtcgagc agtgggtgga aggcccgggc gagcgctggg 3300
ccgaacacat cgaggagacc ggggaggtac ccgaggcggt gtggaccgag ctcaacgagc 3360
tcgggttcct gcggctcgcc gccccggtgg agtacggcgg ccagggcatc gggttcaccc 3420
ggtggatgga actgatggag gtcttctccc gctcccacgg ttccctgcgc atgatcgtgc 3480
acgtcgtcaa cggcatctgg cggtcgatgg acggtcacgc ctccgacgac cagcgcaagc 3540
ggttcgtcgt gccgtcggtc ctgggcgaga tcaagatcgc gttcgccctg accgagccgg 3600
gcaacggcac cggagcggac atcacgacct cggtggtccg cgagggcgac acctactacc 3660
tgtcgggccg caagcacttg atcacgttcg ggatgcgctg tgactactac ctcctggcgg 3720
cgcgcgtcgc gggcacgacg ggccacgagg gcaccgtcgc cctgctggtg ccgcgggacg 3780
cgcccggagt acgggccgaa gactcctccc acacgatggg cgtcaccggt accgaccacg 3840
cctcgatcgt cttcgaccgc accccggtgc ccgtggacca ccggctcggc gaggagggcc 3900
aggggctgga ggtgtttctc ggcggattcc tcaccccgtc gcggatatcc gtcgcgatga 3960
gctgcgtcgg cctcgcccaa cgcgcccagc aactcgccgt cgactacgcc cgctcccggg 4020
tgaccttcgg caaagcgctg acagagcgtc aggccattca gttcatgctc gccgagaacg 4080
cggcggacat cgaggcggcc cgacagctcg tgctgcacgc cgcgcgccgc ttcgaggagg 4140
gcgccgacga cgcatccatg cagtcctcga tggccaagat gcacgcggtg acgatgctga 4200
ccaccgtcac cgacaaggcg ctgcaggtcc acggcgggct cggctactgg aagtcccaga 4260
agatcgagcg cgtctaccgt gacgcccgcg cgcagcgctt cgaggagggc cccaacgagg 4320
tgcaaaaggc cgtggtcttc cgcgaactgc tgcaacaggc cgcaaccgtc accccaggag 4380
ccgcgcgatg aacgacaacc gacaggacga cgcgaccggc ggcgccaaga tctccctcat 4440
cacgggcgct tcgcgcggca tcggacgcgc ccttgcgctc accctggccc gccagggcgg 4500
caccgtggtc gtcaactaca agaagaacgt cggcctggcc gagaagaccg tcgccgacat 4560
cgaggaggcc ggcggccggg gcattgccgt ccaggccgac gtcgaaacga cggaaggcgt 4620
cacggcgttg ttcgacgagg tctcccgtcg ctacggacgc ctggaccact tcgtctccaa 4680
cgccgccgcc ggcgtcttca agaacatcct cgacctcggc cggcaccacc tggaccgctc 4740
ctacgccatg aacctgcgcc ccttcgtgct gggcgcccag caggcggtca ggctcatgga 4800
cgacggcggc cggatcgtcg cgctgtcgtc ctacggctcg atccgcgcct acccgacgta 4860
cgccgcgctc ggcgggatga aggcggcgat cgaggcgtgg gtgcgttata tggcggtcga 4920
gttcgcgccg tacggcatca acgtcaatgc ggtgaacggc ggcctgatcg attccgactc 4980
gctcgcctac ttctacggcg tcgacggcat gcccgacatg cgcggcgtcc tggatcgcat 5040
cccggccggg cgacccggca ccgtgcagga gatggccgac accgtcgcct tcctgctggg 5100
tgcgggcgcc ggatatgtca caggccagac tctggtcgtc gacgggggcc tgagcgtggt 5160
cgcaccgccg ttcttcgccg acgccggcga agcactgcgc ctgcctcccc gcccgacacg 5220
ggaaacctga gggaagaggg gcgtgcgcca tgtacgacca tgtcttccgg gccggcgcca 5280
tcggcaccat gatgctcccg catcgcatcg tcatgggcgc catgcacctc aacctggaga 5340
cccgagacga tgacggcgct gccctggccg cgttctacgc cgagcgcgcg gcagcgggcg 5400
cggcgctcat catcaccggt ggcgtggcag tgaacgccgt gggcgcgggc ggcccggggt 5460
acgcggtgat cggcgactcc cgggacaggg cggccctctc ggcggcggtg cgtgcggtgc 5520
acgaccgcgg tggacggatc gcgcttcagc tctttcacgc cgggcggtac gcgcggcccg 5580
gcgccggcga ggtggttgct cgccccgtcg ccccgtcgcc ggtgtacagc ggcttctccc 5640
gctgcacccc ccgggagatg acccccgggg acatcaccac cacgatcgac gacttcgccg 5700
agggtgcgtg caccgcgcgc gccctcggat tcgacgccgt ggaggtgatg ggctccgagg 5760
gctatctgat caaccagttc acggcaccgc tgaccaacct gcgcgacgac gcgtggggcg 5820
gtgacgccga acgacggcgc aggtttccgc tggaggtgct gcgcgcggtc cgcgcagccg 5880
tgggccggga tttcccggtg ctcttccgca tctccgggaa cgacctggtg gcaggtggca 5940
ctccgcccgg tgaggtcacc gcgctcgcgg tggcgcttgc cgacgccggg gccgacgcgc 6000
tctccgtcgg cgtggggtgg cacgagtcac cggtgcccac cgtgcagtcc caggtccccg 6060
ccggtacctg ggcggccgtc ggcaaggcac tgaaacgcgc gctgctggca gtcggccaca 6120
aggatgtcgc ggtgatcgcc gccaaccgca tccacgactt cgcgcaggcc gacgaagccc 6180
tgtccggtgg cgagttggat ttcgtggcca tggcgcggcc gttcctggcc gacccggaga 6240
tcgtcgccaa gtcggccgca ggccgccccg accaggtcaa tctctgcctc gcctgcaacg 6300
aggcgtgcat cgaccgctcc ttcggcaccg acagggtgtc ctgcctcgtc aaccccagag 6360
ccggccacga agaggagttt cccccaccgc agccggcccg ccaccgcagt gggctgttcg 6420
gccgctacgc cgtgatcggt gcgggcatcg ccggactgga ggccgcccgc acactggccg 6480
gcctcggcca ccatgtcgag gtgtacgaag cggcggcccg cgtcggcggt caattccggc 6540
tggcctccca ggtgccggga aaggcggaat tcgccgagac catccgccac cacgtacggg 6600
aactacggga agccggcgtg ccgttgcacc tgggccgtcg catcggcggc gaggacgtcc 6660
cggagctgcg gtcgttcgac ggtgtggtgc tggccacggg tgtgcgcccc gccaggttgt 6720
ccctgtccgg cgcggagctg ccccatgtac gggactacgc ccaggcgttc gccgatccgg 6780
aggcgctcca cggacggctc gtcatcatcg gcgggggtgg catcgcagtc gatctggccc 6840
acaccctgac cacggacccg gatgccaagg tgacgccgga ggagttcctg gcccggcatg 6900
ccgtgcccga gaacgctccg cggcccggga cggaacccgc cgtccgtggc gcgctgcgcg 6960
aggtgacgct gctgcggcgc ggcccacgga tcggagcggg catcggcccc agcacccgct 7020
gggtcgtcct gcggaacctg aagtccgccg gtgtacagat gctcaccggc gtgacctgcc 7080
gggagatcac acccgccggc gtcatcgtca ctgacgcgga agggagagac cgctacctcg 7140
acgccgacca cgtcgtgctt gcggtcggcc aggtcccggt acgacacctg gccccgctgt 7200
tgcacgaggc cggtgtcccc gtcgtcaccg cgggcggcgc cgcgggaacg gacgggctca 7260
acgccgtacg ggccacagcc gaaggcctgc gagcggcgca ccggataacc cgcgtcgccg 7320
tggggtga 7328。
2. the method for claim 1 is characterized in that comprising the steps: with the encoding gene of Avrmectin polyketide synthases starting module in the encoding gene pnAT1ACP1 gene substitution deinsectization streptomycete of phosphorus azomycin polyketide synthases starting module
(1): take the genome of deinsectization streptomycete as template, the left arm that adopts PCR method to obtain gene substitution institute palpus comprises upstream sequence and the about 2kb of partial response gene order;
(2): take the genome of deinsectization streptomycete as template, the right arm that adopts PCR method to obtain gene substitution institute palpus comprises downstream sequence and the about 2kb of partial response gene order;
(3): take the clay of the encoding gene pnAT1ACP1 that comprises phosphorus azomycin polyketide synthases starting module as template, adopt PCR method to obtain the about 1.2Kb of goal gene of gene substitution institute palpus;
(4): the left and right arms sequence that obtains and target gene fragment be cloned into by corresponding restriction enzyme site be built into the gene substitution recombinant plasmid in the pKC1139 carrier;
(5): the recombinant plasmid that (4) are obtained changes in the deinsectization streptomycete with the method for conjugal transfer, through integrate, lax cultivate to induce finish target gene after double exchange and Screening and Identification occur and replace.
3. the method for claim 1 is characterized in that biosynthesis gene pnP1-pnP4 heterogenous expression in replacement mutant of synthesizing cyclohexyl formyl coenzyme A in the phosphorus azomycin gene cluster is comprised the steps:
(1): take the clay that comprises this gene as template, adopt PCR method to obtain the about 1kb of partial sequence of the pnP1 of gene heterogenous expression institute palpus;
(2): take the clay that comprises this gene as template, adopt PCR method to obtain the about 0.8kb of pnP4 partial sequence of gene heterogenous expression institute palpus;
(3): take the clay that comprises this gene as template, the pnP1 and the pnP4 gene remaining part sequence that adopt the enzyme blanking method to obtain gene heterogenous expression institute palpus also have complete pnP2, the about 5.2Kb of pnP3 sequence;
(4): the sequence that obtains and erythromycin promoter gene sequence be cloned into by corresponding restriction enzyme site be built into the gene substitution recombinant plasmid in the pSET152 carrier;
(5): the recombinant plasmid that (4) are obtained changes in the mutant strain that Avrmectin biosynthesizing starting module replaces with the method for conjugal transfer, finishes the target gene heterogenous expression after resistance screening is identified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010568764 CN102093997B (en) | 2010-12-01 | 2010-12-01 | Method for producing doramectin by modifying streptomyces avermitilis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010568764 CN102093997B (en) | 2010-12-01 | 2010-12-01 | Method for producing doramectin by modifying streptomyces avermitilis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102093997A CN102093997A (en) | 2011-06-15 |
| CN102093997B true CN102093997B (en) | 2013-03-13 |
Family
ID=44127264
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010568764 Expired - Fee Related CN102093997B (en) | 2010-12-01 | 2010-12-01 | Method for producing doramectin by modifying streptomyces avermitilis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102093997B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540603B (en) * | 2012-07-09 | 2015-10-28 | 中国科学院上海有机化学研究所 | The biological synthesis gene cluster of gold alloy hydride and application thereof |
| CN103834605B (en) * | 2012-11-21 | 2018-09-21 | 中国科学院上海有机化学研究所 | A kind of Abamectin producing bacterium and its preparation method and application |
| CN109161559A (en) * | 2018-09-04 | 2019-01-08 | 浙江大学 | A kind of building and application of efficient streptomyces gene group simplified system |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1630712A (en) * | 2002-02-12 | 2005-06-22 | 辉瑞产品公司 | Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins |
| CN1687403A (en) * | 2005-06-06 | 2005-10-26 | 中国农业大学 | Engineering bacterium of producing ivermectin, its constructing method and application |
-
2010
- 2010-12-01 CN CN 201010568764 patent/CN102093997B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1630712A (en) * | 2002-02-12 | 2005-06-22 | 辉瑞产品公司 | Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins |
| CN1687403A (en) * | 2005-06-06 | 2005-10-26 | 中国农业大学 | Engineering bacterium of producing ivermectin, its constructing method and application |
Non-Patent Citations (3)
| Title |
|---|
| Barrie Wilkinson等.Biosynthetic engineering of polyketide synthases.《Expert Opin.Ther.Patents》.2003,第13卷(第10期),1579-1606. * |
| S. Weist等.Mutational biosynthesis—a tool for the generation of structural diversity in the biosynthesis of antibiotics.《Appl Microbiol Biotechnol》.2005,(第68期),141-150. * |
| 陈允亮.磷氮霉素生物合成基因簇的克隆及功能研究.《上海师范大学硕士学位论文》.2010,全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102093997A (en) | 2011-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pan et al. | Improvement of spinosad production by overexpression of gtt and gdh controlled by promoter PermE* in Saccharopolyspora spinosa SIPI-A2090 | |
| CN103468625B (en) | Gene disruption mutant of streptomyces bingchenggensis as well as preparation method and application thereof | |
| KR100945483B1 (en) | An enhancing method of the polyketide production in streptomyces venezuelae using the expression vector of pikD regulator | |
| CN101649326B (en) | Gene recombination mediated by phiC312 and inheritance reformation for erythrocin producing bacterium | |
| CN102093997B (en) | Method for producing doramectin by modifying streptomyces avermitilis | |
| CN101386829B (en) | Avid kyowamycin genetic engineering bacterium and use thereof | |
| CN103820474A (en) | Biosynthesis gene cluster of polyenoid and polyol macrolide compound | |
| CN106191155A (en) | A kind of method improving escherichia coli allos polyketone synthesis compounds and purposes | |
| CN101724646B (en) | Method for increasing yield of avermectin by using regulatory protein gene | |
| CN101717778B (en) | Method for improving yield of abamectin by using primary glucose metabolism genes | |
| US7851190B2 (en) | Biosynthetic gene cluster for leptomycins | |
| CN100393867C (en) | Construction of geldanamycin gene engineering high yield strain | |
| CN105061537B (en) | A kind of nucleoside antibiotic and its application in antibacterials are prepared | |
| CN105018514A (en) | Construction method for efficient biosynthesis of streptomycete drugs | |
| CN113846041B (en) | Method for enhancing expression of transporter genes to increase salinomycin fermentation levels | |
| CN110343650A (en) | A kind of recombination streptomyces nodocus of high yield amphotericin B and its application | |
| CN103409358A (en) | Gene engineering strain of high-yield autolytic mycin | |
| IL158095A (en) | Biosynthetic genes for butenyl-spinosyn insecticide production and process for its preparation | |
| CN102174539A (en) | Piericidin A1 biosynthetic gene cluster | |
| Zhao et al. | Construction of a doramectin producer mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis | |
| CN102010871A (en) | Biosynthesis gene cluster of xantholipin | |
| CN114437155B (en) | Two macrolide compounds and application thereof in preparation of antibacterial and anticancer drugs | |
| CA2354030A1 (en) | Micromonospora echinospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto | |
| CN110396521B (en) | Application of SAV6604 gene in increasing yield of abamectin | |
| CN105237544A (en) | Xantholipin B and preparation method and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130313 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |