CN117721042A - Strain for producing tunicamycin complex and application thereof - Google Patents
Strain for producing tunicamycin complex and application thereof Download PDFInfo
- Publication number
- CN117721042A CN117721042A CN202311736426.9A CN202311736426A CN117721042A CN 117721042 A CN117721042 A CN 117721042A CN 202311736426 A CN202311736426 A CN 202311736426A CN 117721042 A CN117721042 A CN 117721042A
- Authority
- CN
- China
- Prior art keywords
- tunicamycin
- streptomyces mobaraensis
- powder
- soybean
- seed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- MEYZYGMYMLNUHJ-UHFFFAOYSA-N tunicamycin Natural products CC(C)CCCCCCCCCC=CC(=O)NC1C(O)C(O)C(CC(O)C2OC(C(O)C2O)N3C=CC(=O)NC3=O)OC1OC4OC(CO)C(O)C(O)C4NC(=O)C MEYZYGMYMLNUHJ-UHFFFAOYSA-N 0.000 title claims abstract description 45
- YJQCOFNZVFGCAF-UHFFFAOYSA-N Tunicamycin II Natural products O1C(CC(O)C2C(C(O)C(O2)N2C(NC(=O)C=C2)=O)O)C(O)C(O)C(NC(=O)C=CCCCCCCCCC(C)C)C1OC1OC(CO)C(O)C(O)C1NC(C)=O YJQCOFNZVFGCAF-UHFFFAOYSA-N 0.000 title claims abstract description 44
- ZHSGGJXRNHWHRS-VIDYELAYSA-N tunicamycin Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](O)[C@@H](CC(O)[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C(NC(=O)C=C2)=O)O)O1)O)NC(=O)/C=C/CC(C)C)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O ZHSGGJXRNHWHRS-VIDYELAYSA-N 0.000 title claims abstract description 36
- 241001495137 Streptomyces mobaraensis Species 0.000 claims abstract description 33
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- 239000000843 powder Substances 0.000 claims description 33
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 21
- 238000000855 fermentation Methods 0.000 claims description 20
- 230000004151 fermentation Effects 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 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 14
- 239000008103 glucose Substances 0.000 claims description 14
- 238000011218 seed culture Methods 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 240000008042 Zea mays Species 0.000 claims description 12
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 12
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 12
- 235000005822 corn Nutrition 0.000 claims description 12
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 11
- 235000010216 calcium carbonate Nutrition 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 244000068988 Glycine max Species 0.000 claims description 9
- 235000010469 Glycine max Nutrition 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- -1 tunicamycin compound Chemical class 0.000 claims description 9
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 239000003549 soybean oil Substances 0.000 claims description 8
- 235000012424 soybean oil Nutrition 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- 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 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 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 claims description 5
- 235000019764 Soybean Meal Nutrition 0.000 claims description 5
- 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 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- 239000000600 sorbitol Substances 0.000 claims description 5
- 235000010356 sorbitol Nutrition 0.000 claims description 5
- 239000004455 soybean meal Substances 0.000 claims description 5
- 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 claims description 4
- 239000004375 Dextrin Substances 0.000 claims description 4
- 229920001353 Dextrin Polymers 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 235000019425 dextrin Nutrition 0.000 claims description 4
- 235000001727 glucose Nutrition 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 235000019698 starch Nutrition 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
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 3
- 244000105624 Arachis hypogaea Species 0.000 claims description 3
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 3
- 235000018262 Arachis monticola Nutrition 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 3
- 229930091371 Fructose Natural products 0.000 claims description 3
- 239000005715 Fructose Substances 0.000 claims description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 3
- 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 claims description 3
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 claims description 3
- 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 claims description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-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
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 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 claims description 3
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 3
- 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 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 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 claims description 3
- 229960000367 inositol Drugs 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 235000020232 peanut Nutrition 0.000 claims description 3
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose 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[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 108010068370 Glutens Proteins 0.000 claims description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 239000011790 ferrous sulphate Substances 0.000 claims description 2
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 2
- 235000021312 gluten Nutrition 0.000 claims description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- 229940099596 manganese sulfate Drugs 0.000 claims description 2
- 239000011702 manganese sulphate Substances 0.000 claims description 2
- 235000007079 manganese sulphate Nutrition 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 235000002639 sodium chloride Nutrition 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims description 2
- 229940083466 soybean lecithin Drugs 0.000 claims description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 2
- 229940038773 trisodium citrate Drugs 0.000 claims description 2
- 235000019263 trisodium citrate Nutrition 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- 229960001763 zinc sulfate Drugs 0.000 claims description 2
- 235000012054 meals Nutrition 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241000233866 Fungi Species 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 241000222122 Candida albicans Species 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 229940095731 candida albicans Drugs 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 3
- PMMURAAUARKVCB-UHFFFAOYSA-N alpha-D-ara-dHexp Natural products OCC1OC(O)CC(O)C1O PMMURAAUARKVCB-UHFFFAOYSA-N 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 2
- 241000193388 Bacillus thuringiensis Species 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 241000187130 Streptomyces chartreusis Species 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940097012 bacillus thuringiensis Drugs 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 241000186650 Clavibacter Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 101000610620 Homo sapiens Putative serine protease 29 Proteins 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 240000000103 Potentilla erecta Species 0.000 description 1
- 235000016551 Potentilla erecta Nutrition 0.000 description 1
- 102100040345 Putative serine protease 29 Human genes 0.000 description 1
- 241001467568 Rathayibacter rathayi Species 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- YJQCOFNZVFGCAF-LLXUUZRASA-N tunicamycin A1 Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](O)[C@@H](C[C@@H](O)[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C(NC(=O)C=C2)=O)O)O1)O)NC(=O)/C=C/CCCCCCCCC(C)C)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O YJQCOFNZVFGCAF-LLXUUZRASA-N 0.000 description 1
- MEYZYGMYMLNUHJ-DIRMKAHISA-N tunicamycin B2 Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](O)[C@@H](C[C@@H](O)[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C(NC(=O)C=C2)=O)O)O1)O)NC(=O)/C=C/CCCCCCCCCC(C)C)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O MEYZYGMYMLNUHJ-DIRMKAHISA-N 0.000 description 1
- ZOCXUHJGZXXIGQ-NPXWYGMKSA-N tunicamycin C1 Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](O)[C@@H](C[C@@H](O)[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C(NC(=O)C=C2)=O)O)O1)O)NC(=O)/C=C/CCCCCCCCCCC(C)C)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O ZOCXUHJGZXXIGQ-NPXWYGMKSA-N 0.000 description 1
- XCEPHNBEHQJSSB-LGJGITPNSA-N tunicamycin D2 Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](O)[C@@H](C[C@@H](O)[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C(NC(=O)C=C2)=O)O)O1)O)NC(=O)/C=C/CCCCCCCCCCCC(C)C)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O XCEPHNBEHQJSSB-LGJGITPNSA-N 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
Abstract
The invention discloses Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.25567. The Streptomyces mobaraensis (Streptomyces mobaraensis) (CGMCC No. 25567) is a brand new tunicamycin complex producing strain, has high production capacity, greatly improves the capability of producing tunicamycin complex compared with other strains in the prior art, has the potency of the tunicamycin complex reaching more than 1000mg/L, and is beneficial to realizing industrial production.
Description
Technical Field
The invention relates to the technical field of industrial microbial fermentation, in particular to Streptomyces mobaraensis and a method for producing tunicamycin complex by fermentation.
Background
Tunicamycin (tunicamycin) belongs to a nucleoside antibiotic and consists of four units, namely uracil, tunicamycin, N-acetylglucosamine and a fatty chain. Tunicamycin was originally isolated by Japanese scholars from Streptomyces s. Strains Clavibacter species, S.chartreusis and Corynebacterium rathayi were both shown to produce this class of antibiotic. At present, 37 tunicamycin monomer compounds are obtained through total separation and named as tunicamycins, streptovirudins, mycospocidins, MM 19290 and corynetoxins. Tigatsuki et al, to demonstrate that the four major factors of the tunicamycin complex (A.Takatsuki et al, agric.biol. Chem.41,2307-2309, 1977) have the general structure:
(1) Tunicamycin A, n＝9
(2) Tunicamycin B, n＝10
(3) Tunicamycin C, n＝8
(4) Tunicamycin D, n＝11
the tunicamycin has good inhibition effect on most gram-positive bacteria and fungi. Wherein, the average value of the Minimum Inhibitory Concentration (MIC) of bacillus thuringiensis (Bacillus thuringiensis) is 1.27 mug/mL, which is superior to positive controls such as vancomycin, kanamycin and the like. MIC for Candida albicans (Candida albicans) was 2-32. Mu.g/mL. Because of the unique pharmacological actions, the tunicamycin is hopeful to be developed into a novel antibacterial and bactericidal medicament.
AKIRA TAK ATSUKI et al (1970) were first isolated as Streptomyces dysosuperificus nov.sp strain and fermented for 100h to give a total of about 110mg/L of the tunicamycin complex, 65mg/L intracellular and 45mg/L extracellular.
U.S. patent No. 4336333a, which was published in 6 of 1982, discloses a method for preparing a tunicamycin complex by producing the tunicamycin complex by Streptomyces chartreusis (NRRL 12338) with a total yield of about 105mg/L.
In summary, the invention aims at a series of problems of low fermentation yield, long synthesis step, low yield, relatively unsafe and the like in the chemical synthesis preparation process of the tunicamycin compound obtained by fermentation in the prior art, so that the invention seeks a novel microorganism, and the tunicamycin compound with relatively high fermentation yield can be obtained by simple fermentation, thereby realizing relatively low production cost of the tunicamycin compound.
Disclosure of Invention
In order to solve the problem of the deficiency of the existing preparation method of the tunicamycin complex, the first aspect of the invention is to provide a novel tunicamycin complex producing strain which is Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 and is preserved in China general microbiological culture Collection center (CGMCC), beijing, china with the preservation number of CGMCC NO.25567, the preservation date of 2022, 8 months and 20 days, and the preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The second aspect of the invention is to provide an application of Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 (CGMCC NO. 25567) in preparing a tunicamycin complex or a pharmaceutical composition containing the tunicamycin complex.
In a third aspect, the present invention provides a method for preparing a tunicamycin complex, which comprises the step of aerobically fermenting Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 (CGMCC No. 25567) in a nutrient medium containing a carbon source and/or a nitrogen source.
In a preferred embodiment, the above carbon source is selected from one of starch, dextrin, glucose, raffinose, sorbitol, glycerol, maltose, fructose, sucrose, L-arabinose, D-xylose, rhamnose, inositol, D-mannitol, soybean oil or a combination of the above; preferably from one of starch, dextrin, glucose, sorbitol or a combination of the above.
In a preferred embodiment, the nitrogen source is selected from one of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cotton seed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, ammonium salt, potassium salt, urea, or a combination of the foregoing; the assimilable nitrogen source is preferably selected from one of yeast extract powder, soybean meal, corn steep liquor dry powder or a combination of the above.
In a preferred embodiment, the above described nutrient medium further comprises an inorganic salt selected from the group consisting of trisodium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate, or a combination of any of the above, preferably calcium carbonate, dipotassium phosphate, magnesium sulfate, potassium chloride, or a combination of any of the above.
In a preferred embodiment, the nutrient medium contains 5-50g/L of corn starch, 5-50g/L of maltodextrin, 5-60g/L of glucose, 5-50g/L of sorbitol, 1-15g/L of yeast extract powder, 5-20g/L of soybean cake powder, 5-20g/L of corn steep liquor dry powder, 1-10g/L of magnesium sulfate, 2-8g/L of dipotassium hydrogen phosphate, 1-5g/L of potassium chloride and 3-50g/L of calcium carbonate.
In a preferred embodiment, the aerobic fermentation is at a temperature of 20-35 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the incubation time is 24-240 hours, preferably 180-240 hours; the ventilation is from 0.1 to 2.0vvm, preferably from 0.5 to 1.2vvm.
In a preferred embodiment, the Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 (CGMCC No. 25567) is inoculated into the nutrient medium by a seed solution for the fermentation culture; wherein the seed liquid is obtained by seed culturing streptomycete HDCC00224 in a seed culture medium.
In a preferred embodiment, the seed culture medium contains soybean oil 1-10g/L, glucose 5-100g/L, soybean cake powder 5-50g/L, corn steep liquor powder 5-50g/L, and calcium carbonate 1-10g/L; preferably, soybean oil is 5g/L, glucose is 70g/L, soybean cake powder is 18.5g/L, corn steep liquor powder is 5.8g/L, and calcium carbonate is 2.5g/L.
In a preferred embodiment, the seed culture conditions are: the temperature of seed culture is 20-30 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the incubation time is 24 to 80 hours, preferably 24 to 60 hours.
The tunicamycin complexes of the invention were tested by HPLC under the following conditions:
the liquid phase detection method for detecting the valence of the tunicamycin complex comprises the following conditions:
chromatographic column: c18 (250 mm. Times. 460mm,3.5 μm), mobile phase A: water, mobile phase B: acetonitrile, retention time: 20min, flow rate: 1mL/min, sample injection amount: 5 μl, detection wavelength: 260nm, column temperature: (25+ -1) deg.C.
Table 1: elution procedure volume ratio of mobile phases A and B
| Retention time min | A％ | B％ |
| 0 | 50 | 50 |
| 10 | 30 | 70 |
| 10.01 | 50 | 50 |
| 15 | 50 | 50 |
The Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 of the invention refers to the "Bojie's bacteria identification Manual" for physiological and biochemical experiments, and the results show that: the strain is white round and flat colony on Gaoshi culture medium, has branched base wire and gas wire, and has no transverse septum and no rupture of hypha to produce chain spore. Spores do not swim, light grey, chain flex, spore circles or ovals.
The streptomyces mobaraensis (Streptomyces mobaraensis) (CGMCC NO. 25567) serving as the production strain of the tunicamycin compound is a brand-new tunicamycin compound producing strain, has high production capacity, greatly improves the capacity of producing the tunicamycin compound compared with other strains in the prior art, and can reach the titer of more than 1000 mg/L. Furthermore, the tunicamycin compound in the fermentation liquor is mainly distributed in the fungus residues, the ratio of the supernatant is lower than 0.5%, the downstream extraction and purification process can be obviously simplified, the solvent consumption is greatly reduced, the production cost of tunicamycin is reduced, the green process is realized, and the method has great significance. Is beneficial to realizing industrial production.
Drawings
Fig. 1: GYQL-296# colony morphology
Detailed Description
EXAMPLE 1 original Strain Source
Adding 4g of a surface soil sample of Qianlingshan park in Guiyang, guizhou, into 100mL of sterile water, oscillating for 10min at 250rpm, standing for slight sedimentation of soil, taking 5mL of clear liquid, inoculating into a Gao's first liquid culture medium containing 5mg/L penicillin, carrying out enrichment culture at 28 ℃ and 250rpm for 72h, diluting the bacterial liquid with sterile water in a gradient of 10:1-10:5, taking 100 mu L of each gradient diluted bacterial liquid, coating the bacterial liquid on a Gao's first solid plate containing 3mg/L penicillin, and placing the bacterial liquid in a 28 ℃ incubator for culturing for 10 days. After bacterial colony grows out, the bacterial colony is transferred to a fresh ISP2 solid culture medium by a pair of toothpicks, the bacterial colony is placed in an incubator with 28 ℃ and 55% relative humidity for 20 days, then a lawn agar block is dug by an open sterile suction head and placed on the surface of an agar plate coated with staphylococcus aureus and candida albicans respectively, the bacterial colony is placed in the incubator with 65% humidity for culture, and the generation condition of a bacteriostasis ring is observed regularly. As a result, it was found that GYQL-296# strain exhibits an inhibitory effect against Staphylococcus aureus and Candida albicans at the same time. The single colony purified by the strain is shown in figure 1, and the single colony is amplified and cultivated for seed preservation, and the original preservation number is GYQL-296#.
The strain is further inoculated into a liquid high-grade first culture medium, the culture medium is placed at 28 ℃ and 250rpm for 8 days, after pigment is produced, the culture solution is soaked in ethanol and concentrated for LCMS detection, and the series of products are found to be the tunicamycin complex.
Example 2 identification of original species
The GYQL-296# strain is used as a test strain, and physiological and biochemical experiments are carried out by referring to the Bojie's bacteria identification manual, and the results show that:
the strain is white round and flat colony on Gaoshi culture medium, and has aerial hypha and spore, spore wire rotting, loose ring or bending, spore round and oval;
the optimal growth temperature of the strain is 27-30 ℃, and the growth tolerance temperature range is 20-41 ℃;
the optimal growth pH of the strain is 6.3-7.0, and the growth tolerance pH range is 5.5-8.7;
the carbon source available by the bacteria is selected from one of glucose, raffinose, sorbose, glycerol, maltose, fructose, sucrose, L-arabinose, D-xylose, rhamnose, inositol, D-mannitol and soybean oil or a combination of the above substances; the nitrogen source available to the bacteria is selected from one of ammonium sulfate, potassium nitrate, urea or a combination of the above.
Collecting fresh thalli of the strain to be tested, extracting a total DNA template by using a Piccher method (Piccher et al, 1989) modified by a solution bacterial enzyme method, amplifying a 16S rRNA gene by using universal primers (27F and 1495R), detecting and purifying a PCR product, and directly carrying out sequence determination. Sequencing was performed by Nanjing Jinsri Biotechnology Co.
The 16S rDNA sequence of the strain is compared with sequences of related species and genus in GenBank database by BLAST, the sequence has high homology with Streptomyces mobaraensis (Streptomyces mobaraensis strain DSM 40587) and the highest homology is 99.18 percent, and meanwhile, the strain is identified as Streptomyces mobaraensis (Streptomyces mobaraensis) by combining a physiological biochemical test and an apparent characteristic test of the strain, and the classification parameters of the strain and the Streptomyces mobaraensis (Streptomyces mobaraensis) are very similar.
EXAMPLE 3 mutant screening of strains
The original strain GYQL-296# is taken as an original strain, 5-bromouracil mutagenesis is carried out, and 2-deoxyglucose resistance screening is combined to finally obtain the high-yield mutant strain of the tunicamycin compound, wherein the original number of the strain is HDCC00224, and the strain is preserved in the China general microbiological culture collection center (CGMCC) for 20 months in 2022, and the preservation number is CGMCC No.25567. The DNA sequence of the strain HDCC00224 is shown as SEQ ID NO. 1.
The specific obtaining scheme of the strain is as follows:
1. preparing a bacterial suspension: the original strain GYQL-296# is taken as an inclined spore of an original strain, and after scattering, spore suspension with uniform dispersion is obtained for standby.
2. Mutagenesis: transferring the bacterial suspension in the step 1 into a 250mL triangular flask, adding 5-bromouracil mother liquor prepared in advance, controlling the final concentration to be 120 mug/mL, and stopping the reaction after the bacterial suspension is placed in an oscillating box to oscillate for 30min.
3. Resistance screening culture: taking the final mutagenesis sample in the step 2, coating the final mutagenesis sample on a solid medium added with 0.1% of 2-deoxyglucose after gradient dilution, and placing the final mutagenesis sample in an incubator with 30 ℃ and 55% of humidity for 12 days for culturing.
The formula of the resistance screening culture medium comprises the following components: 0.1% of 2-deoxyglucose, 0.1% of ammonium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate and 2% of agar. pH7.0, and sterilizing at 121deg.C for 20min.
4. Dibbling and culturing: and selecting the grown resistance screening isolated colonies, and inoculating one pair of the isolated colonies to a fresh solid plate culture medium, wherein 6-12 colonies are inoculated on each plate, and culturing the plates in an incubator at 30 ℃ and 55% humidity for 8 days. The formula of the dibbling culture medium comprises the following components: glucose 0.8%, wort 1%, yeast extract 0.2%, dipotassium hydrogen phosphate 0.05%, agar powder 1.8%, pH7.0, and sterilizing at 121deg.C for 30min.
5. Seed culture: taking the cultured colony, taking a proper amount of spores by a shovel, inoculating the spore into a seed culture medium, wrapping the seed culture medium, and placing the seed culture medium on a shaking table at 28 ℃ for culturing at 250rpm for 40 hours to obtain seed liquid. The shake flask seed culture medium comprises the following components: glucose 1.4%, soybean cake powder 1.5%, corn steep liquor powder 0.23%, pH7.0, and packaging volume 20mL/250mL, and sterilizing at 121deg.C for 30min.
6. Fermentation culture: transferring the cultured shake flask seeds into a fermentation medium with an inoculum size of 10%, wrapping and placing the shake flask seeds on a shaking table at 30 ℃ for culture at 250rpm for 5 days to obtain fermentation liquor containing the tunicamycin complex. The formula of the fermentation medium comprises the following components: 7% of glucose, 1.85% of soybean meal, 0.58% of corn steep liquor powder and 0.25% of calcium carbonate. pH7.0, 30mL/250mL, and sterilizing at 121deg.C for 30min.
7. Sample processing and detection methods: mixing 1mL of fermentation broth with 4mL of absolute ethanol, performing ultrasonic treatment for 30min, centrifuging at 14000rpm for 10min, and detecting by HPLC (high performance liquid chromatography) method as follows:
column: c18 column
Wavelength: 260nm of
Column temperature: 60 DEG C
Flow rate: 1mL/min
Sample injection amount: 5 mu L
Mobile phase: A-Water, B-acetonitrile
Table 2: elution procedure volume ratio of mobile phases A and B
| Retention time min | A％ | B％ |
| 0 | 50 | 50 |
| 10 | 30 | 70 |
| 10.01 | 50 | 50 |
| 15 | 50 | 50 |
8. Experimental results
As detected, among 372 strains tested in this example, 11 strains had a total yield significantly higher than that of the forward mutant strain of the starting strain, wherein the 296# strain had a total yield of the tunicamycin complex of 1008mg/L at most, which was 3.34 times that of the starting strain (302 mg/L). The strain PCB has the original preservation number of HDCC00224 and is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC) No.25567 in the year 08 and 22.
Example 4 confirmation of the ability of CGMCC No.25567 Strain to produce tunicamycin Complex
500mL of the fermentation broth obtained in example 3 was collected, and after thoroughly mixing, 1g of the whole broth was taken into 5 different centrifuge tubes, each numbered Q1 to Q5. The rest fermentation liquor is frozen and centrifuged at 10000rpm for 15min, the supernatant and the fungus dreg are respectively collected, 1g of the supernatant is respectively taken in 5 different centrifuge tubes with the serial numbers of S1-S5, and 1g of the fungus dreg is respectively taken in 5 different centrifuge tubes with the serial numbers of M1-M5. All centrifuge tubes are added with 4mL of absolute ethyl alcohol, a sealing cover is screwed up, the mixture is fully and evenly vibrated, the ultrasonic treatment is carried out for 30min, the centrifugation is carried out for 10min at 14000rpm, and the HPLC detection is adopted after the microporous filtration. The test method was the same as in example 3, and the experimental results are shown in Table 3 below.
Table 3: parallel test of tunicamycin complex content in fermentation liquor
From the results listed in the table, the tunicamycin composition in the fermentation broth of the strain CGMCC No.25567 is mainly distributed in the fungus residues, and the ratio of the tunicamycin composition in the supernatant is lower than 0.5%, which indicates that the tunicamycin composition synthesized by the strain CGMCC No.25567 is not secreted outside cells in the fermentation process, which is different from the production strain in the prior art. It is worth emphasizing that the excellent characteristic can obviously simplify the downstream extraction and purification process, greatly reduce the solvent consumption, be favorable for reducing the production cost of the tunicamycin and realizing the green process, and has great significance.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
Claims (11)
1. Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC NO.
25567, a date of preservation of 2022, 8 months and 20 days.
2. Use of streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 according to claim 1 for the preparation of a tunicamycin complex or a pharmaceutical composition comprising a tunicamycin complex.
3. A preparation method of a tunicamycin compound is characterized by comprising the following steps:
comprising the step of aerobically fermenting Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 according to claim 1 in a nutrient medium comprising a carbon source and/or a nitrogen source.
4. A method according to claim 3, characterized in that:
the carbon source is selected from one of starch, dextrin, glucose, raffinose, sorbitol, glycerol, maltose, fructose, sucrose, L-arabinose, D-xylose, rhamnose, inositol, D-mannitol and soybean oil or a combination of the above; preferably from one of starch, dextrin, glucose, sorbitol or a combination of the above.
5. A method according to claim 3, characterized in that:
the nitrogen source is selected from one or a combination of yeast extract powder, yeast extract, soybean lecithin, soybean cake powder, cotton seed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, ammonium salt, potassium salt and urea; preferably from one of yeast extract powder, soybean meal, cotton seed meal, peanut meal, corn steep liquor dry powder or a combination of the above.
6. A method according to claim 3, characterized in that:
the nutrient medium also comprises inorganic salt, wherein the inorganic salt is selected from one or a combination of any of trisodium citrate, monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride and manganese sulfate; the inorganic salt is preferably one or a combination of any of calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate and potassium chloride.
7. A method according to claim 3, characterized in that:
the nutrient medium contains 1-10g/L of soybean oil, 5-30g/L of glucose, 5-20g/L of soybean cake powder, 1-10g/L of corn steep liquor powder and 1-5g/L of calcium carbonate.
8. A method according to claim 3, characterized in that:
the fermentation temperature is 20-41 ℃, preferably 27-30 ℃; the pH of the culture medium is 5.5-8.7, preferably 6.3-7.0; the incubation time is 24-240 hours, preferably 180-240 hours; the ventilation is from 0.1 to 2.0vvm, preferably from 0.5 to 1.2vvm.
9. The method according to any one of claims 3-8, wherein:
the Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 is inoculated into the nutrient medium through seed liquid for the fermentation culture; the seed solution is obtained by seed culturing the Streptomyces mobaraensis (Streptomyces mobaraensis) HDCC00224 in a seed culture medium.
10. The method according to claim 9, wherein:
the seed culture medium contains soybean oil 1-10g/L, glucose 5-100g/L, soybean cake powder 5-50g/L, corn steep liquor powder 5-50g/L and calcium carbonate 1-10g/L; preferably, soybean oil is 5g/L, glucose is 70g/L, soybean cake powder is 18.5g/L, corn steep liquor powder is 5.8g/L, and calcium carbonate is 2.5g/L.
11. The method according to claim 10, wherein:
the seed culture conditions are as follows: the temperature of seed culture is 20-30 ℃, preferably 23-28 ℃; the pH of the culture medium is 5.0-8.0, preferably 5.0-7.0; the incubation time is 24 to 80 hours, preferably 24 to 60 hours.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2022117328713 | 2022-12-30 | ||
| CN202211732871 | 2022-12-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117721042A true CN117721042A (en) | 2024-03-19 |
Family
ID=90206591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311736426.9A Pending CN117721042A (en) | 2022-12-30 | 2023-12-15 | Strain for producing tunicamycin complex and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117721042A (en) |
-
2023
- 2023-12-15 CN CN202311736426.9A patent/CN117721042A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108676757B (en) | Streptomyces strain and application thereof in producing staurosporine | |
| CN111172045B (en) | Trichoderma africanum MU153 and application thereof | |
| CN112831421B (en) | Cephalosporin compound production strain and application thereof | |
| US9689017B2 (en) | Method of semi-solid state fermentation for producing surfactin from a mutant strain of Bacillus subtilis subsp | |
| CN105154382B (en) | Engineering strain streptomyces tsukubaensis L20 and its application | |
| US20070142424A1 (en) | Process for producing tacrolimus (FK-506) using vegetable oil as sole source of carbon | |
| US20080318289A1 (en) | Fermentation Processes for the Preparation of Tacrolimus | |
| IL104111A (en) | Microbial process for the production of trans-4-hydroxy-l-proline | |
| WO2023025292A1 (en) | Bacillus coagulans and method for catalytic production of 2'-deoxyadenosine by using bacillus coagulans | |
| WO2022262874A1 (en) | Burkholderia sp. and method for producing fr901464 by fermentation thereof | |
| CN105176904A (en) | Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof | |
| CN117721042A (en) | Strain for producing tunicamycin complex and application thereof | |
| CN112574918B (en) | Ammonia nitrogen degrading bacteria, microbial agent and application thereof | |
| CN110468051B (en) | K252A fermentation medium and preparation method thereof | |
| CN109554321B (en) | Genetically engineered bacterium for high-yield lipopeptide and application thereof | |
| CN107058137B (en) | Penicillium wortmannii and method for producing wortmannin by penicillium wortmannii | |
| CN116004750B (en) | Method for producing angustin by using erwinia persicae | |
| RU2495937C1 (en) | Method of preparation of tacrolimus by microbiological synthesis method | |
| KR850001230B1 (en) | Process for preparing narasin | |
| CN112795522B (en) | Novel species of acinetobacter and uses thereof | |
| Rafique et al. | Optimization of fermentation parameters for enhanced aphe production from streptomyces griseocarneus through submerged fermentation | |
| CN114854613A (en) | Streptomyces albus strain, application thereof and method for producing neomycin | |
| SU1705347A1 (en) | Strain of bacteria escherichia coli for @@@-deoxyadenosine preparation | |
| WO2004003214A1 (en) | Solid state fermentation and fed batch for the production of an immunosuppressant | |
| WO2004069845A1 (en) | Solid state fermentation and fed batch for the production of an immunosuppressant |
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 |