CN117210520A - Method for improving fermentation level of daptomycin - Google Patents
Method for improving fermentation level of daptomycin Download PDFInfo
- Publication number
- CN117210520A CN117210520A CN202311155790.6A CN202311155790A CN117210520A CN 117210520 A CN117210520 A CN 117210520A CN 202311155790 A CN202311155790 A CN 202311155790A CN 117210520 A CN117210520 A CN 117210520A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- daptomycin
- powder
- added
- precursor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 149
- 230000004151 fermentation Effects 0.000 title claims abstract description 149
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 title claims abstract description 50
- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 49
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000002243 precursor Substances 0.000 claims abstract description 46
- LADGBHLMCUINGV-UHFFFAOYSA-N tricaprin Chemical compound CCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCC)COC(=O)CCCCCCCCC LADGBHLMCUINGV-UHFFFAOYSA-N 0.000 claims abstract description 34
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000000463 material Substances 0.000 claims abstract description 22
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 14
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 9
- 239000010452 phosphate Substances 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 5
- 239000000843 powder Substances 0.000 claims description 61
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 21
- 240000008042 Zea mays Species 0.000 claims description 21
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 21
- 235000005822 corn Nutrition 0.000 claims description 21
- 229920000742 Cotton Polymers 0.000 claims description 17
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 14
- 229920001353 Dextrin Polymers 0.000 claims description 9
- 239000004375 Dextrin Substances 0.000 claims description 9
- 235000019425 dextrin Nutrition 0.000 claims description 9
- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 235000012054 meals Nutrition 0.000 claims 2
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 abstract description 30
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 abstract description 14
- 230000001502 supplementing effect Effects 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 abstract description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 abstract description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 230000006378 damage Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 125000005456 glyceride group Chemical group 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 36
- 239000002028 Biomass Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- FIWQZURFGYXCEO-UHFFFAOYSA-M sodium;decanoate Chemical compound [Na+].CCCCCCCCCC([O-])=O FIWQZURFGYXCEO-UHFFFAOYSA-M 0.000 description 11
- 241001052560 Thallis Species 0.000 description 10
- 239000002585 base Substances 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000958215 Streptomyces filamentosus Species 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 229940073769 methyl oleate Drugs 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- GYSCBCSGKXNZRH-UHFFFAOYSA-N 1-benzothiophene-2-carboxamide Chemical compound C1=CC=C2SC(C(=O)N)=CC2=C1 GYSCBCSGKXNZRH-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- -1 cyclic beta-amino acid Chemical class 0.000 description 2
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 125000005313 fatty acid group Chemical group 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 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
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A method for improving fermentation level of daptomycin belongs to the technical field of microbial fermentation. In the method, capric triglyceride is used as a precursor substance for fermentation of daptomycin, and phosphate is added into a fermentation medium to maintain the pH value in the fermentation process and simultaneously improve the nitrogen source proportion in the fermentation medium. According to the invention, the capric triglyceride is used as a precursor substance for fermentation of daptomycin, so that the damage effect on mycelium is reduced, the capric acid is decomposed from glyceride chains according to the utilized degree, the utilization rate of the capric acid is improved, and the precursor is added at one time, so that the complex operation of supplementing the precursor in a fed-batch mode is avoided; and phosphate is added into the base material to maintain the pH value of the fermentation liquid, and ammonia water is not required to be additionally added in the whole fermentation process to control the pH value, so that the operation of frequently adding ammonia water into a shake flask is avoided; and meanwhile, the proportion of the nitrogen source in the base material is increased to replace ammonia water to be supplied as the nitrogen source. The method simplifies the addition method of the precursor, does not need to control the pH value in the fermentation process, and is beneficial to the amplified production of fermentation; the maximum titer of the shake flask reaches 1500mg/L.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for improving fermentation level of daptomycin.
Background
Daptomycin is a novel cycloaliphatic peptide antibiotic produced by fermentation of streptomyces roseosporus, and the daptomycin molecule is formed by connecting a decaalkane side chain with tryptophan at the N-terminal end of a cyclic beta-amino acid peptide chain consisting of 13 amino acid residues. Because of the unique structure and the special drug mechanism, the compound has an inhibiting effect on 15 gram-positive bacteria of 35 species, and is regarded as the last line of defense against severe infection caused by multi-drug resistant gram-positive bacteria. At the end of 2003, the U.S. Food and Drug Administration (FDA) approved daptomycin for injection (trade name cubiciin) for the treatment of concurrent skin and skin structure infections caused by some gram-positive sensitive strains, and was approved for infectious disease treatment in month 3 of 2006.
At present, daptomycin is mainly obtained through microbial fermentation, and two approaches mainly exist, namely, a mother nucleus compound formed by 13 amino acid residues is separated from fermentation liquor, a fatty acid side chain is hydrolyzed, and then the daptomycin is semi-synthesized through a series of chemical reactions; and secondly, adding precursor capric acid in the fermentation process of the streptomyces roseosporus, and separating daptomycin from the fermentation liquor. The method of adding the decanoic acid precursor is relatively simple and time-saving, and most of the current methods are used for obtaining daptomycin. In a batch fermentation system for producing daptomycin, when the concentration of a precursor is high, highly toxic precursors can inhibit the growth of thalli to a certain extent, and when the concentration of the precursor is low, the supply is insufficient, the concentration of a product in a fermentation liquid is correspondingly low, and the addition of the precursor becomes a step for restricting the biosynthesis of the daptomycin, so that the addition mode and the type of the precursor become research hot spots.
The invention patent with publication number of CN111892647A discloses a method for supplementing a precursor of daptomycin fermentation, namely, feeding a 1:1 volume ratio of capric acid-methyl oleate solution into fermentation liquor in a constant velocity gradient mode at the time of beginning differentiation of mycelium and transferring to a metabolic stage. The invention patent with publication number of CN108342435A discloses a feeding method, wherein a carbon source is fed in the whole fermentation process, when the fermentation is carried out for 18-30 hours, a capric acid precursor compound is fed in a feeding rate of 0.12mL/L/h, and when the fermentation is carried out for 28-40 hours, the capric acid precursor compound is fed in a feeding rate of 0.24mL/L/h until the fermentation is finished. In the method disclosed in publication No. CN101824452A, after 30h of fermentation, a solution of methyl caprate-oleate (1:1) is fed at a rate of 0.10mL/h, and glycerin is fed at a rate of 96h as a main carbon source in the later stage of the thalli, so that the synthesis time of the daptomycin is prolonged. In the above disclosed method, the precursor is added by the fed-batch method, and the precursor is basically capric acid, and the capric acid is toxic to the cells when excessive, so that the amount of the capric acid added in the fed-batch process and the healthy growth of the cells are difficult to reach an equilibrium state, and the cells are easy to die due to excessive capric acid addition in the fermentation process.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to design a technical scheme of a method for improving the fermentation level of daptomycin. The method uses capric triglyceride as precursor, and solves the problem of toxic and side effects of precursor on thalli.
In order to achieve the above purpose, the present invention is specifically implemented by the following technical scheme:
a method for increasing fermentation level of daptomycin, which uses capric triglyceride as precursor material of fermentation of daptomycin, and adds phosphate into fermentation medium to maintain pH during fermentation, and at the same time increases nitrogen source proportion in fermentation medium.
Further, the precursor substance capric triglyceride is added in the following manner: the additive is added into the base material at one time without feeding; the precursor capric triglyceride is added at a concentration of 3-4%, preferably 3.5%.
Further, the phosphate is one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, preferably dipotassium hydrogen phosphate.
Further, the phosphate is added at a concentration of 0.2 to 2%, preferably 0.5%.
Further, the nitrogen source in the fermentation medium comprises one or more of yeast powder, corn steep liquor powder and cotton seed powder, preferably three of the yeast powder, the corn steep liquor powder and the cotton seed powder.
Further, the nitrogen source is added to the fermentation medium at a concentration of 0.25-6.5%.
Further, when the nitrogen source in the fermentation medium is three kinds of yeast powder, corn steep liquor powder and cotton seed powder, the adding concentration of the yeast powder is 2-4%, the adding concentration of the corn steep liquor powder is 0.5-1.5%, the adding concentration of the cotton seed powder is 0.5-1%, preferably the adding concentration of the yeast powder is 3%, the adding concentration of the corn steep liquor powder is 1.0%, and the adding concentration of the cotton seed powder is 0.75%.
Further, the fermentation medium comprises the following components in mass concentration: every 100ml, 10-14g of dextrin, 2-4g of yeast powder, 0.5-1.5g of corn steep liquor powder, 0.5-1g of cotton seed powder, 0.2-2g of dipotassium hydrogen phosphate, 0.15-0.25g of ferrous ammonium sulfate and 3-4g of capric triglyceride.
The invention has the beneficial effects that:
(1) The precursor capric triglyceride is an oil compound, has small stimulation and injury effects on thalli, and capric triglyceride molecules can moderately decompose capric molecules for synthesizing daptomycin according to the utilization degree of capric acid in fermentation liquor, and meanwhile, the decomposed residual glycerol molecules can be used as a carbon source for the thalli, so that the situation of cell death caused by excessive capric acid is not easy to occur;
(2) The capric triglyceride is added at one time, and a precursor is not required to be fed in during the fermentation process, so that the control method in the fermentation process is simpler and more convenient, and the energy and personnel cost can be reduced in industrial production;
(3) The pH of the fermentation liquor is basically kept at 6.5 or above due to the fact that the dipotassium hydrogen phosphate is added into the base material, so that ammonia water or liquid alkali is not required to be added in the fermentation process to control the pH of the fermentation liquor, and a control method in the fermentation process is simplified;
(4) The method simplifies the addition method of the precursor, does not need to control the pH value in the fermentation process, and is beneficial to the amplified production of fermentation; the maximum titer of the shake flask reaches 1500mg/L.
Detailed Description
The present invention will be further described more fully with reference to the following specific examples for better understanding of the present technical solution.
The HPLC method is adopted for detecting the content of daptomycin in the fermentation liquor. Sample pretreatment: shaking the fermentation liquor, sucking 2ml fermentation liquor into a 10ml test tube, adding 4ml absolute ethyl alcohol, carrying out ultrasonic treatment for 10min, centrifuging for 10min at 12000rpm by a high-speed centrifuge, taking out, filtering by a 0.45 mu m organic microporous filter membrane, and obtaining filtrate, and analyzing the filtrate by HPLC. HPLC mobile phase formulation: (1) 0.05M disodium hydrogen phosphate solution: weighing 17.9g of disodium hydrogen phosphate (containing 12 crystal water) into 1000ml of purified water, regulating the pH to 3.15+/-0.05 by using phosphoric acid after dissolving, and carrying out suction filtration for use; (2) And measuring 370ml of acetonitrile by using a measuring cylinder, placing the measuring cylinder into a reagent bottle, measuring 630ml of 0.05M disodium hydrogen phosphate solution, pouring the solution into the reagent bottle, shaking the solution uniformly, and performing ultrasonic degassing to obtain the nano-meter. HPLC analysis conditions: 4.6mm×250mm×5mu. m C18 (Welch) column, column temperature 40 ℃, flow rate 1.0ml/min, sample volume 10. Mu.l, detection wavelength 214nm.
The method for detecting the biomass in the fermentation broth comprises the following specific operations: 10ml of the fermentation broth was centrifuged at 3500r/min for 10min in a centrifuge tube, and the volume of the supernatant was measured. Biomass% = [ (10 ml-supernatant volume/ml)/(10 ml ] ×100%).
The daptomycin producing strain used in the examples was Streptomyces roseosporus (Streptomyces roseosporus).
Example 1: shaking flask fermentation (capric acid as precursor)
Preparation of single colonies: (1) The solid culture medium is prepared according to the following proportion, wherein each 100ml of the solid culture medium comprises 0.4g of malt extract powder, 0.4g of yeast extract powder, 0.4g of glucose, 0.2g of calcium carbonate and 1.8g of agar powder, the pH value is adjusted to 7.0-7.4 by using 20% NaOH solution, and the solid culture medium is sterilized for 30min at 120-122 ℃; (2) And (3) carrying out gradient dilution on the preserved glycerol freezing tube, coating the glycerol freezing tube on a flat plate, and placing the flat plate in a constant temperature incubator for culturing for 8-10d at 30 ℃ to obtain single colonies.
Preparing shake flask seeds: (1) The seed culture medium is prepared according to the following proportion, wherein each 100ml of the seed culture medium comprises 2g of dextrin, 1g of yeast powder, 1g of yeast extract powder and 0.5g of corn steep liquor powder, the pH value is adjusted to 7.0-7.4 by using 20% NaOH solution, and the seed culture medium is sterilized for 30min at 120-122 ℃; (2) And 1 single colony after the culture is picked up and put into a shake flask seed culture medium, the seed culture medium is filled with 20ml/250ml, the temperature is 30 ℃, the rpm is 250, and the shaking culture is carried out on a shaking table for 22-24 hours, so that seed liquid is prepared.
Preparing a fermentation medium: the fermentation medium is prepared according to the following proportion, wherein each 100ml of the medium contains 12g of dextrin, 3g of yeast powder, 1.0g of corn steep liquor powder, 0.75g of cotton seed powder, 0.5g of dipotassium hydrogen phosphate and 0.2g of ferrous ammonium sulfate; the materials are weighed and dissolved together, the volume is fixed, the materials are then filled into a fermentation shaking flask, the filling amount of a fermentation medium is 25ml/250ml, the pH value is regulated to 7.0-7.4 by Tris, and the materials are sterilized for 30min at 120-122 ℃.
Preparing a precursor: the decanoic acid and methyl oleate were mixed in a volume ratio of 1:1 and were filtered through a 0.22 μm needle filter for sterilization and ready for use.
Inoculating: the seed solution was inoculated into the shake flask fermentation medium at an inoculum size of 8%, at 29.5℃and 250rpm, and cultured on a shaker for 144 hours.
The method for supplementing the precursor comprises the following steps: the precursors were each performed using three additional strategies.
Strategy one: fermenting for 22-46h, and adding 40 μl/time every 12 h; fermenting for 46-70h, and adding 80 μl/time every 12 h; after 70h of fermentation, 125. Mu.l/time was added every 12 h.
Strategy II: fermenting for 18-30h, and adding 36 μl/time every 6 h; after fermentation for 30h, 36 μl/time is added every 12 h;
strategy III: fermenting for 48h, and adding 80 μl; after which 80 μl/time was added every 24 h.
After fermentation, the daptomycin content, biomass and pH were measured in bottles and the results are shown in Table 1:
TABLE 1
| Strategy one | Policy two | Strategy three | |
| Fermentation time | 144h | 144h | 144h |
| pH | 5.76 | 5.90 | 5.88 |
| Biomass (volume fraction%) | 11 | 12 | 9 |
| Daptomycin content (μg/ml) | 269 | 307 | 216 |
The results show that: when the mixture of the capric acid and the methyl oleate is used as a precursor, the pH of the fermentation liquor is lower, and the toxicity of the capric acid to thalli causes the thalli in the fermentation liquor to basically die in the later period, so that the daptomycin content is lower.
Example 2: shake flask fermentation culture (sodium caprate as precursor)
Single colony and shake flask seed preparation were the same as in example 1.
Preparing a fermentation medium: the fermentation medium is prepared according to the following proportion, wherein each 100ml of the medium contains 12g of dextrin, 3g of yeast powder, 1.0g of corn steep liquor powder, 0.75g of cotton seed powder, 0.5g of dipotassium hydrogen phosphate and 0.2g of ferrous ammonium sulfate; the materials are weighed and dissolved together, the volume is fixed, the materials are then filled into a fermentation shaking flask, the filling amount of a fermentation medium is 25ml/250ml, the pH value is regulated to 7.0-7.4 by Tris, and the materials are sterilized for 30min at 120-122 ℃.
Preparing a precursor: preparing 25% sodium caprate, accurately weighing 10g of sodium caprate, adding a proper amount of water, stirring until the sodium caprate is completely dissolved, finally fixing the volume to 40ml, and sterilizing at 120-122 ℃ for 30min.
Inoculating: the seed solution was inoculated into a shake flask fermentation medium at an inoculum size of 8%, at 29.5℃and 250rpm, and cultured on a shaker for 120 hours.
The method for adding the precursor comprises the following steps: the precursors were added using three strategies, respectively.
Strategy one: the base material is added in a proportion of 3%, namely 3g of sodium caprate is added into every 100ml of fermentation liquor.
Strategy II: the base material and the supplementary material are added, the adding proportion of the base material is 1.4 percent, namely, 1.4g of sodium caprate is added into each 100ml of fermentation liquor; 1.1ml of 25% sodium caprate was then added at 48h of fermentation, followed by 0.5ml of 25% sodium caprate every 24 h.
Strategy III: the base material is not added and is only added in the supplementary material; 1.8ml of 25% sodium caprate is added after fermentation for 24h and 48h respectively, and then 0.9ml of 25% sodium caprate is added every 24 h.
After fermentation, the daptomycin content, biomass and pH were measured in bottles and the results are shown in Table 2:
TABLE 2
| Strategy one | Policy two | Strategy three | |
| Fermentation time | 144h | 144h | 144h |
| pH | 7.94 | 8.25 | 8.39 |
| Biomass (volume fraction%) | 10 | 8 | 9 |
| Daptomycin content (μg/ml) | 124 | 205 | 179 |
The results show that: sodium caprate is used as a precursor, the pH of fermentation liquor is higher and reaches about 8.0, the fermentation liquor is thinner, and mycelia basically die, so that the content of daptomycin is extremely low.
Example 3: shake flask fermentation culture (capric triglyceride as precursor)
Single colony and shake flask seed preparation were the same as in example 1.
Preparing a fermentation medium: the capric triglyceride is in a solid state at normal temperature, and is completely dissolved into a liquid state by hot water in advance. The fermentation medium is prepared according to the following proportion: every 100ml, 12g of dextrin, 3g of yeast powder, 1.0g of corn steep liquor powder, 0.75g of cotton seed powder, 0.5g of dipotassium hydrogen phosphate, 0.2g of ferrous ammonium sulfate and 3.5g of capric triglyceride; wherein, the capric triglyceride is dissolved and then is added into each fermentation shake flask separately, the rest materials are weighed and dissolved together and then are filled into the fermentation shake flasks filled with the capric triglyceride, the filling amount of the fermentation medium is 25ml/250ml, the pH value is regulated to 7.0-7.4 by Tris, and the fermentation medium is sterilized for 30min at 120-122 ℃.
Inoculating: the seed solution was inoculated into a shake flask fermentation medium at an inoculum size of 8%, at 29.5℃and 250rpm, and cultured on a shaker for 120 hours.
The samples were bottled for 96h, 120h and 144h fermentation, and the daptomycin content, biomass and pH were measured and the results are shown in Table 3:
TABLE 3 Table 3
| Fermentation time | 96h | 120h | 144h |
| pH | 6.53 | 6.63 | 6.54 |
| Biomass (volume fraction%) | 36 | 40 | 39 |
| Daptomycin content (μg/ml) | 1130 | 1254 | 1503 |
The results show that: the capric triglyceride is used as a precursor, the pH of the fermentation liquor is basically maintained above 6.5, and the fermentation level of daptomycin reaches 1503 mug/ml when 144 hours.
Example 4: shake flask fermentation culture (capric triglyceride as precursor and supplement)
Single colony and shake flask seed preparation were the same as in example 1.
Preparing a fermentation medium: the capric triglyceride is in a solid state at normal temperature, and is completely dissolved into a liquid state by hot water in advance. The fermentation medium is prepared according to the following proportion: every 100ml, 12g of dextrin, 3g of yeast powder, 1.0g of corn steep liquor powder, 0.75g of cotton seed powder, 0.5g of dipotassium hydrogen phosphate, 0.2g of ferrous ammonium sulfate and 3.5g of capric triglyceride; wherein, the capric triglyceride is dissolved and then is added into each fermentation shake flask separately, the rest materials are weighed and dissolved together and then are filled into the fermentation shake flasks filled with the capric triglyceride, the filling amount of the fermentation medium is 25ml/250ml, the pH value is regulated to 7.0-7.4 by Tris, and the fermentation medium is sterilized for 30min at 120-122 ℃.
Inoculating: the seed solution was inoculated into a shake flask fermentation medium at an inoculum size of 8%, at 29.5℃and 250rpm, and cultured on a shaker for 120 hours.
And (3) supplementing the precursor capric triglyceride into the fermentation shake flask at regular time in the fermentation process, wherein two supplementing strategies are adopted respectively.
Strategy one: 100 μl was added at 72h and 96h of fermentation, respectively.
Strategy II: the fermentation was completed by adding 100. Mu.l for 48h and 100. Mu.l for 24 h.
After fermentation, the daptomycin content, biomass and pH were measured in bottles and the results are shown in Table 4:
TABLE 4 Table 4
| Strategy one | Policy two | |
| Fermentation time | 144h | 144h |
| pH | 6.87 | 6.73 |
| Biomass (volume fraction%) | 42 | 44 |
| Daptomycin content (μg/ml) | 1407 | 1368 |
The results show that: under the two precursor supplementing strategies, the fermentation level is not obviously improved, which indicates that the precursor capric triglyceride added in the fermentation medium is enough for the thalli to synthesize the daptomycin, and the additional supplementing precursor does not promote the improvement of the fermentation level in the process. Therefore, when capric triglyceride is used as a precursor, no additional supplementation during fermentation is required.
Comparative example 1: shake flask fermentation culture (base stock phosphate free comparison)
Single colony and shake flask seed preparation were the same as in example 1.
Preparing a fermentation medium: the preparation is carried out according to the following proportion: every 100ml, 12g of dextrin, 3g of yeast powder, 1.0g of corn steep liquor powder, 0.2g of ferrous ammonium sulfate and 3.5g of capric triglyceride; wherein, the capric triglyceride is dissolved and then is added into each fermentation shake flask separately, and the rest materials are weighed and dissolved together and then are filled into the fermentation shake flasks filled with the capric triglyceride; the fermentation medium is filled in an amount of 25ml/250ml, the pH value is regulated to 7.0-7.4 by Tris, and the fermentation medium is sterilized for 30min at 120-122 ℃.
Inoculating: the seed solution was inoculated into a shake flask fermentation medium at an inoculum size of 8%, at 29.5℃and 250rpm, and cultured on a shaker for 120 hours.
The samples were bottled for 96h, 120h and 144h fermentation, and the daptomycin content, biomass and pH were measured and the results are shown in Table 5:
TABLE 5
| Fermentation time | 96h | 120h | 144h |
| pH | 5.58 | 5.48 | 5.46 |
| Biomass (volume fraction%) | 24 | 26 | 32 |
| Daptomycin content (μg/ml) | 808 | 901 | 917 |
The results show that: the base material is not added with dipotassium hydrogen phosphate, the pH value of the fermentation liquor is basically maintained at about 5.5, the content of daptomycin 144h is 917 mug/ml, and the content is reduced by 39 percent compared with the content of dipotassium hydrogen phosphate.
Comparative example 2: shake flask fermentation culture (ratio of base nitrogen source)
The preparation method and inoculation method of single colony and shake flask seeds are the same as in example 1.
Preparation of fermentation medium 1: the preparation is carried out according to the following proportion: every 100ml, 12g of dextrin, 3g of yeast powder, 1.0g of corn steep liquor powder, 0.75g of cotton seed powder, 0.5g of dipotassium hydrogen phosphate, 0.2g of ferrous ammonium sulfate and 3.5g of capric triglyceride; wherein, the capric triglyceride is dissolved and then is added into each fermentation shake flask separately, and the rest materials are weighed and dissolved together and then are filled into the fermentation shake flasks filled with the capric triglyceride; the fermentation medium is filled in an amount of 25ml/250ml, the pH value is regulated to 7.0-7.4 by Tris, and the fermentation medium is sterilized for 30min at 120-122 ℃.
Preparation of fermentation medium 2: as in the above method, the yeast powder ratio was adjusted to 2.5g/100ml, and the corn steep liquor powder ratio was adjusted to 0.75g/100ml.
Preparation of fermentation medium 3: as in the above method, the yeast powder ratio was adjusted to 2g/100ml, and the corn steep liquor powder ratio was adjusted to 0.5g/100ml.
After fermentation, the daptomycin content is detected in a bottle, and the result is shown in Table 6:
TABLE 6
| Fermentation 1 | Fermentation 2 | Fermentation 3 | |
| Fermentation time | 144h | 144h | 144h |
| pH | 6.71 | 6.79 | 6.82 |
| Biomass (volume fraction%) | 36 | 26 | 20 |
| Daptomycin content (μg/ml) | 1421 | 876 | 752 |
The results show that: under the condition of controlling the pH value without adding ammonia water, the nitrogen source of the base material should be increased in proportion to meet the growth requirement of thalli. When the proportion of nitrogen source is low, the biomass accumulation of the thalli is less, and the content of daptomycin is also obviously reduced.
Claims (8)
1. A method for improving the fermentation level of daptomycin, which is characterized in that capric triglyceride is used as a precursor substance of the fermentation of the daptomycin, and phosphate is added into a fermentation medium to maintain the pH value in the fermentation process and improve the nitrogen source proportion in the fermentation medium.
2. A method for increasing fermentation levels of daptomycin according to claim 1, wherein the precursor substance capric triglyceride is added by: the additive is added into the base material at one time without feeding; the precursor capric triglyceride is added at a concentration of 3-4%, preferably 3.5%.
3. A method of increasing fermentation levels of daptomycin according to claim 1, wherein the phosphate is one or more of dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, preferably dipotassium hydrogen phosphate.
4. A method of increasing the fermentation level of daptomycin according to claim 1 or 3, wherein the phosphate is added at a concentration of 0.2-2%, preferably 0.5%.
5. A method of increasing the fermentation level of daptomycin according to claim 1 wherein the nitrogen source in the fermentation medium comprises one or more of yeast powder, corn steep liquor powder, cotton seed meal, preferably three of yeast powder, corn steep liquor powder and cotton seed meal.
6. A method of increasing the fermentation level of daptomycin according to claim 1 or 5, wherein the nitrogen source is added to the fermentation medium at a concentration of 0.25% to 6.5%.
7. The method for improving the fermentation level of daptomycin according to claim 5, wherein when the nitrogen source in the fermentation medium is three of yeast powder, corn steep liquor powder and cotton seed powder, the adding concentration of the yeast powder is 2-4%, the adding concentration of the corn steep liquor powder is 0.5-1.5%, the adding concentration of the cotton seed powder is 0.5-1%, the adding concentration of the preferred yeast powder is 3%, the adding concentration of the corn steep liquor powder is 1.0%, and the adding concentration of the cotton seed powder is 0.75%.
8. A method of increasing the fermentation level of daptomycin as claimed in claim 1 wherein the fermentation medium comprises the following components in mass concentrations: every 100ml, 10-14g of dextrin, 2-4g of yeast powder, 0.5-1.5g of corn steep liquor powder, 0.5-1g of cotton seed powder, 0.2-2g of dipotassium hydrogen phosphate, 0.15-0.25g of ferrous ammonium sulfate and 3-4g of capric triglyceride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311155790.6A CN117210520A (en) | 2023-09-08 | 2023-09-08 | Method for improving fermentation level of daptomycin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311155790.6A CN117210520A (en) | 2023-09-08 | 2023-09-08 | Method for improving fermentation level of daptomycin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117210520A true CN117210520A (en) | 2023-12-12 |
Family
ID=89034645
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311155790.6A Pending CN117210520A (en) | 2023-09-08 | 2023-09-08 | Method for improving fermentation level of daptomycin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117210520A (en) |
-
2023
- 2023-09-08 CN CN202311155790.6A patent/CN117210520A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105112476B (en) | Method for producing lipopeptide biosurfactant by fermentation | |
| US20240102058A1 (en) | Caproate-producing bacterium with multiple substrate utilization capabilities and its applications | |
| CN112608861A (en) | Composite preparation containing clostridium butyricum and pediococcus acidilactici as well as preparation method and application of composite preparation | |
| CN102220396B (en) | Simple fermentation method for acarbose | |
| RU2381270C1 (en) | STRAIN OF BACTERIA Clostridium acetobutylicum-PRODUCER OF BUTANOL, ACETONE AND ETHANOL | |
| CN104651427A (en) | Method for preparing doramectin | |
| CN117210520A (en) | Method for improving fermentation level of daptomycin | |
| CN106520871B (en) | Method for producing A40926 by fermentation method | |
| CN114276937B (en) | Method for fermenting paecilomyces hepialid by using Chinese yam as carbon source | |
| CN111434775A (en) | Method for preparing daptomycin by fermentation | |
| CN111303248B (en) | Material supplementing method for improving teicoplanin fermentation yield | |
| EP1613759B1 (en) | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients | |
| RU2404247C2 (en) | Method of obtaining butanol | |
| CN109943610B (en) | Natamycin fermentation process based on exogenous saturated fatty acid addition | |
| CN112553132A (en) | Optimized fermentation method of SacC gene knockout saccharopolyspora erythraea | |
| CN114875100B (en) | Method for improving fermentation yield of natamycin by activating natamycin synthesis in advance | |
| CN111979278B (en) | Method for reducing spectinomycin impurity E through fermentation | |
| CN114940950B (en) | Method for recycling clostridium butyricum fermentation waste liquid | |
| CN112410386B (en) | Process for improving content of monensin A component | |
| CN116751822A (en) | Method for promoting fermentation production of polylysine by adding tea polyphenol | |
| CN1070228A (en) | Method with producing Meiling-mycin by fermenting Nanchang streptomycete | |
| CN115478088A (en) | Application of calcium lactate in improvement of bacillus subtilis synthetic bacillomycin D, calcium lactate fermentation culture medium and method | |
| KR0169061B1 (en) | A process for preparing xylitol by controlling fermenting condition | |
| CN117778292A (en) | Method for improving fermentation yield of L-isoleucine | |
| CN112226475A (en) | Production method for improving neomycin yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |