CN117089586A - Culture medium and method for fermentative production of compounds of formula (I) - Google Patents
Culture medium and method for fermentative production of compounds of formula (I) Download PDFInfo
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- CN117089586A CN117089586A CN202210508392.7A CN202210508392A CN117089586A CN 117089586 A CN117089586 A CN 117089586A CN 202210508392 A CN202210508392 A CN 202210508392A CN 117089586 A CN117089586 A CN 117089586A
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- betaine
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000012262 fermentative production Methods 0.000 title claims description 15
- 239000001963 growth medium Substances 0.000 title abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 167
- 230000004151 fermentation Effects 0.000 claims abstract description 167
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 66
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 38
- 229960003237 betaine Drugs 0.000 claims abstract description 33
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- HKSZLNNOFSGOKW-FYTWVXJKSA-N staurosporine Chemical compound C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1[C@H]1C[C@@H](NC)[C@@H](OC)[C@]4(C)O1 HKSZLNNOFSGOKW-FYTWVXJKSA-N 0.000 claims description 65
- HKSZLNNOFSGOKW-UHFFFAOYSA-N ent-staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(C)O1 HKSZLNNOFSGOKW-UHFFFAOYSA-N 0.000 claims description 64
- CGPUWJWCVCFERF-UHFFFAOYSA-N staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(OC)O1 CGPUWJWCVCFERF-UHFFFAOYSA-N 0.000 claims description 64
- 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 claims description 31
- 239000008101 lactose Substances 0.000 claims description 31
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 30
- 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 23
- 239000008103 glucose Substances 0.000 claims description 23
- 239000000843 powder Substances 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 18
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 16
- 239000001301 oxygen Substances 0.000 claims description 16
- 229910052760 oxygen Inorganic materials 0.000 claims description 16
- 239000001888 Peptone Substances 0.000 claims description 15
- 108010080698 Peptones Proteins 0.000 claims description 15
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 15
- 235000019319 peptone Nutrition 0.000 claims description 15
- 235000010469 Glycine max Nutrition 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 244000068988 Glycine max Species 0.000 claims description 12
- 230000001133 acceleration Effects 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 241000921781 Lentzea albida Species 0.000 claims description 6
- 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 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 5
- 238000005273 aeration Methods 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000002518 antifoaming agent Substances 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 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
- 229930006000 Sucrose Natural products 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
- 235000012054 meals Nutrition 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 239000005696 Diammonium phosphate Substances 0.000 claims description 2
- 108010068370 Glutens Proteins 0.000 claims description 2
- 239000005913 Maltodextrin Substances 0.000 claims description 2
- 229920002774 Maltodextrin Polymers 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- 229960000355 copper sulfate Drugs 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
- 235000005822 corn Nutrition 0.000 claims description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 2
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 2
- 229960001781 ferrous sulfate Drugs 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
- 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
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 235000011147 magnesium chloride Nutrition 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 229960003390 magnesium sulfate Drugs 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 229940035034 maltodextrin Drugs 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
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 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
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- 235000015393 sodium molybdate Nutrition 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 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
- 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
- 239000000047 product Substances 0.000 abstract description 11
- 239000012535 impurity Substances 0.000 abstract description 9
- 239000006227 byproduct Substances 0.000 abstract description 6
- 241001052560 Thallis Species 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 150000003839 salts Chemical class 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 32
- 239000007788 liquid Substances 0.000 description 24
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 239000013589 supplement Substances 0.000 description 13
- 239000002054 inoculum Substances 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 9
- 239000013530 defoamer Substances 0.000 description 9
- 238000011068 loading method Methods 0.000 description 9
- 238000009423 ventilation Methods 0.000 description 9
- 238000012258 culturing Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 241000187747 Streptomyces Species 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 4
- 235000019764 Soybean Meal Nutrition 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 230000000694 effects Effects 0.000 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 description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 4
- BMGQWWVMWDBQGC-IIFHNQTCSA-N midostaurin Chemical compound CN([C@H]1[C@H]([C@]2(C)O[C@@H](N3C4=CC=CC=C4C4=C5C(=O)NCC5=C5C6=CC=CC=C6N2C5=C43)C1)OC)C(=O)C1=CC=CC=C1 BMGQWWVMWDBQGC-IIFHNQTCSA-N 0.000 description 4
- 229950010895 midostaurin Drugs 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000003204 osmotic effect Effects 0.000 description 4
- 239000004455 soybean meal Substances 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 208000035404 Autolysis Diseases 0.000 description 3
- 206010057248 Cell death Diseases 0.000 description 3
- 201000008736 Systemic mastocytosis Diseases 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000028043 self proteolysis Effects 0.000 description 3
- 230000001502 supplementing effect Effects 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 2
- 241000235575 Mortierella Species 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 239000006052 feed supplement Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 208000000516 mast-cell leukemia Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 101710175516 14 kDa zinc-binding protein Proteins 0.000 description 1
- 201000008217 Aggressive systemic mastocytosis Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-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
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 101000932478 Homo sapiens Receptor-type tyrosine-protein kinase FLT3 Proteins 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
- 229930195725 Mannitol Natural products 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 229940123924 Protein kinase C inhibitor Drugs 0.000 description 1
- 102100020718 Receptor-type tyrosine-protein kinase FLT3 Human genes 0.000 description 1
- 208000032488 Systemic mastocytosis with associated hematologic neoplasm Diseases 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000012820 cell cycle checkpoint Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000011254 conventional chemotherapy Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 150000002337 glycosamines Chemical class 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000019691 hematopoietic and lymphoid cell neoplasm Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 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
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000003881 protein kinase C inhibitor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 208000026901 systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disease Diseases 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a culture medium and a method for producing a compound of formula (I) by fermentation, wherein the culture medium comprises a carbon source, a nitrogen source, inorganic salts and betaine. The fermentation method can improve the tolerance of thalli to the fermentation environment, improve the concentration and the purity of the fermentation product, reduce the proportion of impurity byproducts, has simple operation and low cost, and is suitable for large-scale industrial production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium and a method for producing a compound of a formula (I) through fermentation.
Background
Staurosporine (staurosporine) is an alkaloid and was isolated in Streptomyces (Streptomyces AM-2282) in 1977, and has the chemical structural formula shown below:
formula (I)
The staurosporine is a broad-spectrum non-specific protein kinase C inhibitor, has the biological activities of antifungal, cell proliferation inhibition, apoptosis induction, cell cycle checkpoint abrogation, vascular proliferation inhibition and the like, and has extremely strong anti-tumor activity.
The semisynthetic product midostaurin (midostaurin), which is an intermediate of staurosporine, was approved by the FDA for the united states at 28, 2017 and for the european at 18, 9, 2017, and has the chemical structure shown below:
midostaurin is marketed under the name RYDAPT, and has been indicated for the treatment of FLT3 positive acute myelogenous leukemia (AML, acute Myeloid Leukemia), systemic mastocytosis (ASM, 25mg Aggressive Systemic Mastocytosis) and its concomitant hematologic tumors (SM-AHN, systemic Mastocytosis with Associated Hematological Neoplasm) or mast cell leukemia (MCL, mast Cell Leukemia) by chemotherapy in combination, breaking the situation of treating AML by conventional chemotherapy for decades. The staurosporine is used as a precursor raw material and has very broad market prospect.
At present, the research reports of the domestic and foreign literature on the fermentation production of staurosporine are less, and the fermentation yield is lower.
Patents US4107297a and US4973552a disclose processes for fermentative production of staurosporine using streptomyces staurosporine, but the yields are low and patent US4973552a discloses a fermentation yield of 130mg/L. Pu Xiaoming, et al, by optimizing the fermentation process of the producing strain so that the fermentation yield reaches 286mg/L (fermentation process conditions of the staurosporine producing strain H41-38, microbiology report, 2009, 36 (11): 1631-1637, pu Xiaoming, etc.).
Patent CN101397540B optimizes the fermentation formulation based on US4107297a, and the disclosed staurosporine production method can improve the staurosporine yield to a certain extent, but the staurosporine yield is still at a lower level, and has no industrial value.
Patent CN108048369A and CN107603922A disclose methods for obtaining staurosporine by fermentation of streptomyces capable of producing staurosporine, but the production strains belong to the genus Streptomyces, seawater is required to be added in the process, macroporous resin is required to be used in the process of patent CN107603922A, the highest yield is 320mg/L, and the practical industrial application is poor.
The fermentation yield of the patent CN113122591A is improved by feeding carbon sources such as glucose, sucrose, glycerol or mannitol, and nitrogen sources such as yeast extract, peptone and soybean meal, and the yield of the 10L fermentation tank disclosed by the patent CN113122591A reaches 0.6-1 g/L. However, the source of the feed is wide and has no specificity, the nutrition is too rich, the utilization rate of the biosynthesis staurosporine is not high, the accumulation efficiency of a target product is influenced, the synchronous accumulation proportion of byproduct impurities is also increased, and the separation and purification efficiency is influenced.
The patent CN108676757A breeds a brand-new staurosporine producing strain, CN112048530B is added with a vitamin composition such as nicotinic acid, thiamine hydrochloride and the like in a fermentation culture medium on the basis of the technology, and glycerol is further added as a carbon source to improve the fermentation yield, and the yield of the disclosed fermentation bottle reaches 1.2-2.2 g/L through shaking culture of a shaking table. However, the fermentation process does not solve the problems of poor tolerance of the staurosporine-producing bacteria to stirring shear force, osmotic pressure and other factors under the condition of a fermentation tank, insufficient bacterial activity, easy autolysis, product degradation and increased proportion of byproduct impurities, and has poor practical industrial application.
In conclusion, the current staurosporine fermentation still has the problems of low fermentation yield, poor environmental tolerance of thalli, easy autolysis, degraded products and more byproduct impurities in large-scale industrialization.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects in the prior art and providing a culture medium and a method for producing a compound shown as a formula (I) by fermentation.
In order to solve the technical problems, the invention adopts the following technical scheme:
a medium for the fermentative production of a compound of formula (I) comprising a carbon source, a nitrogen source, an inorganic salt, the medium further comprising betaine.
Formula (I)
Preferably, the betaine accounts for 0.1 to 0.5 percent by mass and volume.
Preferably, the carbon source is a nutrient source capable of providing carbon elements required by the growth of microorganisms, the mass and volume percentage content of the carbon source is 3-7%, and the carbon source is one or more selected from glycerol, glucose, maltodextrin, sucrose and lactose. More preferably, the carbon source is selected from glucose and lactose combinations.
Preferably, the nitrogen source is a nutrition source capable of providing nitrogen elements required by the growth of microorganisms, the mass and volume percentage content of the nitrogen source is 1-4%, the nitrogen source comprises a slow-acting nitrogen source and a quick-acting nitrogen source, and the slow-acting nitrogen source is one or more selected from soybean cake powder, cotton seed cake powder, yeast powder and corn gluten meal; the quick-acting nitrogen source is selected from one or more of yeast extract powder, peptone and ammonium sulfate. More preferably, the nitrogen source is selected from the group consisting of soy meal and peptone.
Preferably, the inorganic salt provides nutrients such as trace elements, ions, and electrolytes necessary for the growth of microorganisms, and can constitute a cell component or maintain an enzymatic activity, and adjust osmotic pressure, pH, and the like. The inorganic salt is 0.1-0.5% by mass and volume, and is selected from one or more of zinc sulfate, magnesium sulfate, ferrous sulfate, copper sulfate, manganese sulfate, diammonium phosphate, potassium chloride, sodium chloride, magnesium chloride, cobalt chloride, sodium molybdate, trisodium citrate, calcium carbonate and calcium chloride. More preferably, the inorganic salt is selected from calcium carbonate.
Preferably, the culture medium further comprises an antifoaming agent capable of inhibiting foam generation or eliminating foam that has been generated when the staurosporine is produced by fermentation. The mass and volume percentage content of the defoaming agent is 0.05-0.15%.
Preferably, the culture medium comprises the following components in percentage by mass and volume:
in order to solve the technical problems, the invention adopts the following technical scheme:
a medium for fermentative production of a compound of formula (I) as described above for culturing a staurosporine-producing bacterium to produce staurosporine.
Preferably, the staurosporine producer is the strain Lentzea albida.
In order to solve the technical problems, the invention adopts the following technical scheme:
a process for the fermentative production of a compound of formula (I), comprising the steps of:
(1) Fermenting and culturing staurosporine-producing bacteria by using the culture medium to obtain a fermentation broth;
(2) Separating and obtaining the staurosporine from the fermentation liquor.
Preferably, the culture medium in the step (1) comprises the following components in percentage by mass:
preferably betaine and/or lactose are supplemented during the fermentation of step (1).
Preferably, the betaine and/or lactose is fed continuously or intermittently.
Preferably, in step (1), the betaine is fed starting from 12 to 36 hours of fermentation, the feeding duration being 48 to 96 hours.
Preferably, the betaine is continuously added, and the flow acceleration is 0.1-0.2%/day in terms of mass volume percent.
Preferably, when betaine is intermittently fed, the feeding amount per day is 0.1-0.2% by mass and volume percent, and the feeding mode is divided into 1 feeding or more feeding times per day.
Preferably, in the step (1), the fermentation starts to be supplemented with lactose 48-96 hours until the fermentation is finished, and the total sugar concentration of the fermentation broth is 0.5-3% in terms of mass volume percent. When the concentration of the staurosporine is not increased any more after the fermentation culture is carried out for 5 to 7 days, the fermentation is ended.
Preferably, the flow acceleration is 0.5-1.5%/day in mass volume percent when lactose is continuously added.
Preferably, when lactose is intermittently fed, the feeding amount per day is 0.5-1.5% by mass and volume percent, and the feeding mode is divided into 1 feeding or more feeding per day.
Preferably, in the fermentation process of the step (1), the temperature is 26-30 ℃, the aeration ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, the dissolved oxygen is more than or equal to 20%, the stirring speed is 50-150 RPM during the fermentation process, and the fermentation culture period is 5-7 days.
Preferably, the staurosporine producer is the strain Lentzea albida.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:
1. the culture medium provides sugar residues, O-methyl and N-methyl donors for the biosynthesis of staurosporine, specifically improves the biosynthesis efficiency of the staurosporine in the fermentation process, improves the fermentation yield, reduces the proportion of impurity byproducts, and is easy to separate and refine, thereby greatly reducing the production cost;
2. the fermentation method of the invention can lead mycelium to be aggregated into uniform small mycelium pellets in the fermentation tank culture process, improves the tolerance capability of the mycelium to factors such as stirring shearing force, osmotic pressure and the like of fermentation environment stress, leads the activity of the mycelium to be improved, and the growth and metabolism to be more stable, simultaneously reduces the viscosity of fermentation liquor, improves the transmission efficiency of nutrient substances and oxygen, reduces the autolysis of the mycelium and the degradation of products in the later period, and improves the fermentation yield and the purity of the products of staurosporine.
Drawings
FIG. 1 is a diagram showing the mycelium morphology of a fermentation broth obtained by the fermentation method of comparative example 1;
FIG. 2a is a diagram showing the mycelium morphology of a fermentation broth obtained by the fermentation method of example 1 of the present invention;
FIG. 2b is a diagram showing the mycelium morphology of the fermentation broth obtained by the fermentation method of example 5 of the present invention;
FIG. 2c is a diagram showing the mycelium morphology of the fermentation broth obtained by the fermentation method of example 6 of the present invention;
FIG. 3 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of example 1 of the present invention;
FIG. 4 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of example 2 of the present invention;
FIG. 5 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of example 3 of the present invention;
FIG. 6 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of example 4 of the present invention;
FIG. 7 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of example 5 of the present invention;
FIG. 8 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of example 6 of the present invention;
FIG. 9 is a high performance liquid chromatogram of a fermentation broth obtained by the fermentation process of prior art CN 110642872A.
Detailed Description
In order to make the technical scheme and the beneficial effects of the invention more obvious and understandable, the following detailed description is given by way of example only with reference to the accompanying drawings. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which specific conditions are not noted in the examples below, generally follows conventional experimental conditions. The reagents and materials used in the present invention are commercially available unless otherwise specified.
The mass volume percent (%) in the present invention refers to a plurality of g of solute per 100ml of solution.
The sources of the drugs and reagents used in the examples are:
| sequence number | Material name | Manufacturing factories |
| 1 | Glucose | SHANDONG XIWANG FOOD Co.,Ltd. |
| 2 | Lactose and lactose | Leprino Foods in the United states |
| 3 | Soybean cake powder | QINGDAO HAIKE BIOTECHNOLOGY Co.,Ltd. |
| 4 | Peptone | Shanghai peptide biotechnology Co Ltd |
| 5 | Betaine (betaine) | Hunan Ochi Biotechnology Co Ltd |
| 6 | Calcium carbonate | Jiang You silver-tay calcium Co.Ltd |
| 7 | Defoaming agent | YANTAI THINKING FINECHEM TECHNOLOGY Co.,Ltd. |
| 8 | Soluble starch | HUZHOU ZHANWANG PHARMACEUTICAL Co.,Ltd. |
| 9 | Magnesium sulfate heptahydrate | CHONGQING CHUANDONG CHEMICAL (Group) Co.,Ltd. |
| 10 | Bean pulp powder | QINGDAO HAIKE BIOTECHNOLOGY Co.,Ltd. |
In the present invention, staurosporine producing strain refers to any strain of the prior art that is suitable for fermentative production of staurosporine. The examples of the present invention are described by taking the staurosporine producer as the strain Lentzea albida (ATCC 55006), which is commercially available from ATCC (American Type Culture Collection), but is not limited thereto.
Studies of the staurosporine biosynthetic pathway have been referred to: the sugar residues in the staurosporine structure are derived from glucose, and the modified O-methyl and N-methyl carbon atoms and protons after amino sugar ligation are derived from methionine ("research progress of staurosporine biosynthesis pathway", chinese medical biotechnology, 2011, 274-277, zhao Xueer, etc.).
Thus, the inventors tested whether staurosporine production could be improved by the addition of a precursor substance. However, the precursor is directly added into the culture medium, so that the cells cannot be simply utilized, and excessive precursor can inhibit the activity of the cells, and the tolerance of the cells and the yield cannot be improved.
Through a number of experiments, the inventors have surprisingly found that betaine is specifically suitable for use by a staurosporine producer to provide a methyl group for staurosporine biosynthesis; lactose is also specifically suitable for use by the staurosporine producer to provide sugar residues for staurosporine biosynthesis; and further, the biosynthesis efficiency of the staurosporine in the fermentation process is specifically improved, and the fermentation yield is improved.
Meanwhile, the inventors have found through researches that: in the culture process of the staurosporine producing strain in the fermentation tank, the strain has poor adaptability to factors such as stirring shearing force, osmotic pressure and the like of fermentation environment, so that the strain has poor growth and development in the fermentation tank, mycelium forms are fine meshes and are easy to break and autolyze, the yield of a fermentation product staurosporine is low, the product is easy to degrade, and byproduct impurities are more. This negative effect is more serious as the fermenter size is enlarged, limiting large-scale industrial production.
The inventor further optimizes the fermentation process for specific precursor compound combination suitable for being utilized by the mortierella-producing bacteria, and the method provided by the invention can be used for gathering mycelium in a dispersion state in fermentation liquor into uniform small mycelium pellets, so that the tolerance of the mycelium to a fermentation environment can be enhanced, the activity of the mycelium is improved, the viscosity of the fermentation liquor can not be obviously increased, the mass transfer efficiency of the fermentation liquor is higher, the diffusion and transmission of nutrients and dissolved oxygen are facilitated, the higher yield and the product purity of the mortierella can be obtained, and the impurity is reduced.
A typical staurosporine fermentation liquid obtained in the prior art is obtained as in patent CN110642872A, the purity of the staurosporine is 87.70% through HPLC detection, the number of impurities with the peak area of more than 0.1% is 18, and a high performance liquid chromatogram is shown in figure 9.
The fermentation method provided by the invention can ensure that the fermentation yield of the staurosporine reaches 1.1-1.3 g/L on a fermentation tank with 5000L and 20000L specifications, the purity of the product reaches more than 97%, and the impurity quantity with the peak area of more than 0.1% is reduced to be within 10. The high performance liquid chromatogram of the fermentation method provided by the invention in typical staurosporine fermentation liquid fermented by the staurosporine is shown in figures 3-8.
The present invention will be described in further detail with reference to specific examples.
The concentration of staurosporine was detected by High Performance Liquid Chromatography (HPLC) during the fermentation process of the present invention, the chromatographic conditions were as follows:
chromatographic column: xbridge-C18 column (4.6 mm. Times.250 mm. Times.5 um), column temperature 40 ℃; the flow rate was 1.0. 1.0 mL/min, the measurement wavelength was 292nm, and the sample injection amount was 10uL. The mobile phase A was 0.42% aqueous perchloric acid (pH 2.5 adjusted with ammonia) and the phase B was 100% acetonitrile.
The gradient elution table is as follows:
| time (min) | Phase A (%) | Phase B (%) |
| 0 | 65 | 35 |
| 20 | 65 | 35 |
| 30 | 30 | 70 |
| 35 | 30 | 70 |
| 36 | 65 | 35 |
| 40 | 65 | 35 |
The concentration or content thereof is determined by an external standard method. The external standard method is a conventional technical means and will not be described in detail herein.
Wherein, comparative examples 1 to 3 are fermented in a 30L-500L-sized fermenter according to the prior art. Comparative examples 4 to 6 were prepared by fermenting the basic medium of the present invention in a fermenter having a loading of 30L to 20000L.
In the fermentation process of the present invention, a Lentzea albida (ATCC 55006) strain is first cultured in a seed medium. The seed medium may be a liquid medium conventionally used in the art, for example, a seed medium disclosed in patent CN101397540B, CN113122591A, CN112048530B and the like.
Preparation example 1
The Lentzea albida (ATCC 55006) strain is inoculated on a proper amount of shake flask seed culture medium (described by referring to CN101397540B, glucose 2.0%, peptone 0.5%, beef extract 0.5%, yeast powder 0.3%, calcium carbonate 0.4%, pH7.0, sterilizing at 120 ℃ for 30 min), and culturing for 24h at 26-30 ℃ at 220-250 rpm to obtain shake flask seeds.
Example 1
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 10% by volume into a fermentation medium having a loading of 3000L at 120℃for 30min after sterilization and cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 3% of glucose, 3% of lactose, 1.5% of soybean cake powder, 0.8% of peptone, 0.3% of betaine, 0.3% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
And fermenting for 24 hours, and starting to supplement betaine in a continuous feeding mode, wherein the feeding acceleration is 0.15 percent/day in terms of mass and volume percent, and the feeding duration is 72 hours.
And (3) fermenting for 72 hours, starting to supplement lactose, wherein the total sugar concentration of the fermentation liquid is controlled to be 0.5-3% in terms of mass volume percent until the fermentation is finished, and the supplementing mode is continuous flow feeding, wherein the flow acceleration is 0.5-1.5%/day in terms of mass volume percent.
After fermentation culture for 139 hours, the final concentration of staurosporine is 1114mg/L and the purity is 97.69 percent by HPLC detection, the high performance liquid chromatogram is shown in figure 3, the mycelium morphology diagram of the fermentation broth is shown in figure 2a, and mycelium in a dispersed state in the fermentation broth is aggregated into uniform small mycelium pellets, so that the tolerance of the thalli to a fermentation environment can be enhanced.
Example 2
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 10% by volume into a fermentation medium having a loading of 3000L at 120℃for 30min after sterilization and cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 2.5% of glucose, 3.5% of lactose, 1% of soybean cake powder, 1.2% of peptone, 0.2% of betaine, 0.2% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
And fermenting for 18 hours, and starting to supplement betaine in an intermittent feeding mode, wherein the feeding amount per day is 0.1 percent by mass and volume percent, the feeding mode is 1 time per day, and the feeding duration is 96 hours.
And (3) fermenting for 64 hours, starting to supplement lactose, wherein the total sugar concentration of the fermentation liquid is controlled to be 0.5-3% by mass and volume percent until the fermentation is finished, the supplement mode is intermittent supplement, the daily feed supplement amount is 0.5-1.5% by mass and volume percent, and the feed supplement mode is divided into 2 times per day.
Fermenting and culturing for 165 hr, and detecting final staurosporine concentration by HPLC as 1124mg/L and purity as 97.51%, wherein high performance liquid chromatogram is shown in figure 4.
Example 3
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 7% by volume into a fermentation medium having a loading of 15000L at 120℃for 30 minutes and then cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 2.5% of glucose, 2.5% of lactose, 2% of soybean cake powder, 1.5% of peptone, 0.2% of betaine, 0.3% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
The fermentation is started until 30 hours, the betaine is fed in a continuous feeding mode, the feeding acceleration is 0.2 percent/day in terms of mass and volume percent, and the feeding duration is 48 hours.
And (3) fermenting for 80 hours, starting to supplement lactose, wherein the total sugar concentration of the fermentation liquid is controlled to be 0.5-3% in terms of mass volume percent until the fermentation is finished, and the supplementing mode is continuous flow feeding, wherein the flow acceleration is 0.5-1.5%/day in terms of mass volume percent.
After fermentation culture for 162 hours, the final concentration of staurosporine is 1263mg/L and the purity is 97.90% by HPLC, and the high performance liquid chromatogram is shown in figure 5.
Example 4
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 7% by volume into a fermentation medium having a loading of 15000L at 120℃for 30 minutes and then cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: glucose 3%, lactose 3.5%, soybean cake powder 1%, peptone 1%, betaine 0.3%, calcium carbonate 0.2%, defoamer 0.1%, and water the rest.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
And fermenting for 30 hours, and starting to supplement betaine in an intermittent feeding mode, wherein the daily feeding amount is 0.2 percent by mass and volume percent, the feeding mode is 2 times per day, and the feeding duration is 48 hours.
And fermenting for 72 hours, and starting to supplement lactose, wherein the total sugar concentration of the fermentation liquid is controlled to be 0.5-3% in terms of mass and volume percent until the fermentation is finished. The feeding mode is intermittent feeding, the feeding amount per day is 0.5-1.5% in terms of mass volume percentage, and the feeding mode is divided into 3 feeding steps per day.
After fermentation culture for 137 hours, the final concentration of staurosporine is 1238mg/L and the purity is 97.97% by HPLC, and the high performance liquid chromatogram is shown in figure 6.
Example 5
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 8% by volume into a fermentation medium having a loading of 3000L at 120℃for 30min after sterilization and cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 1% of glucose, 2% of lactose, 1% of soybean cake powder, 1% of peptone, 0.5% of betaine, 0.2% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
And fermenting for 32 hours, and starting to supplement betaine in an intermittent feeding mode, wherein the feeding amount per day is 0.1 percent by mass and volume percent, the feeding mode is 2 times per day, and the feeding duration is 48 hours.
And fermenting for 48 hours, and starting to supplement lactose, wherein the total sugar concentration of the fermentation liquid is controlled to be 0.5-3% in terms of mass and volume percent until the fermentation is finished. The feeding mode is intermittent feeding, the feeding amount per day is 0.5-1.5% in terms of mass volume percentage, and the feeding mode is divided into 2 feeding steps per day.
Fermenting and culturing for 164 hours, wherein the final concentration of staurosporine is 1168mg/L and the purity is 97.72% by HPLC, the high performance liquid chromatogram is shown in figure 7, and the mycelium morphology of the fermentation broth is shown in figure 2 b.
Example 6
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 8% by volume into a fermentation medium having a loading of 3000L at 120℃for 30min after sterilization and cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: glucose 3%, lactose 4%, soybean cake powder 1%, peptone 1%, betaine 0.1%, calcium carbonate 0.2%, defoamer 0.1% and water the rest.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
And fermenting for 16 hours, and starting to supplement betaine in a continuous feeding mode, wherein the feeding acceleration is 0.2 percent/day in terms of mass and volume percent, and the feeding duration is 96 hours.
And (3) fermenting for 80 hours, starting to supplement lactose, wherein the total sugar concentration of the fermentation liquid is controlled to be 0.5-3% in terms of mass volume percent until the fermentation is finished, and the supplementing mode is continuous flow feeding, wherein the flow acceleration is 0.5-1.5%/day in terms of mass volume percent.
Fermenting and culturing for 167 hours, detecting final staurosporine concentration by HPLC to be 1108mg/L, purity to be 97.08%, high performance liquid chromatogram of the final staurosporine concentration is shown in figure 8, and mycelium morphology of the fermentation broth is shown in figure 2 c.
Comparative example 1
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 8% by volume into 30L of fermentation medium sterilized at 120℃for 30min and then cooled to 28 ℃. As described in example 1 with reference to patent CN101397540B, the fermentation medium consists of, in weight percent: 1% of glucose, 3% of soluble starch, 0.2% of magnesium sulfate heptahydrate, 1.5% of soybean meal powder, 0.4% of calcium carbonate and the balance of water.
In the fermentation process, the temperature is controlled at 28 ℃, the aeration ratio is 1.0VVM, the tank pressure is 0.05MPa, the stirring speed is 100-300 RPM, and the dissolved oxygen is more than or equal to 20%. And (3) fermenting and culturing for 113h, wherein the concentration of the final staurosporine is 104mg/L and the purity is 82.79% by HPLC detection, the mycelium form of the fermentation liquor is in a fine net shape as shown in the figure 1, and the mycelium is easy to break and autolyze.
Comparative example 2
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 8% by volume into 30L of fermentation medium sterilized at 120℃for 30min and then cooled to 28 ℃. As described in example 1 with reference to patent CN113122591a, the fermentation medium consists of, in weight percent: 1% of glucose, 3% of soluble starch, 0.2% of magnesium sulfate heptahydrate, 1.5% of soybean meal powder and the balance of water.
Glucose and yeast extract are fed in after fermentation for 24 hours, wherein the glucose feeding rate is 1%/day, and the yeast extract feeding rate is 0.2%/day.
In the fermentation process, the temperature is controlled at 28 ℃, the aeration ratio is 1.0VVM, the tank pressure is 0.05MPa, the stirring speed is 100-300 RPM, and the dissolved oxygen is more than or equal to 20%.
The fermentation culture is carried out for 191 hours, and the final concentration of the staurosporine is 233mg/L and the purity is 83.33 percent by HPLC detection.
Comparative example 3
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 10% by volume into 300L of a fermentation medium sterilized at 120℃for 30 minutes and then cooled to 28 ℃. As described in example 1 with reference to patent CN113122591a, the fermentation medium consists of, in weight percent: 1% of glucose, 3% of soluble starch, 0.2% of magnesium sulfate heptahydrate, 1.5% of soybean meal powder and the balance of water.
Glucose and yeast extract are fed in after fermentation for 24 hours, wherein the glucose feeding rate is 1%/day, and the yeast extract feeding rate is 0.2%/day.
In the fermentation process, the temperature is controlled at 28 ℃, the aeration ratio is 1.0VVM, the tank pressure is 0.05MPa, the stirring speed is 100-300 RPM, and the dissolved oxygen is more than or equal to 20%.
Fermenting and culturing for 137h, wherein the final concentration of staurosporine is 77mg/L and the purity is 82.16 percent by HPLC detection.
Comparative example 4
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 8% by volume into 30L of fermentation medium sterilized at 120℃for 30min and then cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 3% of glucose, 3% of lactose, 1.5% of soybean cake powder, 1% of peptone, 0.2% of betaine, 0.3% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
The fermentation culture is carried out for 187 hours, and the final concentration of the staurosporine is 788mg/L and the purity is 96.91 percent by HPLC detection.
Comparative example 5
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 8% by volume into a fermentation medium having a loading of 3000L at 120℃for 30min after sterilization and cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 3% of glucose, 3% of lactose, 2.0% of soybean cake powder, 1.5% of peptone, 0.2% of betaine, 0.3% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
Fermentation culture is carried out for 118 hours, and the final concentration of staurosporine is 405mg/L and the purity is 96.95 percent by HPLC detection.
Comparative example 6
The seed solution prepared in preparation example 1 was inoculated in an inoculum size of 7% by volume into a fermentation medium having a loading of 15000L at 120℃for 30 minutes and then cooled to 28 ℃. The fermentation medium comprises the following components in percentage by mass and volume: 2.5% of glucose, 2.5% of lactose, 1% of soybean cake powder, 1.5% of peptone, 0.3% of betaine, 0.3% of calcium carbonate, 0.1% of defoamer and the balance of water.
In the fermentation process, the temperature is controlled at 26-30 ℃, the ventilation ratio is 0.5-1.0 VVM, the tank pressure is 0.04-0.06 MPa, and the stirring speed is 50-150 RPM during the fermentation process, so that the dissolved oxygen is more than or equal to 20%.
Fermentation culture is carried out for 184 hours, and the final concentration of staurosporine is 561mg/L and the purity is 94.78 percent by HPLC detection.
It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.
Claims (20)
1. A medium for the fermentative production of a compound of formula (I), comprising a carbon source, a nitrogen source, an inorganic salt, characterized in that the medium further comprises betaine.
2. The medium for the fermentative preparation of a compound of formula (I) according to claim 1, characterized in that the betaine is present in a mass volume percentage of 0.1 to 0.5%.
3. The medium for fermentative production of a compound of formula (I) according to claim 1, wherein the carbon source is present in an amount of 3-7% by mass and volume, and wherein the carbon source is selected from one or more of glycerol, glucose, maltodextrin, sucrose and lactose.
4. The medium for fermentative production of a compound of formula (I) according to claim 1, wherein the mass volume percentage content of the nitrogen source is 1-4%, the nitrogen source comprises a slow-acting nitrogen source and a fast-acting nitrogen source, the slow-acting nitrogen source is selected from one or more of soybean cake powder, cotton seed cake powder, yeast powder and corn gluten meal; the quick-acting nitrogen source is selected from one or more of yeast extract powder, peptone and ammonium sulfate.
5. The medium for fermentative production of a compound of formula (I) according to claim 1, wherein the inorganic salt is present in an amount of 0.1-0.5% by mass and volume, and the inorganic salt is selected from one or more of zinc sulfate, magnesium sulfate, ferrous sulfate, copper sulfate, manganese sulfate, diammonium phosphate, potassium chloride, sodium chloride, magnesium chloride, cobalt chloride, sodium molybdate, trisodium citrate, calcium carbonate and calcium chloride.
6. The medium for fermentative production of a compound of formula (I) according to claim 1, wherein the medium further comprises an antifoaming agent in an amount of 0.05-0.15% by mass volume.
7. The medium for the fermentative production of a compound of formula (I) according to claim 1, characterized in that it comprises, in mass volume percentages:
the balance being water.
8. A medium for the fermentative production of a compound of formula (I) as claimed in any one of claims 1 to 7, characterized in that it is used for the cultivation of staurosporine-producing bacteria for the production of staurosporine.
9. The use of a medium for the fermentative preparation of a compound of formula (I) according to claim 8, characterized in that the producer of the compound of formula (I) is the strain Lentzea albida.
10. A process for the fermentative production of a compound of formula (I), comprising the steps of:
(1) Fermenting a producer of a compound of formula (I) using a medium according to any one of claims 1 to 7 to obtain a fermentation broth;
(2) Separating the compound (I) from the fermentation broth.
11. The method for fermentatively producing a compound of formula (I) according to claim 10, characterized in that betaine and/or lactose are supplemented during the fermentation of step (1).
12. The method for the fermentative preparation of a compound of formula (I) according to claim 11, characterized in that the betaine and/or lactose is fed in a continuous or intermittent manner.
13. The method for fermentatively producing a compound of formula (I) according to claim 12, characterized in that in said step (1) the betaine is fed starting at 12 to 36 hours of fermentation, the feeding duration being 48 to 96 hours.
14. The process for the fermentative preparation of a compound of formula (I) according to claim 13, characterized in that the continuous flow of betaine is carried out with a flow acceleration of 0.1 to 0.2%/day in mass volume percent.
15. The method for fermentative preparation of a compound of formula (I) according to claim 13, wherein the amount of betaine fed intermittently is 0.1-0.2% by mass volume per day, in 1 or more feeds per day.
16. The process for fermentatively producing a compound of formula (I) according to claim 12, characterized in that in said step (1) the addition of lactose is started at 48-96 hours of fermentation until the fermentation is completed, the total sugar concentration of the fermentation broth being 0.5-3% in mass volume percent.
17. The process for the fermentative preparation of a compound of formula (I) according to claim 16, characterized in that the continuous flow of lactose is carried out with a flow acceleration of 0.5 to 1.5%/day in mass volume percent.
18. The method for fermentative preparation of a compound of formula (I) according to claim 16, wherein the intermittent feeding of lactose is performed in a mass volume percentage of 0.5 to 1.5% per day by feeding in 1 or more feeds per day.
19. The method for fermentatively producing a compound of formula (I) according to claim 10, wherein in the fermentation process of step (1), the temperature is 26 to 30 ℃, the aeration ratio is 0.5 to 1.0VVM, the tank pressure is 0.04 to 0.06MPa, the dissolved oxygen is not less than 20%, the stirring is carried out during the fermentation process, the stirring speed is 50 to 150RPM, and the fermentation culture period is 5 to 7 days.
20. The method for fermentative preparation of a compound of formula (I) according to claim 10, wherein the producer of the compound of formula (I) is the strain luntzea albida.
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