CN115927033B - Saccharopolyspora spinosa strain and method for high-yield spinosad thereof - Google Patents
Saccharopolyspora spinosa strain and method for high-yield spinosad thereof Download PDFInfo
- Publication number
- CN115927033B CN115927033B CN202210636375.1A CN202210636375A CN115927033B CN 115927033 B CN115927033 B CN 115927033B CN 202210636375 A CN202210636375 A CN 202210636375A CN 115927033 B CN115927033 B CN 115927033B
- Authority
- CN
- China
- Prior art keywords
- saccharopolyspora spinosa
- spinosad
- fermentation
- culture medium
- strain
- 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.)
- Active
Links
- JFLRKDZMHNBDQS-UCQUSYKYSA-N CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C(=C[C@H]3[C@@H]2CC(=O)O1)C)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C.CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C=C[C@H]3C2CC(=O)O1)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C Chemical compound CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C(=C[C@H]3[C@@H]2CC(=O)O1)C)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C.CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C=C[C@H]3C2CC(=O)O1)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C JFLRKDZMHNBDQS-UCQUSYKYSA-N 0.000 title claims abstract description 45
- 239000005930 Spinosad Substances 0.000 title claims abstract description 45
- 229940014213 spinosad Drugs 0.000 title claims abstract description 45
- 241000868102 Saccharopolyspora spinosa Species 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 43
- 230000004151 fermentation Effects 0.000 claims abstract description 43
- 239000001963 growth medium Substances 0.000 claims abstract description 41
- 229930185156 spinosyn Natural products 0.000 claims abstract description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 239000011573 trace mineral Substances 0.000 claims abstract description 8
- 235000013619 trace mineral Nutrition 0.000 claims abstract description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims description 24
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 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 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 238000010564 aerobic fermentation Methods 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 229920002472 Starch Polymers 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 235000012054 meals Nutrition 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 229920000742 Cotton Polymers 0.000 claims description 5
- 240000008042 Zea mays Species 0.000 claims description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 5
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 5
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 5
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 4
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 claims description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 4
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 claims description 4
- 229940073769 methyl oleate Drugs 0.000 claims description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 235000019764 Soybean Meal Nutrition 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000004455 soybean meal Substances 0.000 claims description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 3
- 229930003270 Vitamin B Natural products 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 235000019156 vitamin B Nutrition 0.000 claims description 2
- 239000011720 vitamin B Substances 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 9
- 239000001301 oxygen Substances 0.000 abstract description 9
- 229910052760 oxygen Inorganic materials 0.000 abstract description 9
- 238000002703 mutagenesis Methods 0.000 abstract description 7
- 231100000350 mutagenesis Toxicity 0.000 abstract description 7
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 3
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 239000003905 agrochemical Substances 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- 239000000523 sample Substances 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 239000002689 soil Substances 0.000 description 18
- 241000186361 Actinobacteria <class> Species 0.000 description 16
- 230000001954 sterilising effect Effects 0.000 description 16
- 239000003480 eluent Substances 0.000 description 15
- 238000001514 detection method Methods 0.000 description 13
- 238000011218 seed culture Methods 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- 238000004809 thin layer chromatography Methods 0.000 description 10
- 229920001817 Agar Polymers 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 101000610620 Homo sapiens Putative serine protease 29 Proteins 0.000 description 8
- 102100040345 Putative serine protease 29 Human genes 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 8
- 238000000926 separation method Methods 0.000 description 8
- 239000000741 silica gel Substances 0.000 description 8
- 229910002027 silica gel Inorganic materials 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 238000011068 loading method Methods 0.000 description 7
- 238000004659 sterilization and disinfection Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 241000233866 Fungi Species 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000000123 paper Substances 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 229910000019 calcium carbonate Inorganic materials 0.000 description 4
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000013530 defoamer Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 238000002953 preparative HPLC Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000007480 spreading Effects 0.000 description 3
- 238000003892 spreading Methods 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 241001446247 uncultured actinomycete Species 0.000 description 3
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229910052564 epsomite Inorganic materials 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000000749 insecticidal effect Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 231100000225 lethality Toxicity 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229910052603 melanterite Inorganic materials 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- SRJQTHAZUNRMPR-SFGMUSMWSA-N spinosyn-a Chemical compound C([C@@H](OC(=O)C[C@H]1[C@@H]2C=C[C@@H]3C[C@H](C[C@H]3[C@@H]2C=C1C(=O)[C@@H]1C)O[C@H]2[C@@H]([C@H](OC)[C@@H](OC)[C@H](C)O2)OC)CC)CCC1O[C@H]1CC[C@H](N(C)C)[C@@H](C)O1 SRJQTHAZUNRMPR-SFGMUSMWSA-N 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- VRYALKFFQXWPIH-PBXRRBTRSA-N (3r,4s,5r)-3,4,5,6-tetrahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)CC=O VRYALKFFQXWPIH-PBXRRBTRSA-N 0.000 description 1
- RBCOYOYDYNXAFA-UHFFFAOYSA-L (5-hydroxy-4,6-dimethylpyridin-3-yl)methyl phosphate Chemical compound CC1=NC=C(COP([O-])([O-])=O)C(C)=C1O RBCOYOYDYNXAFA-UHFFFAOYSA-L 0.000 description 1
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 1
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 description 1
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 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
- 206010044565 Tremor Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- PMMURAAUARKVCB-UHFFFAOYSA-N alpha-D-ara-dHexp Natural products OCC1OC(O)CC(O)C1O PMMURAAUARKVCB-UHFFFAOYSA-N 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- -1 compound vitamin Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000013265 extended release Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000021312 gluten Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000004021 humic acid Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 1
- 239000002655 kraft paper Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 229960000210 nalidixic acid Drugs 0.000 description 1
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical compound [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 description 1
- 229960003212 sodium propionate Drugs 0.000 description 1
- 235000010334 sodium propionate Nutrition 0.000 description 1
- 239000004324 sodium propionate Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 which is preserved in China Center for Type Culture Collection (CCTCC) in 2022 and 04 month 11, wherein the preservation number is CCTCC No. M2022404. The spinosyn H-2203 is obtained by combining resistance breeding and screening of original strains through physical and chemical mutagenesis treatment such as atmospheric pressure plasma, nitrosoguanidine, ultraviolet rays and the like. The spinosyn H-2203 can produce the agricultural chemical spinosad with high yield by fermentation under the condition of oxygen supply in a fermentation culture medium containing assimilable carbon source, nitrogen source and trace elements. The strain and the fermentation method provided by the invention can realize the industrialized amplification production of spinosad.
Description
Technical Field
The invention relates to a spinosyn strain and a method for producing spinosad with high yield, belonging to the technical field of microorganisms and fermentation.
Background
The spinosad belongs to macrolide agricultural antibiotics, and the main active components are spinosad A and D, and has the advantages of wide insecticidal spectrum, easy degradation, low residue, no drug resistance, no harm to human and livestock, less environmental pollution and the like, thus having wide application in agriculture and animal husbandry. The insecticidal mechanism of spinosad is mainly as follows: firstly, by acting on the nervous system of insects, it causes nonfunctional muscle contraction, failure, and with tremors and paralysis, it appears that the sustained activation of nicotinic acetylcholine receptors (nachrs) causes an extended release response of acetylcholine (Ach). Secondly, by acting on gamma-aminobutyric acid (GABA) receptors, the function of GABA-gated channels is altered. The spinosad has unique sterilization mechanism, safety of biological pesticides, no harm to environment, crops and mammals, and easy degradability. Due to these superior properties and applications, spinosad has been obtained three times in the united states "president green chemical challenge prize".
Currently spinosad is produced by aerobic fermentation from spinosad. Although patents and literature report that the yield of spinosad is improved through optimization of a culture medium and a fermentation process, the effect is not good all the time, and the industrial production of spinosad is severely restricted. The production of the strain is key to realize fermentation amplification of spinosad and make the product have economic competitiveness. Therefore, it is necessary to select and breed microorganism strains capable of high-yield spinosad and to establish efficient fermentation processes.
Disclosure of Invention
The invention aims to provide spinosad with high spinosyns yield and a fermentation method thereof.
The main components of spinosad produced by the strain of the invention are spinosad A and spinosad D, and the structural formula is as follows:
Spinosad a, r=h
Spinosad, r=ch 3
In order to achieve the above purpose, the technical scheme provided by the invention is as follows: saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 was preserved in China Center for Type Culture Collection (CCTCC) on the 11 th month 04 of 2022, and the preservation number is CCTCC No. M2022404.
The main biological characteristics of the strain are as follows: the bacterial colony is white or off-white, the center is slightly gray brown, the bacterial colony is round and smooth, the surface is protruded, and aerial hyphae are developed and easy to pick up.
The invention describes morphological and molecular level characteristics of strain H-2203, and by comparing the morphological and molecular level characteristics with those of known spinosyn-producing bacteria, strain H-2203 can be identified as belonging to spinosyns according to the taxonomy of microorganisms.
To achieve the above and other related objects, the present invention provides the following technical solutions: a process for preparing spinosyn by fermenting the spinosyn includes such steps as preparing a culture medium containing assimilable carbon source, assimilable nitrogen source and assimilable trace elements, inoculating spinosyn to said culture medium, and aerobic fermenting.
The preferable technical scheme is as follows: the assimilable carbon source is at least one of glycerol, sucrose, glucose, starch, molasses, lactose, maltose, mannitol, sorbitol, maltodextrin, vegetable oil and methyl oleate.
The preferable technical scheme is as follows: the assimilable nitrogen source is at least one of soybean meal, corn steep liquor powder, peptone, yeast powder, yeast extract powder, cotton seed meal, peanut meal and gluten powder.
The preferable technical scheme is as follows: the assimilable trace elements are derived from at least one of magnesium sulfate heptahydrate, zinc sulfate heptahydrate, ferrous sulfate heptahydrate, manganese chloride tetrahydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, anhydrous calcium chloride, sodium molybdate dihydrate, ferric trichloride hexahydrate, tyrosine, valine, methionine, vitamin B 1, vitamin B 9 and vitamin PP.
The preferable technical scheme is as follows: the strain is a spontaneous mutant strain of Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203, saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 and a high-yield mutant strain of Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 obtained by mutagenesis or genetic engineering.
The preferable technical scheme is as follows: the pH of the culture medium is 7.0-8.0, the aerobic fermentation temperature is 25-30 ℃, the aerobic fermentation time is 8-12d, and the relative humidity is 45-65%.
The aerobic fermentation mode is liquid submerged fermentation, and the aeration ratio is 0.5-1.0vvm (the ratio of the aeration amount per minute to the actual feed liquid volume of the tank body).
The liquid phase detection method of spinosad comprises the following steps:
Chromatographic column: agilent Eclipse XDB-C 18, 4.6X105 mm,5 μm
Mobile phase: 400mL of water+2000 mL of acetonitrile+1800 mL of methanol
Flow rate: 1mL/min
Wavelength: 254nm
Sample injection amount: 10 mu L
Column temperature: 35 DEG C
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
1. Improving spinosad yield. Compared with the reported strain and fermentation method, the strain and fermentation method have the advantages that the yield of spinosad is greatly improved, and the industrial production is facilitated.
2. The production cost is reduced. The strain and the fermentation method are beneficial to reducing the production cost of spinosad and have the potential of realizing industrialized mass production.
Drawings
Fig. 1: HPLC diagram of the prepared spinosad.
Fig. 2: 1 H-NMR spectra of spinosad.
Fig. 3: 13 C-NMR spectra of spinosad.
Fig. 4: LC-MS profile of spinosad.
Fig. 5: the original strain was treated with an atmospheric argon plasma jet.
Fig. 6: the lethality of the atmospheric plasma treatment time to the spinosyns.
Fig. 7: plasma mutagenesis procedure of high yielding strain.
Fig. 8: colony morphology of spinosad (Saccharopolyspora spinosa) H-2203.
Detailed Description
Further advantages and effects of the present invention will be readily apparent to those skilled in the art from the following disclosure of the present invention by reference to the specific embodiments.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
In describing the preferred embodiment, specific terminology may be resorted to for the sake of clarity; however, the disclosure of the present specification is not intended to be limited to the specific terminology so selected; and it is to be understood that each specific element includes all equivalent techniques for performing the same function, operating in a similar manner, and achieving a similar effect.
Example 1: isolation of spinosyns and identification of spinosad
1. Isolation of strains
(1) Soil sample collection
Soil samples are collected from rhizosphere soil samples of plants around the Huangshan of Anhui mountain, soil 5-10cm deep from different positions of the roots of wild plants is dug by a medicine spoon, and the soil is placed in a kraft paper bag. 100 parts of the paper bags are collected, the paper bags are sealed and brought back to a laboratory, and the paper bags are placed at a ventilation place for a week, so that the moisture in the paper bags is volatilized as much as possible.
(2) Preparation of soil suspension
Small pieces of soil in the air-dried soil sample are selected, 5g of the soil is accurately weighed, and the soil is ground into powder by a pestle. Poured into a 250mL conical flask containing 50mL of sterile water and small glass beads, and sonicated for 30min to give a stock solution. 100 mu L of the soil sample stock solution is sucked into an EP pipe filled with 900 mu L of sterile water by a sterile pipetting gun, and the soil suspension with the concentration of 10 -1 is obtained by fully shaking. Dilution was performed as described above to obtain soil suspensions having concentrations of 10 -2、10-3、10-4 and 10 -5 in sequence, and separation of actinomycetes was performed on the separation medium with the 4-concentration dilutions.
(3) Isolation of actinomycetes from soil
Selecting GS culture medium: soluble starch 2％,KNO3 0.1％,NaCl 0.05％,K2HPO4·3H2O 0.05％,MgSO4·7H2O 0.05％,FeSO4·7H2O 0.001％, agar 2%, pH 7.2-7.4 before elimination; HV medium: humic acid 0.1％,KCl 0.17％,MgSO4·7H2O 0.05％,FeSO4·7H2O 0.01％,Na2HPO4 0.05％,CaCO30.02％, compound vitamin 0.1% (vitamin B 1 0.00005.00005%, vitamin B 2 0.00005.00005%, inositol 0.00005%, vitamin B 5 0.00005.00005%, vitamin B 6 0.00005%, para-aminobenzoic acid 0.00005%, vitamin H0.000025%, niacin 0.00005%, fungus polysaccharide 2%), agar 2%, pre-sterilization pH 7.2-7.4, and can be used as separation culture medium for actinomycetes.
The two prepared separation media were sterilized in a high pressure steam kettle, and when the temperature was reduced to 40-50 ℃, 100. Mu.L of nalidixic acid (10 mg/L) which inhibits the growth of bacteria and 100. Mu.L of cycloheximide (10 mg/L) which inhibits the growth of fungi were added with a sterile pipette, and after shaking thoroughly, poured into a plate. After the culture medium is solidified, 100 mu L of 10 -2、10-3、10-4 and 10 -5 concentration diluted solution are respectively taken by a sterile pipetting gun to two separated culture mediums, the culture mediums are evenly spread by a spreading rod, and the culture dishes are sealed and then placed in a constant temperature incubator at 28 ℃ for culturing for about 5-10 d.
(4) Purification of actinomycetes in soil
During the culture process, the growth vigor of actinomycetes on the culture medium is observed at any time, so that more colonies are prevented from growing later or bacteria are prevented from covering the culture medium or being infected by fungi to affect the picking work. If the strain growth amount in the culture medium is large, actinomycetes which can be cultivated on the culture medium need to be picked up and transferred to a blank ISP2 culture medium (yeast extract powder 0.4%, malt extract powder 1.0%, glucose 0.4%, agar 1.8% and pre-digestion pH 7.2-7.4) in time for further purification.
After the colony grows out, picking out the colony with different morphological and color characteristics, and transferring the colony to an ISP2 culture medium for purification. If there are other bacteria in the actinomycetes grown on ISP2 medium, it is necessary to pick up actinomycetes and transfer them to blank ISP2 medium for further purification.
(5) Antibacterial activity detection of actinomycetes in soil
Preliminary activity detection is carried out on the actinomycete strain 125 strain obtained by separation, and agar blocks of the strain to be detected with the diameter of about 5mm are prepared on ISP2 culture medium by using a sterile puncher for standby.
Bacteria are prepared by an agar block diffusion method:
The bacterial suspension is added into MYG culture medium (maltose 0.5%, yeast extract powder 0.5%, glucose 1%, agar 1.5%, pre-digestion pH 6.5) or NA culture medium (peptone 1%, beef extract 0.3%, sodium chloride 0.5%, agar 1.5%, pre-digestion pH 7.2-7.4) at about 40deg.C, mixed well, and poured into a culture dish to prepare a bacteria-carrying plate. After the culture medium is solidified, the beaten bacteria blocks to be tested are equidistantly placed on the culture medium by using sterile forceps, and are cultured overnight at 37 ℃, and whether the antibacterial activity and the relative strength of the activity are judged according to the existence and the diameter of the antibacterial zone.
The filamentous fungi are prepared by a plate counter method:
placing agar blocks of fungi in the center of PDA culture medium, placing agar blocks of bacteria to be tested on two opposite sides of the bacteria blocks and at the same position apart from the bacteria blocks, culturing at 28deg.C for 3-5d, and determining antifungal activity of actinomycetes strain according to growth condition of mycelium.
(6) Fermentation of actinomycetes in soil
Selecting a strain with better antibacterial activity, scraping and inoculating purified single colony spores or mycelia into a seed culture medium (malt extract powder 1%, yeast powder 0.4%, glucose 0.4%, calcium carbonate 0.4%, pH adjusted before digestion 7.2-7.4), shaking a triangular flask with the filling amount of 50mL/250mL, and inoculating 1 colony/flask. Shaking culture at constant temperature of 28deg.C for 3d at shaking table rotation speed of 250 r/min.
After hypha is mature, and microscopic examination is carried out for sterile bacteria, 5mL of bacterial liquid is sucked from a seed culture medium and transferred to a GYM culture medium (yeast extract powder 0.4%, malt extract powder 1.0%, glucose 0.4%, corn starch 4%, caCO 3 0.2.2%, pH adjusted before digestion is 7.2-7.4), 3 repeats are arranged for each strain, the shaking bottle filling amount is 50mL/250mL triangular flask, the shaking table rotating speed is 250r/min, and the constant-temperature shaking culture is carried out for 7d at 28 ℃.
(7) Thin Layer Chromatography (TLC) of actinomycetes active ingredient in soil
After 7d of fermentation culture, microscopic examination and observation confirm that the hypha has good growth state and no bacteria infection, ethanol is added into the fermentation liquid and the volume is fixed to 100mL, and the shake flask is placed in an ultrasonic cleaner for ultrasonic treatment for 30min. And (5) carrying out suction filtration on the fermentation liquor after the ultrasonic treatment is completed, filtering out hyphae, and retaining filtrate.
Each strain has 3 bottles of fermentation liquor, the filtrates of the 3 bottles of fermentation liquor are combined together, concentrated to a small amount by using a rotary evaporator, and transferred into a small glass bottle for TLC detection. The concentrate was diluted appropriately and sampled by capillary tube, and spotted one by one on a silica gel plate, after which the silica gel plate was put in a developing solvent, and the sample was developed by TLC. Chloroform in the development system methanol=9:1 (v/v). The TLC development results were observed under UV light having a wavelength of 254 nm.
(8) Preservation of bacterial species
The actinomycete original strain H-2035 with research value is selected by combining various factors such as the form of the strain, the TLC development result and the antibacterial activity detection result, spores are scraped on a single colony of the actinomycete original strain H-2035 and are stored in a freezing tube filled with 1.5mL of 20% glycerol at the temperature of minus 20 ℃ for periodic recovery.
(9) Strains were fermented in 50L fermenters
And (3) resuscitating actinomycetes H-2035 stored in a glycerol tube on an ISP2 culture medium, inoculating the actinomycetes H-2035 to a seed culture medium (formula is as above) after the strain grows well, shaking a triangular flask with the liquid filling amount of 250mL/1000mL, culturing for 3d at 28 ℃, and carrying out microscopic examination to determine that the mycelium has good growth state and no bacteria infection phenomenon, and then feeding the actinomycetes into a fermentation tank. Fermenting in 50L fermenter, preparing fermentation medium 30L (glucose 5%, malt extract 1%, yeast extract 0.4%, corn starch 1%, caCO 3 0.3.3%, pH 7.2-7.4 before sterilizing, sterilizing at 121-125deg.C for 30min, inoculating with 5% (v/v), controlling dissolved oxygen to be greater than 20%, and culturing at 28+ -0.5deg.C for 7d.
(10) Separation and extraction of metabolites
① Pretreatment of fermentation broth
And centrifuging the fermentation broth after the culture is finished for 20min at 4000r/min to obtain fermentation supernatant and thalli. Passing the supernatant through HP-20 resin column (8×110cm, resin loading 1.5L), controlling the elution flow rate at 20mL/min, washing the resin column (2 times of resin volume) with clear water after adsorption, eluting off sugar on the resin surface, and eluting with 95% ethanol to obtain ethanol eluent. The thalli is washed by 95% ethanol, the washing solution is combined with ethanol eluent, then absolute methanol is added into the thalli and soaked overnight at room temperature, and the thalli is concentrated to dryness in vacuum at 50 ℃ after suction filtration. The ethanol eluate was likewise concentrated to dryness in vacuo at 50℃and the two pastes were dissolved separately in a small amount of methanol, and the two were mixed together if their principal points were identical as determined by TLC.
Separating the obtained paste sequentially by normal phase silica gel column chromatography, dextran column chromatography and preparative HPLC separation method to obtain pure compound.
② Normal phase silica gel column chromatography
Sample mixing: dissolving the sample to be separated in a solvent with equal volume of chloroform and methanol, uniformly stirring with a proper amount of silica gel (200-300 meshes) in a mortar, placing in a fume hood, volatilizing the solvent, and grinding the silica gel with a pestle to uniformly distribute the sample, thereby obtaining the column chromatography sample.
And (3) column loading: selecting a glass chromatographic column with proper diameter and length, blocking the outlet at the bottom of the chromatographic column by cotton, adding silica gel with the volume about 5 times of the sample volume, and tapping the outer wall of the chromatographic column by hand to ensure that the silica gel column is uniformly filled.
Loading: and (3) loading samples by a dry method, uniformly spreading the samples on a cylindrical surface, and spreading a layer of absorbent cotton on the samples. Adding proper amount of initial eluent (petroleum ether or chloroform is selected according to TCL detection result of sample to be separated), slowly wetting the sample and silica gel column, adding eluent after the sample and silica gel column are completely wetted,
Eluting: according to the separation effect of the sample to be separated in TLC detection, proper elution system, gradient change amplitude and final gradient size are selected, gradient elution is carried out according to the order from small to large in polarity, and the eluent is collected by a branch pipe. The eluted fractions were combined according to TLC detection results and concentrated to dryness in vacuo at 50 ℃.
③ Sephadex column chromatography
And (3) column loading: the sephadex is mixed with an equal volume of mixed solvent of chloroform and methanol before use, and is subjected to ultrasonic treatment for 30min to fully swell the sephadex and remove bubbles in suspension, and excessive stirring is avoided in the process. And (3) selecting a glass chromatographic column with proper diameter and length, slowly pouring the suspension into the chromatographic column, opening a valve of the chromatographic column, slightly vibrating the outer wall of the chromatographic column when the gel subsides, uniformly settling the gel until the gel subsides completely, and closing the valve.
Loading: after the separated sample is dissolved, directly loading the sample into the liquid, and adding the eluent after the sample completely enters the column.
Eluting: the eluent is selected from the mixed solvent of chloroform and methanol with equal volume, the flow rate is controlled according to the size of the chromatographic column, and the eluent is passed through the column at normal pressure. The eluent is collected by a branch pipe, and the same components are combined according to the TLC detection result, and the eluent is concentrated to be dry in vacuum at 50 ℃.
④ Preparative HPLC
The components to be separated are first subjected to liquid phase analysis (figure 1), the flow rate is fixed at 1.5mL/min, the composition of the eluent (purified water: acetonitrile: methanol=2:10:9, v/v) is adjusted, the target peak can be completely separated from other components, and the proper chromatographic separation conditions, mainly the composition and detection wavelength of the eluent, are determined. The sample injection amount of the preparative HPLC is controlled to be 0-5mL, the flow rate of the mobile phase is 20mL/min, and the components to be separated can be separated in a large amount in a short time by referring to chromatographic separation conditions and detection wavelength determined by liquid phase analysis.
And collecting the eluent according to the peak condition by a branch pipe, combining the same components according to the TLC detection result, and concentrating the eluent in vacuum at 50 ℃ until the eluent is dry.
⑤ Structure identification of spinosad
The isolated compounds are subjected to structural identification mainly by nuclear magnetic resonance NMR and mass spectrum MS analysis technology. The 1 H-NMR spectrum (FIG. 2) and 13 C-NMR spectrum (FIG. 3) of the compounds were analyzed and compared with the nuclear magnetic data of spinosad. The molecular weights of spinosad a (MW 731) and D (MW 745) were then determined based on mass spectrometry MS analysis (fig. 4), and the composition of spinosad was finally determined.
Example 2: breeding of high-yield strain of saccharopolyspora spinosa
(1) Seed selection of strain
The H-2035 separated in example 1 is taken as an original strain, and mutant strain H-2203 is obtained through multiple rounds of breeding by physical and chemical mutagenesis treatment such as atmospheric pressure plasma, nitrosoguanidine, ultraviolet rays and the like.
Preferred embodiments: the atmospheric pressure plasma breeding process is as follows:
① Mature inclined plane of original strain H-2035 is taken, inclined plane spores are washed by sterile water, and diluted to prepare spore suspension.
② 100 Mu L of spore suspension is sucked and uniformly coated on a sterile paper sheet with the diameter of 8mm, and is placed in the center of a sterile plate with the diameter of 90mm, and naturally dried in an aseptic super clean bench for standby.
③ The mutagenesis experiment is carried out on an atmospheric pressure plasma generating device, argon is used as working gas to treat a sample (figure 5), different treatment times (10, 20, 30, 40, 50 and 60 s) are selected for irradiation, the gas flow rate is 21.54L/min, the plasma gas flow temperature is 30-50 ℃, the sample which is not subjected to irradiation is used as a control, and the lethality of the thalli is shown in figure 6 in different treatment times.
④ After irradiation, the treated sample and the control sample were washed with 1mL of sterile water, 100. Mu.L of the treated sample and the control sample were applied to ISP2 solid medium containing 2g/L spinosad (or 0.5% 2-deoxy-glucose, or 2.5mg/L rhamnose, or 1.8% sodium propionate) for resistance breeding, and cultured at 28℃for 7d, and single colonies were picked up with sterilized toothpick into fresh ISP2 solid medium for expansion culture for 10-12d. And selecting mutant strains with more regular colony morphology for shake flask fermentation experiments. The atmospheric pressure plasma breeding flow is shown in figure 7.
⑤ Inoculating the mutant strain into a seed culture medium, wherein the seed culture medium comprises the following formula: TSB (tryptone soy peptone) 4.5%, glucose 1%, yeast powder 0.8%, magnesium sulfate 0.1%, shake flask 25mL/250mL Erlenmeyer flask, and sterilization at 115℃for 30min. Culturing in shaking table at 28deg.C for 3d at 250r/min, and inoculating into fermentation medium with 20% inoculum size, wherein the formula of fermentation medium is as follows: glucose 8%, starch 2%, cottonseed meal 3%, bean meal 1%, corn steep liquor 0.5%, yeast powder 0.5%, calcium carbonate 0.5%, trace elements 2.5mL/L (cobalt chloride hexahydrate 0.28g, zinc sulfate heptahydrate 1.4g, sodium molybdate dihydrate 0.1g, ferrous sulfate heptahydrate 3.8g, constant volume to 1L, shaking uniformly and preserving for 4 ℃ refrigerator standby), methyl oleate 4%, pH 7.5, shake-bottled 25mL/250mL triangular flask, sterilizing at 121 ℃ for 20min. Culturing in shaking table at 28deg.C for 8-12d at 250r/min, collecting fermentation broth 1mL, soaking in 10 times of anhydrous methanol, ultrasonic treating for 30min, filtering, and measuring spinosad yield in liquid phase. Through multiple rounds of mutagenesis and resistance screening, the high-yield mutant strain H-2203 is bred. Through fermentation shake flask verification, the spinosad yield reaches 4152mg/L, which is 19.3 times of the original strain H-2035 yield (215 mg/L), and the spinosad has good industrialized application prospect.
The mutant H-2203 is preserved in China Center for Type Culture Collection (CCTCC) No. M2022404 at the year 04 and 11 of 2022 and registered in a book to prove survival.
(2) Identification of species
The main appearance characteristics of mutant strain H-2203 are: the colony is white or off-white, the center is slightly gray brown, the colony is round, the surface is protruded, the aerial hypha is developed, and the aerial hypha is easy to pick up (figure 8).
Universal primers based on the conserved sequence of actinomycetes 16S rDNA:
Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3' A
Reverse primer: 5'-TACGGTTACCTTGTTACGACTT-3' A
The 16S rDNA sequence of strain H-2203 was PCR amplified using the above primers. PCR cycle conditions: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, renaturation at 55 ℃ for 30s, extension at 72 ℃ for 1.5min, repeating for 31 cycles, extension at 72 ℃ for 10min, and finally heat preservation at 4 ℃. The PCR products were detected by 1% agarose gel electrophoresis. Products with clear bands are selected for sequencing. The result shows that the primary structural sequence of the strain 16S rDNA is 1422 bases. As a result of blast in Genebank database, it was found that H-2203 was 99% similar to the Saccharopolyspora spinosa strain:JCM 9375 gene sequence (GenBank: LC 149867.1). From the above results, it was confirmed that the strain H-2203 belongs to Saccharopolyspora spinosa (Saccharopolyspora spinosa).
Example 3: shaking flask fermentation of spinosad
The formula of the seed culture medium comprises: starch 2%, glucose 3%, soybean cake powder 2%, yeast powder 1.5%, ammonium chloride 0.1%, magnesium sulfate heptahydrate 0.2%, dipotassium phosphate trihydrate 0.1%, shake-flask 25mL/250mL triangular flask, and sterilizing at 115 ℃ for 30min. The mutant strain H-2203 bred in example 2 is inoculated into a seed culture medium by a sterile inoculating shovel and is placed into a constant-temperature shaking table at the temperature of 28 ℃ for 3d at the speed of 250 r/min. The formula of the fermentation medium comprises: glucose 6%, starch 2%, cotton seed cake powder 2%, bean cake powder 1%, corn steep liquor 0.5%, yeast powder 0.5%, magnesium sulfate heptahydrate 0.01%, ferrous sulfate heptahydrate 0.0002%, cobalt chloride hexahydrate 0.001%, sodium molybdate dihydrate 0.0005%, calcium chloride 0.002%, zinc sulfate heptahydrate 0.0005%, copper sulfate pentahydrate 0.0001%, calcium carbonate 0.5%, methyl oleate 4%, soybean oil 4%, pH-adjusted 7.5, shake-bottled 25mL/250mL triangular flask, and sterilization at 121deg.C for 20min. 5mL of seed culture solution is sucked into each fermentation shake flask, and the culture is carried out in a shaking table at the constant temperature of 28 ℃ at 250r/min for 8-12d. After the fermentation broth is extracted by absolute methanol, the yield of spinosad is 8628mg/L by liquid phase measurement.
Example 4: method for producing spinosad by fermentation tank
(1) Preparation of first-level seed liquid
7L of seed culture medium (the strain is the same as in example 3, the proportion of the seed culture medium is shown in example 3, 0.05% of defoamer is added at the same time, w/v) is put into a 10L seed tank, a steam valve is opened, steam is introduced into a jacket, the seed culture medium in the tank is preheated, the temperature of the tank is raised to 80-90 ℃, the steam valve is regulated, and after 20-30min of heat preservation, the jacket steam valve is closed. Opening the bottom valve of the seed tank and the air inlet pipe to enter steam in a double way, and when the tank temperature rises to 121-125 ℃ and the pressure is 0.11-0.14MPa, regulating the steam valves and the exhaust valve in each way, and preserving heat and sterilizing for 30min. After sterilization, adjusting the exhaust valve, closing each path of near-tank valve, closing the corresponding steam valve, introducing sterile air when the tank pressure is lower than 0.08MPa, maintaining the pressure of the tank body, opening the jacket water inlet valve and the jacket water outlet valve, cooling the seed tank, and cooling the tank temperature to 28 ℃. Under the protection of flame, 200mL of shaking bottle seed liquid is connected, the culture temperature is 28+/-0.5 ℃, the ventilation quantity is 1vvm, the dissolved oxygen range is set to be 20-40%, the tank pressure is 0.045Mpa, the initial stirring rotating speed is 200r/min, the stirring rotating speed is linked with the dissolved oxygen, and the culture is carried out for 60-80h, at the moment, the pH is 6.5-7.5, and the mycelium concentration is 30-40%.
(2) Preparation of secondary seed liquid
10L of seed culture medium (the proportion of the seed culture medium is shown in example 3, and 0.08 percent of defoamer is added at the same time, w/v) is put into a 15L seed tank, a steam valve is opened, steam is introduced into a jacket, heating steam sterilization is carried out, the tank pressure is stabilized at 0.11-0.14MPa, the temperature is controlled at 121-125 ℃, and the heat preservation sterilization is carried out for 30min. Cooled to 28 ℃ for inoculation. Maintaining the pressure of the seed transfer pipeline at 0.20-0.40MPa, and sterilizing for 60min. About 2l of seed solution from the primary seed tank was transferred into the secondary seed tank. Stirring is started, air flow is controlled, and tank pressure is controlled. The culture temperature is 28+/-0.5 ℃, the ventilation rate is 1vvm, the dissolved oxygen range is set to be 20-40%, the tank pressure is 0.045Mpa, the initial stirring rotating speed is 200r/min, the stirring rotating speed is linked with the dissolved oxygen, the culture is carried out for 40-60h, the pH value is 7.0-7.5, and the mycelium concentration is 35-40%.
(3) Preparation and culture of fermentation tank culture medium
The formulation of the fermentation medium was the same as in the above-mentioned example 3, and 0.08% (w/v) of polyether defoamer was added, 50L of fermentor was fed in a volume of 30L, and pH7.5 was adjusted before digestion. Opening a steam valve, heating, steam sterilizing, stabilizing the tank pressure at 0.11-0.14MPa, controlling the temperature at 121-125 ℃, and sterilizing for 30min. Cooling to 26-30deg.C for inoculation. Maintaining the pressure of the seed transfer pipeline at 0.20-0.40MPa, and sterilizing for 60min. About 6L of the secondary seed tank was transferred into the fermenter. Stirring is started, air flow is controlled, and tank pressure is controlled. The fermentation culture temperature is 28+/-1 ℃, the ventilation amount is 0.8vvm, the dissolved oxygen range is set to be 30-50%, the tank pressure is 0.045Mpa, the initial stirring rotation speed is 200r/min, the stirring rotation speed is linked with the dissolved oxygen, the fermentation culture is carried out for 8-12d, and the fermentation is ended when the yield is not increased. Sampling treatment, and liquid phase detection of spinosad yield of 7364mg/L.
Example 5: method for producing spinosad by fermentation
Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 was preserved in China Center for Type Culture Collection (CCTCC) on the 11 th month 04 of 2022, and the preservation number is CCTCC No. M2022404.
A process for preparing spinosyn by fermenting the spinosyn includes such steps as preparing a culture medium containing assimilable carbon source, assimilable nitrogen source and assimilable trace elements, inoculating spinosyn to said culture medium, and aerobic fermenting. Other media, as well as other parts of the fermentation media, were the same as in example 4. The fermentation conditions were the same as in example 4.
The preferred embodiments are: the assimilable carbon source is a mixture of starch, glucose and glycerol according to a mass ratio of 1:3:1.
The preferred embodiments are: the assimilable nitrogen source is a mixture composed of cottonseed meal, soybean meal, corn steep liquor powder and yeast powder according to the mass ratio of 2:1:1:1.
The preferred embodiments are: the mass ratio of the assimilable trace elements ferrous sulfate heptahydrate, cobalt chloride hexahydrate, sodium molybdate dihydrate, anhydrous calcium chloride, zinc sulfate heptahydrate and copper sulfate pentahydrate is 2:10:5:10:5:1.
The preferred embodiments are: the strain is a spontaneous mutant strain of Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203, saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 and a high-yield mutant strain of Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 obtained by mutagenesis or genetic engineering.
The preferred embodiments are: the pH value of the culture medium is 7.0-8.0, the aerobic fermentation temperature is 28-30 ℃, and the aerobic fermentation time is 8-12d; the oxygen flow is 0.5-1vvm.
The foregoing description of the preferred embodiment of the invention is provided for the purpose of illustration only and is not intended to limit the invention in any way, but is intended to cover any and all modifications or variations of the invention that fall within the spirit and scope of the invention.
SEQUENCE LISTING
<110> Proc for fertilizer combination
<120> A spinosyn strain and a method for high-yield spinosad thereof
<140> 2022106363751
<141> 2022-06-07
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Forward primer
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 22
<212> DNA
<213> Reverse primer
<400> 2
tacggttacc ttgttacgac tt 22
<210> 3
<211> 1422
<212> DNA
<213> H-2203 Gene sequence
<400> 3
gtgggagtcg gcgtgcttac acatgcagtc gaacgctgaa gcatcttcgg gtgtggatga 60
gtggcgaacg ggtgagtaac acgtgggtaa tctgccctgc actctgggat aagccttgga 120
aacggggtct aataccggat atgacacatt atcgcatggt ggtgtgtgga aagttctggc 180
ggtgcaggat gagtccgcgg cctatcagct tgttggtggg gtgatggcct accaaggcga 240
cgacgggtag ccggcctgag agggtgaccg gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagtg gggaatcttg cgcaatgggc gaaagcctga cgcagcaacg 360
ccgcgtgggg gatgacggcc ttcgggttgt aaacctcttt cgacatcgac gaagccttcg 420
ggtgacggta ggtgtagaag aagcaccggc taactacgtg ccagcagccg cggtaatacg 480
tagggtgcga gcgttgtccg gatttattgg gcgtaaagag ctcgtaggcg gtttgtcgcg 540
tcggccgtga aaacctgcag cttaactgtg ggcttgcggt cgatacgggc agacttgagt 600
tcggtagggg agactggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac 660
accggtggcg aaggcgggtc tctgggccga tactgacgct gaggagcgaa agcgtgggga 720
gcgaacagga ttagataccc tggtagtcca cgccgtaaac gttgggcgct aggtgtgggg 780
atgggttcca ctgtttccgt gccgtagcta acgcattaag cgccccgcct ggggagtacg 840
gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcggcg gagcatgtgg 900
attaattcga tgcaacgcga agaaccttac ctggggtttg acatgcacta gacagctcca 960
gagatggggt ttcccttgtg gttggtgtac aggtggtgca tggctgtcgt cagctcgtgt 1020
cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgccctatgt tgccagcggg 1080
ttatgccggg gactcgtggg ggactgccgg ggtcaactcg gaggaaggtg gggatgacgt 1140
caagtcatca tgccccttat gcccagggct tcacacatgc tacaatggct ggtacagagg 1200
gtggcgatat cgtgaggtgg agcgaatccc ttaaagccgg tctcagttcg gatcggggtc 1260
tgcaactcga cctcgtgaag tcggagtcgc tagtaatcgc agatcagcat tgctgcggtg 1320
aatacgttcc cgggccttgt acacaccgcc cgtcacgtca tgaaagtcgg taacacccga 1380
agcccatggc ccaacccttg tggggggagt ggtcgaaggt ga 1422
Claims (3)
1. Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 was preserved in China Center for Type Culture Collection (CCTCC) on the year 2022, 04 and 11, and the preservation number is CCTCC NO: M2022404.
2. A method for producing spinosad by utilizing saccharopolyspora spinosa fermentation, which is characterized in that: comprises a culture medium containing assimilable carbon source, assimilable nitrogen source and assimilable trace elements, and then inoculating saccharopolyspora spinosa in the culture medium for aerobic fermentation; saccharopolyspora spinosa is Saccharopolyspora spinosa (Saccharopolyspora spinosa) H-2203 which has been preserved in China Center for Type Culture Collection (CCTCC) for 11/04/2022, with the preservation number of CCTCC NO: M2022404;
the assimilable carbon source is at least one of glycerol, glucose, starch, maltose and methyl oleate;
The assimilable nitrogen source is at least one of soybean meal, corn steep liquor powder, peptone, yeast powder, yeast extract powder and cotton seed meal;
The assimilable trace elements are derived from at least one of magnesium sulfate heptahydrate, zinc sulfate heptahydrate, ferrous sulfate heptahydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, anhydrous calcium chloride, sodium molybdate dihydrate, vitamin B 1 and vitamin B 9.
3. The method for producing spinosyns by fermentation using spinosyns according to claim 2, characterized in that: the pH of the culture medium is 7.0-8.0, the aerobic fermentation temperature is 25-30 ℃, the aerobic fermentation time is 8-12 d, and the relative humidity is 45-65%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210636375.1A CN115927033B (en) | 2022-06-07 | 2022-06-07 | Saccharopolyspora spinosa strain and method for high-yield spinosad thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210636375.1A CN115927033B (en) | 2022-06-07 | 2022-06-07 | Saccharopolyspora spinosa strain and method for high-yield spinosad thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115927033A CN115927033A (en) | 2023-04-07 |
| CN115927033B true CN115927033B (en) | 2024-06-11 |
Family
ID=86698136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210636375.1A Active CN115927033B (en) | 2022-06-07 | 2022-06-07 | Saccharopolyspora spinosa strain and method for high-yield spinosad thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115927033B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160015542A (en) * | 2014-07-31 | 2016-02-15 | 우석대학교 산학협력단 | Novel Saccharopolyspora spinosa wsp8209 Producing Spinosad with High Yield and Method for Producing Spinosad |
| CN111849807A (en) * | 2020-06-30 | 2020-10-30 | 北大方正集团有限公司 | Saccharopolyspora spinosa DS190375 and fermentation product, microbial inoculum, fermentation and screening method and application thereof |
| CN113444659A (en) * | 2021-06-17 | 2021-09-28 | 武汉大学 | Saccharopolyspora spinosa for high yield of spinosad |
-
2022
- 2022-06-07 CN CN202210636375.1A patent/CN115927033B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160015542A (en) * | 2014-07-31 | 2016-02-15 | 우석대학교 산학협력단 | Novel Saccharopolyspora spinosa wsp8209 Producing Spinosad with High Yield and Method for Producing Spinosad |
| CN111849807A (en) * | 2020-06-30 | 2020-10-30 | 北大方正集团有限公司 | Saccharopolyspora spinosa DS190375 and fermentation product, microbial inoculum, fermentation and screening method and application thereof |
| CN113444659A (en) * | 2021-06-17 | 2021-09-28 | 武汉大学 | Saccharopolyspora spinosa for high yield of spinosad |
Non-Patent Citations (2)
| Title |
|---|
| 产多杀菌素刺糖多孢菌的诱变选育;王欣荣;张爱雪;唐智超;翟龙飞;张新宜;林家富;黄挺;;中国抗生素杂志;20201231(第09期);全文 * |
| 多杀菌素高产菌株的选育;车可舒;王相晶;向志丹;;东北农业大学学报;20080825(第08期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115927033A (en) | 2023-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101245362B (en) | Method for producing polypeptide antibiotic enramycin by fermentation method | |
| CN112662585B (en) | Bacillus atrophaeus DX-9 and application thereof | |
| CN101402929B (en) | A alkali-fast sorangium cellulosum and uses of the same in producing epothilone | |
| CN115820454B (en) | Streptomyces wetland (Streptomyces paludis) strain 13-3 and application thereof | |
| CN113061551B (en) | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria | |
| CN109868242B (en) | Salt-tolerant acetoin-producing bacillus subtilis and application thereof | |
| CN108823110B (en) | Strain for producing griseofulvin and application thereof | |
| CN108841889B (en) | Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation | |
| CN113005048B (en) | Streptomyces nigricans CYS22, metabolite thereof and application thereof | |
| CN111778172B (en) | Streptomyces for producing antibacterial active compound and separation method and application thereof | |
| CN105985919B (en) | Bacillus and application thereof | |
| CN111363696B (en) | Streptomyces, screening method and application thereof | |
| CN103555636B (en) | Streptomyces violaceorubidus and application thereof | |
| CN101720781B (en) | New phosphorus and nitrogen mycin A for preventing and controlling fungal disease of crop and preparation process thereof | |
| CN115927033B (en) | Saccharopolyspora spinosa strain and method for high-yield spinosad thereof | |
| CN110832066B (en) | Ivermectin B1B producing strain and application thereof | |
| CN1928075B (en) | Strain capable of generating avermectin B component and utilization thereof | |
| CN112680387B (en) | Streptomyces rubiginosus strain for producing actinomycin D through fermentation and application thereof | |
| CN111154679B (en) | Efficient fermentation method of aflatoxin degradation bacteria | |
| CN110092758B (en) | Novel alkaloid compound and wart spore strain for preparing compound by fermentation | |
| CN108410760B (en) | Microbacterium aromaticum for efficiently degrading fenvalerate and application thereof | |
| CN110423790A (en) | A kind of metabolic engineering method of antimycotic tetramycin B orientation high yield | |
| CN112159777B (en) | Cyclopeptide antibiotic valinomycin high-yield strain and application thereof | |
| CN114058557B (en) | Multifunctional bio-control degradation strain of bacillus megatherium and application thereof | |
| CN108456689A (en) | The method for improving ansamitocin P-3 biosynthesis yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |