CN116768971B - Method for producing thiostrepton by microbial fermentation - Google Patents
Method for producing thiostrepton by microbial fermentation Download PDFInfo
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- CN116768971B CN116768971B CN202310947529.3A CN202310947529A CN116768971B CN 116768971 B CN116768971 B CN 116768971B CN 202310947529 A CN202310947529 A CN 202310947529A CN 116768971 B CN116768971 B CN 116768971B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 69
- 230000004151 fermentation Effects 0.000 title claims abstract description 69
- NSFFHOGKXHRQEW-UHFFFAOYSA-N Thiostrepton B Natural products N1C(=O)C(C)NC(=O)C(=C)NC(=O)C(C)NC(=O)C(C(C)CC)NC(C(C2=N3)O)C=CC2=C(C(C)O)C=C3C(=O)OC(C)C(C=2SC=C(N=2)C2N=3)NC(=O)C(N=4)=CSC=4C(C(C)(O)C(C)O)NC(=O)C(N=4)CSC=4C(=CC)NC(=O)C(C(C)O)NC(=O)C(N=4)=CSC=4C21CCC=3C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 NSFFHOGKXHRQEW-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 229930188070 thiostrepton Natural products 0.000 title claims abstract description 18
- NSFFHOGKXHRQEW-AIHSUZKVSA-N thiostrepton Chemical compound C([C@]12C=3SC=C(N=3)C(=O)N[C@H](C(=O)NC(/C=3SC[C@@H](N=3)C(=O)N[C@H](C=3SC=C(N=3)C(=O)N[C@H](C=3SC=C(N=3)[C@H]1N=1)[C@@H](C)OC(=O)C3=CC(=C4C=C[C@H]([C@@H](C4=N3)O)N[C@H](C(N[C@@H](C)C(=O)NC(=C)C(=O)N[C@@H](C)C(=O)N2)=O)[C@@H](C)CC)[C@H](C)O)[C@](C)(O)[C@@H](C)O)=C\C)[C@@H](C)O)CC=1C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 NSFFHOGKXHRQEW-AIHSUZKVSA-N 0.000 title claims abstract description 18
- 229940063214 thiostrepton Drugs 0.000 title claims abstract description 18
- NSFFHOGKXHRQEW-OFMUQYBVSA-N thiostrepton A Natural products CC[C@H](C)[C@@H]1N[C@@H]2C=Cc3c(cc(nc3[C@H]2O)C(=O)O[C@H](C)[C@@H]4NC(=O)c5csc(n5)[C@@H](NC(=O)[C@H]6CSC(=N6)C(=CC)NC(=O)[C@@H](NC(=O)c7csc(n7)[C@]8(CCC(=N[C@@H]8c9csc4n9)c%10nc(cs%10)C(=O)NC(=C)C(=O)NC(=C)C(=O)N)NC(=O)[C@H](C)NC(=O)C(=C)NC(=O)[C@H](C)NC1=O)[C@@H](C)O)[C@](C)(O)[C@@H](C)O)[C@H](C)O NSFFHOGKXHRQEW-OFMUQYBVSA-N 0.000 title claims abstract description 18
- 230000000813 microbial effect Effects 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 51
- 238000000034 method Methods 0.000 claims abstract description 23
- 235000015097 nutrients Nutrition 0.000 claims abstract description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 13
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- 241000187398 Streptomyces lividans Species 0.000 claims abstract description 9
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- 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 39
- 239000008103 glucose Substances 0.000 claims description 39
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 32
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 23
- 240000008042 Zea mays Species 0.000 claims description 21
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- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 21
- 235000005822 corn Nutrition 0.000 claims description 21
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 20
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- 239000012138 yeast extract Substances 0.000 claims description 17
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 16
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 16
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 16
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 16
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 16
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 16
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 16
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 16
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 16
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
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- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 5
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 3
- YNIFQWSXTHTYPX-UHFFFAOYSA-L copper;sulfate;dihydrate Chemical compound O.O.[Cu+2].[O-]S([O-])(=O)=O YNIFQWSXTHTYPX-UHFFFAOYSA-L 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000011684 sodium molybdate Substances 0.000 claims description 3
- 235000015393 sodium molybdate Nutrition 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 3
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241001310047 Streptomyces azureus Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- -1 dihydroimidazole piperidines Chemical class 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 150000003222 pyridines Chemical class 0.000 description 2
- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 description 2
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- 239000011550 stock solution Substances 0.000 description 2
- AKNNEGZIBPJZJG-MSOLQXFVSA-N (-)-noscapine Chemical compound CN1CCC2=CC=3OCOC=3C(OC)=C2[C@@H]1[C@@H]1C2=CC=C(OC)C(OC)=C2C(=O)O1 AKNNEGZIBPJZJG-MSOLQXFVSA-N 0.000 description 1
- IMSODMZESSGVBE-UHFFFAOYSA-N 2-Oxazoline Chemical compound C1CN=CO1 IMSODMZESSGVBE-UHFFFAOYSA-N 0.000 description 1
- BHNHHSOHWZKFOX-UHFFFAOYSA-N 2-methyl-1H-indole Chemical compound C1=CC=C2NC(C)=CC2=C1 BHNHHSOHWZKFOX-UHFFFAOYSA-N 0.000 description 1
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- 241000283086 Equidae Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- FPTCMHOCGKKRGQ-WYOWUDGCSA-N Multhiomycin Natural products CC=C1NC(=O)[C@@H](NC(=O)c2csc(n2)c3cc(O)c(nc3c4csc(n4)[C@H]5CSC(=O)c6[nH]c7cccc(COC(=O)[C@@H](O)C[C@H](NC(=O)c8csc1n8)c9nc(cs9)C(=O)N5)c7c6C)c%10nc(cs%10)C(=O)N[C@@H](C)C(=O)N)[C@H](C)O FPTCMHOCGKKRGQ-WYOWUDGCSA-N 0.000 description 1
- 101800003864 Nosiheptide Proteins 0.000 description 1
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- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
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- 229930185202 Thiopeptin Natural products 0.000 description 1
- IXEQUEFFDUXSRS-UGJHNBEYSA-N Thiopeptin Bb Natural products CC=C1NC(=O)[C@@H](NC(=O)c2csc(n2)[C@]34CCC(=N[C@@H]3c5csc(n5)[C@@H](NC(=S)c6csc(n6)[C@H](NC(=O)[C@@H]7CN=C1S7)[C@@](C)(O)[C@H](C)O)[C@H](C)OC(=O)c8cc([C@@H](C)O)c9C=C[C@@H](N[C@H](C(C)C)C(=O)N[C@H](C)C(=O)NC(=C)C(=O)N[C@H](C)C(=O)N4)[C@@H](O)c9n8)c%10nc(cs%10)C(=O)NC(=C)C(=O)NC(=C)C(=O)O)[C@H](C)O IXEQUEFFDUXSRS-UGJHNBEYSA-N 0.000 description 1
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- 125000000539 amino acid group Chemical group 0.000 description 1
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- 208000022531 anorexia Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
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- 125000002619 bicyclic group Chemical group 0.000 description 1
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- AKFVOKPQHFBYCA-ASKSIGGCSA-N chembl1967607 Chemical compound N=1C2C(N=3)=CSC=3C(C(C)OC(=O)C=3N=C4C(O)C(NC(C(C)C)C(=O)NC(=C)C(=O)NC(=C)C(=O)NC(C)C(=O)N5)C=CC4=C(C(C)O)C=3)NC(=O)C(N=3)=CSC=3C(C(C)(O)C(C)O)NC(=O)C(N=3)CSC=3C(=C/C)/NC(=O)C(C(C)O)NC(=O)C(N=3)=CSC=3C25CCC=1C1=NC(C(=O)NC(=C)C(=O)NC(=C)C(N)=O)=CS1 AKFVOKPQHFBYCA-ASKSIGGCSA-N 0.000 description 1
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 1
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- 238000007710 freezing Methods 0.000 description 1
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- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 208000006443 lactic acidosis Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- PLPRGLOFPNJOTN-UHFFFAOYSA-N narcotine Natural products COc1ccc2C(OC(=O)c2c1OC)C3Cc4c(CN3C)cc5OCOc5c4OC PLPRGLOFPNJOTN-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 229960004708 noscapine Drugs 0.000 description 1
- MQWDKYHFGBWGQZ-JQTJYXGUSA-N nosiheptide Chemical compound N([C@H](C(=O)N\C(C=1SC=C(N=1)C(=O)N[C@@H]1CC(O)C(=O)OCC=2C=CC=C3NC(=C(C3=2)C)C(=O)SC[C@H](NC(=O)C=2N=C1SC=2)C=1SC=C(N=1)C1=N2)=C/C)[C@@H](C)O)C(=O)C(N=3)=CSC=3C1=CC(=O)\C2=C1/NC(C(=O)NC(=C)C(N)=O)=CS1 MQWDKYHFGBWGQZ-JQTJYXGUSA-N 0.000 description 1
- 229950006423 nosiheptide Drugs 0.000 description 1
- 150000002916 oxazoles Chemical class 0.000 description 1
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- 230000003239 periodontal effect Effects 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- LOAUVZALPPNFOQ-UHFFFAOYSA-N quinaldic acid Chemical compound C1=CC=CC2=NC(C(=O)O)=CC=C21 LOAUVZALPPNFOQ-UHFFFAOYSA-N 0.000 description 1
- 229960001404 quinidine Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 108010088584 siomycin Proteins 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 108010057451 thiocillin Proteins 0.000 description 1
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- SRTDKHGZQCTGBY-RVDKCFQWSA-N thiopeptin Chemical compound N1C2C(N=3)=CSC=3C(C(C)OC(=O)C=3N=C4C(O)C(NC(C(C)C)C(=O)NC(C)C(=O)NC(=C)C(=O)NC(C)C(=O)N5)C=CC4=C(C(C)O)C=3)NC(=S)C(N=3)=CSC=3C(C(C)(O)C(C)O)NC(=O)C(N=3)CSC=3C(=C/C)\NC(=O)C(C(C)O)NC(=O)C(N=3)=CSC=3C25CCC1C1=NC(C(N)=O)=CS1 SRTDKHGZQCTGBY-RVDKCFQWSA-N 0.000 description 1
- 108010030742 thiopeptin Proteins 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microbial fermentation, in particular to a method for producing thiostrepton by microbial fermentation, which comprises the steps of carrying out feed supplement culture in the fermentation culture process of Streptomyces lividans seed liquid, wherein the feed supplement culture supplements a carbon source when the carbon source is exhausted and supplements nutrient solution in a fed-batch mode, the feed supplement of the nutrient solution comprises three stages, the feeding rate of the first stage is 0.5-0.65mL/L/h, and the feed supplement time is when the carbon source is exhausted; the feeding rate of the second stage is 0.75-0.9mL/L/h, and the feeding starting time is that the fermentation period is in the range of 85-146 h; the flow rate of the third stage is 0.5-0.75mL/L/h, the feeding starting time is 135-180h, and the total fermentation period is 180-190 h. The invention has the advantages of suitability for industrial production, simple process and short fermentation period.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing thiostrepton by microbial fermentation.
Background
The thiopeptides (thiopeptide) antibiotics are a kind of natural products of polythiazoles (oxazoles) which are rich in sulfur elements and are highly modified, and have unique core aza six-membered rings, thiazole (or oxazoline), dehydrated amino acid residues and other structures. Based on the substitution pattern and the difference in oxidation degree of the core aza six-membered ring, the thiopeptide family members can be divided into 5 subfamilies: group a piperidines, such as thiopeptides (thiopeptin); group b dehydrogenated piperidines, such as salt house mycin (siomycin, SIO); group c dihydroimidazole piperidines, such as Sch40832; trisubstituted pyridines of group d, such as methicillin (thiocillin); group e hydroxylated pyridines, such as noscapine (nosiheptide, NOS). According to the number of rings in the framework structure, the two-ring thiopeptides and the single-ring thiopeptides (type III) can be classified; depending on the side ring precursors, bicyclic thiopeptides can also be subdivided into those comprising methylindole acid (4-methyl-2-indolyl acid, MIA) units (type I) and those comprising quinidine acid (quinaldic acid, QA) units (type II).
The Streptomyces lividans (thiostrepton, TSR) has a unique chemical structure and good biological activity, and is discovered for the first time after Streptomyces lividans (Streptomyces azureus), the TSR is sequentially separated from other Streptomyces lividans (Streptomyces laurentii) and the like. TSR belongs to group b thiopeptides in terms of the structure of the core aza six-membered ring; TSR belongs to type II thiopeptides in terms of the number of rings and side loop precursors.
Thiostrepton has activity against gram-positive bacteria and mycobacteria. Thiostrepton has been widely used as a livestock feed additive in europe, australia and north america to combat lactic acidosis in cattle, sheep and horses. And the thiostrepton is a common screening marker in the field of genetic engineering. In addition, in recent years, research shows that the thiostrepton can inhibit the level of inflammatory cells in periodontal tissues, promote the generation of fibroblasts, contribute to the treatment of periodontitis, and have no side effects of anorexia and growth influence. Therefore, the thiostrepton has wide application prospect.
However, the molecular structure of the thiostrepton is complex and the chemical synthesis is difficult, so that the thiostrepton is mainly synthesized by microbial fermentation. However, there are few studies on the production of thiostrepton by microbial fermentation, and none of the current fermentation processes are suitable for large-scale industrial production.
Therefore, it is necessary to develop a method for producing thiostrepton by microbial fermentation which solves the above-mentioned technical problems.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for producing thiostrepton by microbial fermentation, which is suitable for industrial production, simple in process and short in fermentation period.
The invention is realized by the following technical scheme:
A method for producing thiostrepton by microbial fermentation, comprising the following steps:
(1) Inoculating Streptomyces farinosus into the seed culture medium A for culture to obtain strain liquid containing Streptomyces farinosus;
(2) Inoculating strain liquid containing Streptomyces farinosus into a seed tank for culture to obtain seed liquid;
(3) Fermenting and culturing the seed liquid, wherein the fermenting and culturing comprises feeding and culturing, the feeding and culturing is to supplement the carbon source when the carbon source is exhausted and supplement the nutrient solution in a fed-batch mode, the feeding and culturing of the nutrient solution comprises three stages, the feeding rate of the first stage is 0.5-0.65mL/L/h, and the feeding and culturing time is when the carbon source is exhausted; the feeding rate of the second stage is 0.75-0.9mL/L/h, and the feeding starting time is that the fermentation period is in the range of 85-146 h; the flow rate of the third stage is 0.5-0.75mL/L/h, the feeding starting time is 135-180h, and the total fermentation period is 180-190 h.
Total fermentation period: starting fermentation culture of the seed liquid in the step (3), and ending feeding in the third stage to obtain the final fermentation.
Taking the seed liquid in the step (3) as a fermentation zero point when fermentation culture starts, and carrying out first material feeding when the fermentation culture reaches the exhaustion of carbon sources, wherein the flow rate is 0.5-0.65mL/L/h; fermenting and culturing for 85-146h, wherein the first feeding is finished, and the second feeding is started, and the feeding rate is 0.75-0.9mL/L/h; fermenting and culturing for 135-180h, after the second feeding, starting to perform the third feeding, wherein the feeding rate is 0.5-0.75mL/L/h; fermenting and culturing for 180-190 h, and finishing the third feeding.
Typically fermentation proceeds to 60-96 hours of carbon source depletion.
Preferably, the Streptomyces lividans in the step (1) is inoculated in the form of glycerol seed liquid, the inoculation amount is 2-4% (according to volume percent), the culture mode is shake flask culture, the rotation speed is 200-250rpm, and the shake flask culture is carried out at 25-30 ℃ for 25-35h.
Preferably, the bacterial concentration (according to the volume percentage) of the bacterial liquid in the step (1) is 15-25%.
The "inoculum size" of the invention was calculated as volume percent.
Preferably, the composition of the seed medium a in step (1) comprises: 1.5 to 2.5 percent of glucose (according to mass percent), 12 to 18g/L of bean pulp powder, 18 to 22g/L of corn steep liquor, 1.5 to 2.5g/L of yeast extract powder, 1.5 to 2.5g/L of monopotassium phosphate, 0.8 to 1.2g/L of ammonium sulfate, 1.5 to 2.5g/L of calcium sulfate, 0.2 to 0.6g/L of magnesium sulfate heptahydrate, 0.1 to 0.3g/L of ferrous sulfate heptahydrate and 1 to 3mL/L of microelement liquid.
Preferably, in the step (2), the seed tank is added with a seed culture medium B, and the composition is as follows: 1.5 to 2.5 percent of glucose (according to mass percent), 18 to 22g/L of bean pulp powder, 27 to 33g/L of corn steep liquor, 1.5 to 2.5g/L of yeast extract powder, 1.5 to 2.5g/L of monopotassium phosphate, 0.8 to 1.2g/L of ammonium sulfate, 1.5 to 2.5g/L of calcium sulfate, 0.2 to 0.6g/L of magnesium sulfate heptahydrate, 0.1 to 0.3g/L of ferrous sulfate heptahydrate, 1 to 3mL/L of microelement liquid and 0.5 to 1.0g/L of defoamer.
More preferably, the inoculation amount in the step (2) is 0.2-0.6% (calculated by the volume percentage of the strain liquid containing the streptomyces farinosus to the seed culture medium B), the initial stirring speed of the seed tank is 150-250rpm, the aeration ratio is 0.8-1.2vvm, the culture temperature is 25-27 ℃, the pressure is 0.03-0.07MPa, the dissolved oxygen is controlled to be 20-40%, the dissolved oxygen is associated with stirring, and the culture is carried out for 40-50h.
Preferably, in the step (3), a fermentation medium is used for fermentation culture, and the composition is as follows: 4-8% of glucose (according to mass percent), 18-22g/L of soybean meal powder, 27-33g/L of corn steep liquor, 1.5-2.5g/L of yeast extract powder, 1.5-2.5g/L of sodium dihydrogen phosphate, 0.8-1.2g/L of ammonium sulfate, 1.5-2.5g/L of calcium sulfate, 0.2-0.6g/L of magnesium sulfate heptahydrate, 0.1-0.3g/L of ferrous sulfate heptahydrate, 2-6mL/L of trace element liquid and 0.5-1.0g/L of defoamer.
Preferably, the nutrient solution in step (3) comprises the following components: 160-180g/L of soybean meal hydrolysate, 120-180g/L of corn steep liquor, 6-10g/L of monopotassium phosphate, 15-20g/L of sodium sulfate and 6-10mL/L of microelement liquid.
More preferably, the trace element liquid comprises the following components: cobalt chloride hexahydrate 4-6g/L, zinc sulfate heptahydrate 0.5-1.5g/L, copper sulfate dihydrate 0.5-1.5g/L, boric acid 0.3-0.7g/L and sodium molybdate 0.3-0.7 g/L.
Preferably, during the feed culture in step (3), the concentration of the carbon source (glucose) is controlled to be 0.2-1.0% (in mass percent), and the pH is controlled to be 6.8.
Preferably, in the step (3), the fermentation seed transfer amount is 8-12% (according to the volume percent of the seed solution to the fermentation culture medium), the initial stirring speed is 150-250rpm, the aeration ratio is 0.8-1.2vvm, the culture temperature is 25-27 ℃, the pressure is 0.03-0.07MPa, the dissolved oxygen is controlled to be 15-40%, and the dissolved oxygen is associated with stirring.
The beneficial effects of the invention are as follows:
The invention optimizes the fermentation feeding process, replaces bean pulp powder feeding with bean pulp hydrolysate, avoids the easy deposition of the bean pulp powder feeding in a pipeline, is easier for industrial production, adopts the feeding mode of gradient feeding, adopts low flow rate in the early stage, gradually increases and then gradually decreases in the later stage. Through improvement, the titer of the thiostrepton can be stabilized to be more than 2.30g/L, the industrialization level is achieved, the fermentation process is simple, the cost is low, and the method still has higher titer when being used for industrial production.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
The trace element liquid composition in each of the following examples is: cobalt chloride hexahydrate 5g/L, zinc sulfate heptahydrate 1g/L, copper sulfate dihydrate 1g/L, boric acid 0.5g/L, and sodium molybdate 0.5 g/L.
The defoamer adopts defoamer THIX-298.
The glycerol stock solution was Streptomyces lividans (Streptomyces azureus) glycerol stock solution, and the strain was purchased from the American Type Culture Collection (ATCC) under the accession number ATCC14921. The specific preparation process is as follows: 20mL of sterile water was added to the well grown eggplant bottle slant, after the mycelium was washed off, 1mL of the bacterial suspension was sucked into a 250mL Erlenmeyer flask containing 25mL of seed medium, and shaking culture was performed at 28℃and 220rpm for 48 hours. 0.8mL of seed solution is respectively absorbed into a freezing tube filled with sterilized 0.8mL of 40% glycerol solution, and the frozen tube is stored in a refrigerator at the temperature of minus 20 ℃ for standby. The seed culture medium is as follows: glucose 2%, soybean meal 15g/L, corn steep liquor 20g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L and trace element liquid 2mL/L.
Example 1
1) Strain culture
1ML of glycerol tube seed liquid is inoculated into 25mL of sterile shake flask seed culture medium, wherein the seed culture medium is as follows: glucose 2%, soybean meal 15g/L, corn steep liquor 20g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L and trace element liquid 2mL/L. Shake flask seeds were shake-cultured at 220rpm at 27℃for 30 hours to obtain a strain solution having a cell concentration of 18%.
2) Seed culture
The seed culture medium is as follows: glucose 2%, soybean meal 20g/L, corn steep liquor 30g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L, trace element liquid 2mL/L and defoamer 0.8g/L. Sequentially weighing 10L, dissolving with a certain volume of drinking water, mixing, pouring into a seed tank, fixing volume to 8L, adjusting pH to 7.0 with sodium hydroxide, sterilizing with high temperature and high pressure steam at 0.11-0.13MPa and 121-123 deg.C, maintaining for 30min, and cooling to 26 deg.C, wherein the volume of culture medium is 10L. Transferring 25mL of the strain liquid obtained in the step 1) to a seed tank, initially stirring the seed tank at 200rpm, enabling the aeration ratio to be 0.8vvm, enabling the culture temperature to be 26 ℃, enabling the pressure to be 0.05MPa, controlling the dissolved oxygen to be 20-40%, enabling the dissolved oxygen to be associated with stirring, and culturing for 42h to obtain the seed liquid, wherein the concentration of the thallus is 20%.
3) Fermentation culture
The fermentation medium is as follows: 6% of glucose, 20g/L of soybean meal, 30g/L of corn steep liquor, 2g/L of yeast extract powder, 2g/L of sodium dihydrogen phosphate, 1.0g/L of ammonium sulfate, 2.0g/L of calcium sulfate, 0.4g/L of magnesium sulfate heptahydrate, 0.2g/L of ferrous sulfate heptahydrate, 4mL/L of trace element liquid and 0.8g/L of defoamer. Sequentially weighing 25L, dissolving with a certain volume of drinking water, mixing, pouring into a tank, constant volume of 18L, adjusting pH to 7.0 with sodium hydroxide, sterilizing with high temperature and high pressure steam at 0.11-0.13MPa, maintaining at 121-123 deg.C for 30min, and cooling to 26 deg.C. The fermentation seed transfer amount is 10%, the initial stirring is 200rpm, the aeration ratio is 0.8vvm, the culture temperature is 26 ℃, the pressure is 0.05MPa, the dissolved oxygen is controlled to be 15-40%, and the dissolved oxygen is associated with stirring.
4) Material supplementing method
Glucose solution: preparing 70% (w/v) glucose aqueous solution 8L, sterilizing at 121deg.C for 20 min.
Nutrient solution: 500g of soybean meal hydrolysate (purchased from Shandong Yubao biotechnology Co., ltd.), 450g of corn steep liquor, 250g of yeast extract powder, 25g of monopotassium phosphate, 50g of sodium sulfate and 25mL of trace element liquid, adding a proper amount of drinking water to the total volume of 3L, and sterilizing for 30min at 121 ℃ for later use.
Ammonia water: 3L of 25% ammonia water solution.
When the fermentation culture is completed for 63 hours, the glucose is consumed, the pH value starts to rise back, the glucose solution is supplemented, the flow rate is controlled by a glucose supplementing pump, the sugar content of the fermentation liquid is measured by a sugar meter, and the glucose concentration is controlled to be 0.3% in the whole process. Ammonia water is used for controlling the pH value of the fermentation liquor to be 6.8, meanwhile, nutrient solution is fed in a flowing way (three times of feeding is taken in total), the first feeding is carried out when the fermentation culture is carried out until the glucose is exhausted, and the feeding rate is 0.5mL/L/h; fermenting and culturing for 85h, wherein the first feeding is finished, the second feeding is started, and the feeding rate is 0.75mL/L/h; fermenting and culturing for 135h, wherein the second feeding is finished, and the third feeding is started, and the feeding rate is 0.5mL/L/h; fermenting and culturing for 180h, and finishing the third feeding. Putting the mixture into a tank, and fermenting the product to 3.16g/L.
Example 2
1) Strain culture
1ML of glycerol tube seed liquid is inoculated into 25mL of sterile shake flask seed culture medium, wherein the seed culture medium is as follows: glucose 2%, soybean meal 15g/L, corn steep liquor 20g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L and trace element liquid 2mL/L. Shake flask seeds were shake-cultured at 220rpm at 27℃for 38 hours to obtain a strain solution having a cell concentration of 21%.
2) Seed culture
The seed culture medium is as follows: glucose 2%, soybean meal 20g/L, corn steep liquor 30g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L, trace element liquid 2mL/L and defoamer 0.8g/L. Sequentially weighing 10L, dissolving with a certain volume of drinking water, mixing, pouring into a seed tank, fixing volume to 8L, adjusting pH to 7.0 with sodium hydroxide, sterilizing with high temperature and high pressure steam at 0.11-0.13MPa and 121-123 deg.C, maintaining for 30min, and cooling to 26 deg.C, wherein the volume of culture medium is 10L. Transferring 25mL of the strain liquid obtained in the step 1) to a seed tank, initially stirring the seed tank at 200rpm, enabling the aeration ratio to be 1.0vvm, enabling the culture temperature to be 26 ℃, enabling the pressure to be 0.05MPa, controlling the dissolved oxygen to be 20% -40%, enabling the dissolved oxygen to be associated with stirring, and culturing for 45h to obtain the seed liquid, wherein the concentration of the thallus is 22%.
3) Fermentation culture
The fermentation medium is as follows: 6% of glucose, 20g/L of soybean meal, 30g/L of corn steep liquor, 2g/L of yeast extract powder, 2g/L of sodium dihydrogen phosphate, 1.0g/L of ammonium sulfate, 2.0g/L of calcium sulfate, 0.4g/L of magnesium sulfate heptahydrate, 0.2g/L of ferrous sulfate heptahydrate, 4mL/L of trace element liquid and 0.8g/L of defoamer. Sequentially weighing 25L, dissolving with a certain volume of drinking water, mixing, pouring into a tank, constant volume of 18L, adjusting pH to 7.0 with sodium hydroxide, sterilizing with high temperature and high pressure steam at 0.11-0.13MPa, maintaining at 121-123 deg.C for 30min, and cooling to 26 deg.C. The fermentation seed transfer amount is 10%, the initial stirring is 200rpm, the aeration ratio is 1.2vvm, the culture temperature is 26 ℃, the pressure is 0.05MPa, the dissolved oxygen is controlled to be 15% -40%, and the dissolved oxygen is associated with stirring.
4) Material supplementing method
Glucose solution: preparing 70% (w/v) glucose aqueous solution 8L, sterilizing at 121deg.C for 20 min.
Nutrient solution: 500g of soybean meal hydrolysate (purchased from Shandong Yubao biotechnology Co., ltd.), 450g of corn steep liquor, 25g of monopotassium phosphate, 50g of sodium sulfate and 25mL of microelement liquid, adding a proper amount of drinking water to the total volume of 3L, and sterilizing for 30min at 121 ℃ for later use.
Ammonia water: 25% aqueous ammonia solution 3L
When the fermentation culture is completed for 60 hours, glucose is consumed, pH begins to rise back, glucose solution is supplemented, a sugar supplementing pump is used for controlling the flow rate, a sugar meter is used for measuring the sugar content of the fermentation liquid, and the glucose concentration is controlled to be 0.5% in the whole process. Ammonia water is used to control the pH of the fermentation liquor to 6.8, and meanwhile, nutrient solution is fed in (three times of feeding are taken). The first feeding is carried out when the fermentation culture is carried out until the glucose is exhausted, and the feeding rate is 0.65mL/L/h; fermenting and culturing for 146h, wherein the first feeding is finished, the second feeding is started, and the feeding rate is 0.9mL/L/h; fermenting and culturing for 170h, wherein the second feeding is finished, and the third feeding is started, and the feeding rate is 0.75mL/L/h; fermenting and culturing for 185h, and finishing the third feeding. Putting the mixture into a tank, and fermenting the product to 3.21g/L.
Example 3
1) Strain culture
1ML of glycerol tube seed liquid is inoculated into 25mL of sterile shake flask seed culture medium, wherein the seed culture medium is as follows: glucose 2%, soybean meal 15g/L, corn steep liquor 20g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L and trace element liquid 2mL/L. Shake flask seeds were shake-cultured at 220rpm at 27℃for 40h to obtain a strain solution having a cell concentration of 22%.
2) Seed culture
The seed culture medium is as follows: glucose 2%, soybean meal 20g/L, corn steep liquor 30g/L, yeast extract 2g/L, monopotassium phosphate 2g/L, ammonium sulfate 1.0g/L, calcium sulfate 2.0g/L, magnesium sulfate heptahydrate 0.4g/L, ferrous sulfate heptahydrate 0.2g/L, trace element liquid 2mL/L and defoamer 0.8g/L. Sequentially weighing 10L, dissolving with a certain volume of drinking water, mixing, pouring into a seed tank, fixing volume to 8L, adjusting pH to 7.0 with sodium hydroxide, sterilizing with high temperature and high pressure steam, maintaining at 0.12MPa and 122 deg.C for 30min, and cooling to 26 deg.C, wherein the volume of culture medium is 10L. Transferring 25mL of the strain liquid obtained in the step 1) to a seed tank, initially stirring the seed tank at 200rpm, enabling the aeration ratio to be 1.0vvm, enabling the culture temperature to be 26 ℃, enabling the pressure to be 0.05MPa, controlling the dissolved oxygen to be 20% -40%, enabling the dissolved oxygen to be associated with stirring, and culturing for 43h to obtain the seed liquid, wherein the concentration of the thallus is 20%.
3) Fermentation culture
The fermentation medium is as follows: 6% of glucose, 20g/L of soybean meal, 30g/L of corn steep liquor, 2g/L of yeast extract powder, 2g/L of sodium dihydrogen phosphate, 1.0g/L of ammonium sulfate, 2.0g/L of calcium sulfate, 0.4g/L of magnesium sulfate heptahydrate, 0.2g/L of ferrous sulfate heptahydrate, trace element liquid and 0.8g/L of defoamer. Sequentially weighing 25L, dissolving with a certain volume of drinking water, mixing, pouring into a tank, constant volume of 18L, adjusting pH to 7.0 with sodium hydroxide, sterilizing with high temperature and high pressure steam at 0.11-0.13MPa, maintaining at 121-123 deg.C for 30min, and cooling to 26 deg.C. The fermentation seed transfer amount is 10%, the initial stirring is 200rpm, the aeration ratio is 1.0vvm, the culture temperature is 26 ℃, the pressure is 0.05MPa, the dissolved oxygen is controlled to be 15% -40%, and the dissolved oxygen is associated with stirring.
4) Material supplementing method
Glucose solution: preparing 70% (w/v) glucose aqueous solution 8L, sterilizing at 121deg.C for 20 min.
Nutrient solution: 500g of soybean meal hydrolysate (purchased from Shandong Yubao biotechnology Co., ltd.), 450g of corn steep liquor, 25g of monopotassium phosphate, 50g of sodium sulfate and 25mL of microelement liquid, adding a proper amount of drinking water to the total volume of 3L, and sterilizing for 30min at 121 ℃ for later use.
Ammonia water: 25% aqueous ammonia solution 3L
When the fermentation culture is completed for 63 hours, glucose is consumed, pH begins to rise back, glucose solution is supplemented, a sugar supplementing pump is used for controlling the flow rate, a sugar meter is used for measuring the sugar content of the fermentation liquid, and the glucose concentration is controlled to be 1.0% in the whole process. Ammonia water is used to control the pH of the fermentation liquor to 6.8, and meanwhile, nutrient solution is fed in (three times of feeding are taken). The first feeding is carried out when the fermentation culture is carried out until the glucose is exhausted, and the feeding rate is 0.60mL/L/h; fermenting and culturing for 115 hours, wherein the first material supplementing is finished, and the second material supplementing is started, and the feeding rate is 0.85mL/L/h; fermenting and culturing for 165h, wherein the second feeding is finished, and the third feeding is started, and the feeding rate is 0.60mL/L/h; fermenting and culturing for 190h, and finishing the third feeding. Putting the mixture into a tank, and fermenting the product to 3.30g/L.
Comparative example 1
The difference from example 3 is only that the nutrient solution is added in the feeding method of step 4), and the other conditions are the same, specifically as follows:
and feeding nutrient solution when the glucose is exhausted during fermentation culture, wherein the feeding rate is 0.85mL/L/h, the total fermentation period is 190h, and the fermentation unit of the product is 2.50g/L.
Comparative example 2
The difference from example 3 is only that the composition of the nutrient solution in the feed method of step 4) is different, the soybean meal hydrolysate is replaced by the soybean meal powder with equal mass, and the rest conditions are the same.
The final product fermentation unit was 2.93g/L.
Comparative example 3
The difference from example 3 is only that the nutrient solution is added in the feeding method of step 4), and the other conditions are the same, specifically as follows:
The first feeding is carried out when the fermentation culture is carried out until the glucose is exhausted, and the feeding rate is 0.85mL/L/h; fermenting and culturing for 115 hours, wherein the first material supplementing is finished, and the second material supplementing is started, and the feeding rate is 0.85mL/L/h; fermenting and culturing for 165h, wherein the second feeding is finished, and the third feeding is started, and the feeding rate is 0.60mL/L/h; fermenting and culturing for 190h, and finishing the third feeding. And (5) placing the pot.
The final product fermentation unit was 2.79g/L.
Comparative example 4
The difference from example 3 is only that the nutrient solution is added in the feeding method of step 4), and the other conditions are the same, specifically as follows:
The first feeding is carried out when the fermentation culture is carried out until the glucose is exhausted, and the feeding rate is 0.60mL/L/h; fermenting and culturing for 115 hours, wherein the first material supplementing is finished, and the second material supplementing is started, and the feeding rate is 0.85mL/L/h; fermenting and culturing for 165h, wherein the second feeding is finished, and the third feeding is started, and the feeding rate is 0.85mL/L/h; fermenting and culturing for 190h, and finishing the third feeding. And (5) placing the pot.
The final product fermentation unit was 2.86g/L.
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (7)
1. A method for producing thiostrepton by microbial fermentation, which is characterized by comprising the following steps:
(1) Inoculating Streptomyces lividans into a first seed culture medium for culture to obtain strain liquid containing the Streptomyces lividans;
(2) Inoculating strain liquid containing Streptomyces farinosus into a seed tank for culture to obtain seed liquid;
(3) The seed liquid is subjected to fermentation culture, wherein the fermentation culture comprises feed supplement culture, the feed supplement culture is to supplement a carbon source when the carbon source is exhausted and supplement nutrient solution in a fed-batch mode, the feed supplement of the nutrient solution comprises three stages, the feeding rate of the first stage is 0.5-0.65 mL/L/h, and the feed supplement time is when the carbon source is exhausted; the feeding rate of the second stage is 0.75-0.9 mL/L/h, and the feeding starting time is that the fermentation period is in the range of 85-146 h; the flow rate in the third stage is 0.5-0.75 mL/L/h, the feeding time is 135-180 h, and the total fermentation period is 180 h-190 h;
The nutrient solution in the step (3) comprises the following components: 160-180 g/L of soybean meal hydrolysate, 120-180 g/L of corn steep liquor, 6-10 g/L of monopotassium phosphate, 15-20 g/L of sodium sulfate and 6-10 mL/L of trace element liquid;
The pH value in the feeding culture process of the step (3) is 6.8;
in the step (3), a fermentation medium is adopted for fermentation culture, and the composition is as follows: 4-8% of glucose, 18-22 g/L of bean pulp powder, 27-33 g/L of corn steep liquor, 1.5-2.5 g/L of yeast extract powder, 1.5-2.5 g/L of sodium dihydrogen phosphate, 0.8-1.2 g/L of ammonium sulfate, 1.5-2.5 g/L of calcium sulfate, 0.2-0.6 g/L of magnesium sulfate heptahydrate, 0.1-0.3 g/L of ferrous sulfate heptahydrate, 2-6 mL/L of microelement liquid and 0.5-1.0 g/L of defoamer;
In the feed supplement culture process of the step (3), the mass percentage concentration of the carbon source is controlled to be 0.2-1.0%.
2. The method according to claim 1, wherein the Streptomyces lividans is inoculated in the form of a glycerol seed solution in the step (1) in an amount of 2 to 4% by shaking culture at a rotation speed of 200 to 250 rpm and 25 to 35 h by shaking culture at 25 to 30 ℃; the bacterial concentration of the bacterial liquid in the step (1) is 15-25%.
3. The method of claim 1, wherein the first seed medium composition of step (1) comprises: 1.5 to 2.5 percent of glucose, 12 to 18 g/L of bean pulp powder, 18 to 22 g/L of corn steep liquor, 1.5 to 2.5 g/L of yeast extract powder, 1.5 to 2.5 g/L of monopotassium phosphate, 0.8 to 1.2 g/L of ammonium sulfate, 1.5 to 2.5 g/L of calcium sulfate, 0.2 to 0.6 g/L of magnesium sulfate heptahydrate, 0.1 to 0.3 g/L of ferrous sulfate heptahydrate and 1 to 3 mL/L of microelement liquid.
4. The method of claim 1, wherein the seeding tank in step (2) is supplemented with a second seed medium comprising: 1.5 to 2.5 percent of glucose, 18 to 22 g/L of bean pulp powder, 27 to 33 g/L of corn steep liquor, 1.5 to 2.5 g/L of yeast extract powder, 1.5 to 2.5 g/L of monopotassium phosphate, 0.8 to 1.2 g/L of ammonium sulfate, 1.5 to 2.5 g/L of calcium sulfate, 0.2 to 0.6 g/L of magnesium sulfate heptahydrate, 0.1 to 0.3 g/L of ferrous sulfate heptahydrate, 1 to 3mL/L of microelement liquid and 0.5 to 1.0 g/L of defoamer.
5. The method of any one of claims 3-4, wherein the trace element liquid composition is: cobalt chloride hexahydrate 4-6 g/L, zinc sulfate heptahydrate 0.5-1.5 g/L, copper sulfate dihydrate 0.5-1.5 g/L, boric acid 0.3-0.7 g/L and sodium molybdate 0.3-0.7 g/L.
6. The method according to claim 1, wherein the inoculation amount in the step (2) is 0.2-0.6%, the initial stirring rate of the seed tank is 150-250 rpm, the aeration ratio is 0.8-1.2 vvm, the culture temperature is 25-27 ℃, the pressure is 0.03-0.07 MPa, the dissolved oxygen is controlled to be 20-40%, the dissolved oxygen is associated with stirring, and the culture is 40-50 h.
7. The method according to claim 1, wherein in the step (3), the fermentation and seed transfer amount is 8-12%, the initial stirring rate is 150-250 rpm, the aeration ratio is 0.8-1.2 vvm, the culture temperature is 25-27 ℃, the pressure is 0.03-0.07-MPa, the dissolved oxygen is controlled to be 15-40%, and the dissolved oxygen is associated with stirring.
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