CN113980873A - Streptomyces tsukubaensis SCLX0001 and application thereof in fermentation production of tacrolimus - Google Patents
Streptomyces tsukubaensis SCLX0001 and application thereof in fermentation production of tacrolimus Download PDFInfo
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- CN113980873A CN113980873A CN202111495505.6A CN202111495505A CN113980873A CN 113980873 A CN113980873 A CN 113980873A CN 202111495505 A CN202111495505 A CN 202111495505A CN 113980873 A CN113980873 A CN 113980873A
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- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 title claims abstract description 76
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 title claims abstract description 73
- 229960001967 tacrolimus Drugs 0.000 title claims abstract description 63
- 238000000855 fermentation Methods 0.000 title claims abstract description 49
- 230000004151 fermentation Effects 0.000 title claims abstract description 49
- 241001647839 Streptomyces tsukubensis Species 0.000 title claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 241000187747 Streptomyces Species 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 23
- 239000002904 solvent Substances 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 17
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 17
- 239000008103 glucose Substances 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 229920002472 Starch Polymers 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 13
- 239000008107 starch Substances 0.000 claims description 13
- 235000019698 starch Nutrition 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 10
- 229920001353 Dextrin Polymers 0.000 claims description 9
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- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 235000019425 dextrin Nutrition 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 238000005273 aeration Methods 0.000 claims 2
- 238000012262 fermentative production Methods 0.000 claims 1
- 231100000350 mutagenesis Toxicity 0.000 abstract description 9
- 238000002703 mutagenesis Methods 0.000 abstract description 9
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- 238000012216 screening Methods 0.000 description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 239000012138 yeast extract Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 229930105110 Cyclosporin A Natural products 0.000 description 4
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 4
- 108010036949 Cyclosporine Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 229960001265 ciclosporin Drugs 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000003018 immunosuppressive agent Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229960003444 immunosuppressant agent Drugs 0.000 description 3
- 230000001861 immunosuppressant effect Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- YKMDNKRCCODWMG-UHFFFAOYSA-N 2,5-dinitrobenzoic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC=C1[N+]([O-])=O YKMDNKRCCODWMG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000187180 Streptomyces sp. Species 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- ZDQSOHOQTUFQEM-NURRSENYSA-N ascomycin Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C/C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 ZDQSOHOQTUFQEM-NURRSENYSA-N 0.000 description 2
- ZDQSOHOQTUFQEM-XCXYXIJFSA-N ascomycin Natural products CC[C@H]1C=C(C)C[C@@H](C)C[C@@H](OC)[C@H]2O[C@@](O)([C@@H](C)C[C@H]2OC)C(=O)C(=O)N3CCCC[C@@H]3C(=O)O[C@H]([C@H](C)[C@@H](O)CC1=O)C(=C[C@@H]4CC[C@@H](O)[C@H](C4)OC)C ZDQSOHOQTUFQEM-XCXYXIJFSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000003120 macrolide antibiotic agent Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000187437 Streptomyces glaucescens Species 0.000 description 1
- 241000187132 Streptomyces kanamyceticus Species 0.000 description 1
- 241000309093 Streptomyces tacrolimicus Species 0.000 description 1
- 230000037328 acute stress Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- RQYGKZGKXDOUEO-HHRHWXIDSA-N dihydro-fk 506 Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)OC([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C/C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CCC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 RQYGKZGKXDOUEO-HHRHWXIDSA-N 0.000 description 1
- RQYGKZGKXDOUEO-UHFFFAOYSA-N dihydrotacrolimus Natural products CC1C(O)CC(=O)C(CCC)C=C(C)CC(C)CC(OC)C(C(CC2C)OC)OC2(O)C(=O)C(=O)N2CCCCC2C(=O)OC1C(C)=CC1CCC(O)C(OC)C1 RQYGKZGKXDOUEO-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940124589 immunosuppressive drug Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
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- 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
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
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Abstract
The invention relates to streptomyces tsukubaensis and application thereof in fermentation production of tacrolimus. Belongs to the technical field of microbial fermentation. Streptomyces tsukubaensis (A)Streptomyces tsuknbaenis) SCLX0001, accession number: CCTCC NO: M20211303. The Streptomyces tsukubaensis SCLX0001 has strong ability of synthesizing tacrolimus, the shake flask fermentation yield of a mutagenic starting strain is 50mg/L, the starting strain is subjected to multiple rounds of mutagenesis, the high-yield Streptomyces tsubaensis SCLX0001 of tacrolimus is finally obtained, the shake flask fermentation yield is 127.3mg/L, and the yield is improved by 154%. After optimization on a 5L fermentor,the yield reaches 174.8mg/L, is improved by 37.3 percent compared with the shake flask fermentation, and can relieve the problem of low yield of tacrolimus in the current industrial production.
Description
Technical Field
The invention relates to streptomyces tsukubaensis and application thereof in fermentation production of tacrolimus. Belongs to the technical field of microbial fermentation.
Background
Tacrolimus, also known as FK506, is used as a novel macrolide immunosuppressant, is widely applied to clinical treatment by virtue of the advantages of strong immunosuppressive activity, small nephrotoxicity and the like, can effectively reduce acute stress rejection, improves the success rate of kidney transplantation and kidney function, and plays an important role in the whole immunosuppressive drug market. Prior to the discovery of tacrolimus, cyclosporin A (CsA) was used clinically as an immunosuppressant to treat rejection after organ transplantation. CsA is also a CaN inhibitor, the immunosuppressive mechanism is similar to tacrolimus, but the effect of tacrolimus is about 100 times that of CsA, and the clinical advantage is more. Currently, the number of patients requiring organ transplantation for liver disease, kidney disease, etc. worldwide is increasing year by year. The number of patients requiring organ transplantation is approximately 30 ten thousand in China each year.
FK506 (tacrolimus ) is a fermentation product isolated from Streptomyces (Streptomyces), and is a very effective novel immunosuppressant for 23-membered macrolides. A number of Streptomyces species such asStreptomyces tsukubaensis， Streptomyces tacrolimicus， Streptomyces sp. ATCC 53770 ， Streptomyces sp. MA6949， Streptomyces kanamyceticus KCC-S0436 ，Streptomyces glaucescens，Streptomyces clavuligerusCKD1119 and the like can produce FK 506. Its molecular formula is C44H69NO12The relative molecular mass is 804.02, and the chemical structural formula of FK506 is shown as follows.
FK506 is colorless crystal at normal temperature, is insoluble in water, and is soluble in organic solvents such as methanol, ethanol, ethyl acetate, diethyl ether, chloroform, and acetone. The melting point is 127-129 ℃, and the stability is high under different storage conditions.
During the biosynthesis of FK506, two closely related analogues, FK520 (ascomycin) and FK506D (dihydrotacrolimus), exist, depending on the R group at carbon position 21. The two by-products mentioned above pose difficulties for further increasing the yield of FK 506. At present, the yield of FK506 is generally low, the medical application and the industrial production of the FK506 are severely limited, although scientists optimize the production of the FK506 from the aspect of strain screening and fermentation process control, the high-yield mechanism of the FK506 is lack of understanding, the FK506 has the defects of high cost, long period, complex operation, unobvious effect, difficulty in industrial amplification and the like, and the industrial production level of the FK506 is still lower than the market demand.
Disclosure of Invention
The invention aims to provide a streptomyces tsukubaensis SCLX0001 strain capable of producing tacrolimus at a high yield and application thereof, and provides support for further improving the yield of tacrolimus.
The technical scheme for solving the problems is as follows:
streptomyces tsukubaensis (B)Streptomyces tsuknbaenis) SCLX0001, preserved in China Center for Type Culture Collection (CCTCC), with a preservation date of 2021, 10 months and 21 days, and a preservation number of: CCTCC NO: M20211303, address: wuhan, Wuhan university, post 430072, China.
Another purpose of the invention is to provide the application of the streptomyces tsukubaensis SCLX0001 in producing tacrolimus through fermentation.
Still another object of the present invention is to provide a method for producing tacrolimus by fermentation of the streptomyces tsukubaensis SCLX 0001.
The method for producing tacrolimus by fermenting the streptomyces tsukubaensis SCL0001 comprises the following steps:
inoculating streptomyces tsukubaensis SCLX0001 to a fermentation culture medium, fermenting and culturing at the temperature of 28-32 ℃ and the rpm of 500-700 for 120-250 h to obtain a fermentation liquid containing tacrolimus, and then separating and purifying the fermentation liquid to obtain the tacrolimus.
Preferably, the ventilation volume is 4-8L/min, the tank pressure is 0.04-0.06 MPa, and the dissolved oxygen in the tank is kept at 20-30% by controlling the stirring rotating speed and the ventilation volume in the fermentation process.
Preferably, in the above aspect, the initial fermentation medium comprises: 50-70 g/L of starch, 25-50 g/L of dextrin, 5-15 g/L of glucose, 15-20 g/L of yeast powder, 1-3 g/L of peptone, (NH)4）2SO4 1~2 g/L，MgSO20.5~1 g/L，CaCO34-8 g/L, water as solvent, and pH 6.8-7.2.
Preferably, in the above aspect, the initial fermentation medium comprises: 50-60 g/L of starch, 30-50 g/L of dextrin, 7-15 g/L of glucose, 15-20 g/L of yeast powder, 1-2 g/L of peptone, (NH)4）2SO4 1~1.5 g/L，MgSO20.5~0.8 g/L，CaCO35-8 g/L, water as solvent, and pH 6.8-7.2; and sterilizing at 115 deg.C for 30 min.
Preferably, in the above aspect, the initial medium comprises: 60g/L of starch, 40g/L of dextrin, 12g/L of glucose, 20g/L of yeast powder, 3g/L of peptone, (NH)4）2SO4 2 g/L，MgSO2 1 g/L，CaCO38g/L, water as solvent, pH 7.0.
Compared with the prior art, the invention has the following beneficial effects:
the method selects streptomyces tsukubaensis as an initial strain to carry out mutagenesis to obtain a high-yield strain, the streptomyces tsukubaensis SCLX0001 has strong ability of synthesizing tacrolimus, the shake flask fermentation yield of the mutagenesis initial strain is 50mg/L, and the high-yield streptomyces tsukubaensis tacrolimus is finally obtained after multiple rounds of mutagenesis, wherein the shake flask fermentation yield is 127.3mg/L and the yield is improved by 154%. After optimization on a 5L fermentation tank, the yield reaches 174.8mg/L, which is improved by 37.3 percent compared with the shake flask fermentation. The method can screen the streptomyces tsukubaensis with high tacrolimus yield, and can relieve the problem of low tacrolimus yield in the current industrial production.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. Are protected by the patent laws unless otherwise indicated by the scope of the claims.
Example 1:
first, screening of original strain
(1) Primary screening: inoculating single colony to seed liquid, culturing for 48 hr, diluting bacterial liquid with normal saline 10, 102、103、104And 105After the volume is doubled, the mixture is uniformly coated in a GYM solid culture medium containing 30 mu g/mL 2 and 5-dinitrobenzoic acid, the mixture is cultured for 3-4 days at the temperature of 28 ℃, the color of a bacterial colony is observed, the surface structural characteristics are shown, the bacterial colony of the streptomyces tsukubaensis is in a circular bulge shape, the surface of the streptomyces tsubaensis is like a petal shape, the color of the streptomyces tsubaensis is in a red color, the streptomyces tsubaensis is subjected to fermentation culture, the strain obtained after the culture for 6-8 days at the temperature of 28 ℃ is fermented, the yield of tacrolimus is detected through a liquid phase, and the streptomyces tsubazakii with high tacrolimus yield is screened out. Composition of GyM solid medium: 10g/L of malt extract, 4g/L of glucose, 4g/L of yeast extract, 20g/L of agar powder, 2g/L of calcium carbonate and water as solvent, wherein the pH value is 7.0.
(2) Re-screening: inoculating the streptomyces tsukubaensis strain screened out in the step (1) to a seed liquid culture medium, and culturing for 48h at 28 ℃ to obtain a seed liquid: inoculating the seed solution into a 500 mL shake flask filled with 50 mL initial fermentation medium at an inoculation amount of 5% by volume concentration, and culturing for 168h at 28 ℃ in a shaking table at 200 rpm to obtain fermentation liquid. And (3) measuring the tacrolimus yield by using an HPLC method, and further picking out the streptomyces tsukubaensis strain with high yield.
Initial fermentation medium composition: 60g/L of starch, 40g/L of dextrin, 12g/L of glucose, 20g/L of yeast powder, 3g/L of peptone, (NH)4）2SO4 2 g/L，MgSO2 1 g/L，CaCO3 8g/L, solvent is water (distilled water or tap water), pH 7.0.
(3) Determination of tacrolimus production: measuring by adopting an HPLC method, taking 1mL of fermentation liquor obtained in the step (2), and mixing the fermentation liquor: mixing methanol with a volume ratio of =1:1, shaking for 2.5h at 50 ℃, centrifuging at 6000rpm for 10min, taking the supernatant, using a 0.22 mu m organic filter membrane, detecting the peak area of tacrolimus through High Performance Liquid Chromatography (HPLC), obtaining the concentration of tacrolimus according to a tacrolimus standard curve, and selecting the strain with high tacrolimus yield as the starting strain of streptomyces tsukubaensis, wherein the yield is 127.3 mg/L.
Tacrolimus standard curve: tacrolimus sample is taken and dissolved in methanol to prepare sample solutions (25 mg/L, 50mg/L, 100mg/L, 150mg/L and 200 mg/L) with different concentrations, and HPLC detection is adopted. And drawing a standard curve by taking the peak area as an ordinate and the concentration (mg/L) of tacrolimus as an abscissa.
The specific HPLC measurement conditions were as follows: the column was a C18 column (250 x 4.6mm, 5 μm), mobile phase: pure acetonitrile: 0.1% phosphoric acid in water =65: 35; detecting the column temperature: 60 ℃, detection flow rate: 0.9mL/min, a sample amount of 10 muL, a chromatographic retention time of 25min, and a detection wavelength of 210 nm. The time to peak of tacrolimus was 22.5 min.
Secondly, screening streptomyces tsukubaensis strain with high tacrolimus yield
Ultraviolet mutagenesis treatment is carried out on the Streptomyces tsukubaensis initial strain in the embodiment 1, the strain is coated on a flat plate and cultured for 3 to 4 days to obtain a mutagenesis treatment mutant strain, and the specific method is as follows:
1. 600 mu g/mL 2, 5-dinitrobenzoic acid is used as a resistance screening agent
(1) Inoculating the screened starting strain of the streptomyces tsukubaensis to an activation culture medium, and culturing for 2-3 d at 28 ℃ to obtain an activated strain of the streptomyces tsukubaensis; and selecting a single colony, inoculating the single colony into a seed culture medium, and culturing at 28 ℃ for 48 hours to obtain a seed solution. The activation medium consists of: 10g/L of malt extract, 4g/L of glucose, 4g/L of yeast extract, 20g/L of agar powder, 2g/L of calcium carbonate and water as solvent, and the pH value is 7.0. The seed culture medium comprises the following components: 25 g/L of glycerin, 10g/L of starch, 1.5 g/L of glucose, 10g/L of yeast extract and CaCO31 g/L, solvent is water, pH 7.0.
(2) The chamber of the UV mutagenic apparatus (UV 8AC18H Saybolt) was wiped with alcohol cotton and sterilized by UV irradiation at 18W for 30 min. Sucking 1mL of the seed solution obtained in the step (1), placing the seed solution in a clean sterile plate, placing the plate in an ultraviolet mutagenesis operation table, irradiating by 18W ultraviolet for 60S, coating the treated bacterial solution on a resistant culture medium, and culturing for 3-4 d at 30 ℃. The final concentration composition of the resistant culture medium is as follows: 10g/L of malt extract, 4g/L of glucose, 4g/L of yeast extract, 20g/L of agar powder, 2g/L of calcium carbonate and water as solvent, and the pH value is 7.0. 2. 600 mu g/mL of 5-dinitrobenzoic acid, water as a solvent and natural pH value.
(3) Selecting a larger single colony of the mutagenic strain from the resistant plate in the step (2), inoculating the single colony into a seed culture medium, and culturing at 28 ℃ for 48 hours to obtain a seed solution of the mutagenic strain; the seed culture medium comprises the following components: 25 g/L of glycerin, 10g/L of starch, 1.5 g/L of glucose, 10g/L of yeast extract and CaCO31 g/L, solvent is water, pH 7.0.
(4) Inoculating the mutagenic strain seed liquid obtained in the step (3) to 50 mL of initial fermentation medium by an inoculation amount with a volume concentration of 5%, and performing fermentation culture at a rotation speed of 200 rpm/min and a temperature of 28 ℃ for 168 h; initial fermentation medium composition: 60g/L of starch, 40g/L of dextrin, 12g/L of glucose, 20g/L of yeast powder, 3g/L of peptone, (NH 4)2SO4 2 g/L，MgSO2 1 g/L，CaCO3 8g/L, solvent is water (distilled water or tap water), pH 7.0.
(5) Taking 1mL of fermentation liquor obtained in the step (4), and mixing the fermentation liquor: mixing methanol =1:1 in volume ratio, shaking for 2.5h at 50 ℃, centrifuging at 6000rpm for 10min, taking supernatant, using a 0.22 mu m organic filtering membrane, and detecting filtrate by adopting high performance liquid phase to obtain the concentration of tacrolimus; the mutant strain S0101 with the highest tacrolimus yield is obtained by selecting the strain with the highest tacrolimus yield from 100 mutant strains, and the yield of the tacrolimus is 55.2 mg/L.
Tacrolimus standard curve: tacrolimus sample is taken and dissolved in methanol to prepare sample solutions (25 mg/L, 50mg/L, 100mg/L, 150mg/L and 200 mg/L) with different concentrations, and HPLC detection is adopted. And drawing a standard curve by taking the peak area as an ordinate and the concentration (mg/L) of tacrolimus as an abscissa.
The specific HPLC measurement conditions were as follows: the column was a C18 column (250 x 4.6mm, 5 μm), mobile phase: pure acetonitrile: 0.1% phosphoric acid in water =65: 35; detecting the column temperature: 60 ℃, detection flow rate: 0.9mL/min, a sample amount of 10 muL, a chromatographic retention time of 25min, and a detection wavelength of 210 nm. The time to peak of tacrolimus was 22.5 min.
(6) Mutant strain S0101 was subjected to multiple rounds of mutagenesis
Repeating the operations of the steps (1) to (5) on the strain S0101 obtained in the step (5), and screening to obtain a mutant strain S0210, wherein the yield of tacrolimus is 61.1 mg/L; repeating the operations of the steps (1) to (5) on the mutant strain S0210, and screening to obtain a mutant strain S0314 with the tacrolimus yield of 63.1 mg/L; repeating the steps (1) to (5) on the mutant strain S0314, screening to obtain a mutant strain S0407, wherein the tacrolimus yield is 68.8mg/L, repeating the steps (1) to (5) on the mutant strain S0407, screening to obtain a mutant strain S0520, and the tacrolimus yield is 72.4 mg/L; and (5) repeating the steps (1) to (5) on the mutant strain S0520, and screening to obtain a mutant strain S0632 with the tacrolimus yield of 76.9 mg/L.
2. 300 ug/mL streptomycin as resistance screening agent
Replacing 2, 5-dinitrobenzoic acid in the resistance culture medium in the step (2) with 300 mu g/mL streptomycin, repeating the operations of the steps (1) to (5) on the mutant strain S0632 in the step (6), and screening to obtain a mutant strain S0701 with the tacrolimus yield of 86.9 mg/L; repeating the steps (1) to (5) on the mutant strain S0701, and screening to obtain a mutant strain S0802, wherein the yield of tacrolimus is 93.4 mg/L; repeating the steps (1) to (5) on the mutant strain S0802, and screening to obtain a mutant strain S0910 with the tacrolimus yield of 105.4 mg/L; repeating the steps (1) to (5) on the mutant strain S0910, and screening to obtain the mutant strain SCLX0001 (namely CCTCC NO: M20211303), wherein the yield of tacrolimus is 130.3mg/L (the yield cannot be less than 127.3 of the previous improvement).
3. High-yield strain stability detection
The strain obtained by mutagenesis screening may cause the degeneration phenomenon of the strain through the self-repairing capability of the strain in the subculture process due to the change of the genetic property of the strain caused by human factors. In order to avoid the occurrence of strain degeneration, the experiment continuously cultures the screened high-yield strain SCLX0001 for 10 generations and detects the yield of tacrolimus. Through continuous 10-generation subculture, the strain SCLX0001 has the advantages that during the subculture, the yield of tacrolimus from generation 1 to generation 3 is about 130.1 mg/L, the yield from generation 4 and generation 6 is about 129.2 mg/L, the yield from generation 7 to generation 8 is about 128.4 mg/L, and the yield from generation 9 to generation 10 is about 127.3 mg/L. Through subsequent subculture experiments, the strain SCLX0001 has stable tacrolimus production capability.
Production of Tacrolimus tsukubaensis SCLX0001 in 5L fermentation tank
(1) Inoculating the mutagenized streptomyces tsukubaensis SCLX0001 strain into an activation culture medium, and culturing at 28 ℃ for 3-4 days to obtain a streptomyces tsukubaensis activation strain; the activation medium consists of: 10g/L of malt extract, 4g/L of glucose, 4g/L of yeast extract, 20g/L of agar powder, 2g/L of calcium carbonate and water as solvent, and the pH value is 7.0. The seed culture medium comprises the following components: 25 g/L of glycerin, 10g/L of starch, 1.5 g/L of glucose, 10g/L of yeast extract and CaCO31 g/L, solvent is water, pH 7.0.
(2) And (3) selecting the single colony in the step (1) to be inoculated into a seed culture medium, and culturing at 28 ℃ for 48h to obtain a seed solution. The seed culture medium comprises the following components: 25 g/L of glycerin, 10g/L of starch, 1.5 g/L of glucose, 10g/L of yeast extract and CaCO31 g/L, solvent is water, pH 7.0.
(3) Inoculating the seed solution into a 5L fermentation tank filled with 2L fermentation medium in an inoculation amount with the volume concentration of 10%, controlling the temperature in the fermentation tank to be 28 ℃, the pH value to be 7.0, the stirring speed to be 400-600rpm/min, the ventilation volume to be 4-8L/min and the tank pressure to be 0.05MPa, and supplementing 2g/L of lysine and histidine after fermenting for 24h in the fermentation process. During the fermentation process, samples were taken every 12h to determine the tacrolimus yield and the biological dry weight, and the results (table 1) show that the highest tacrolimus yield is 174.8mg/L after 168h of fermentation culture.
The fermentation medium comprises the following components: 60g/L of starch, 40g/L of dextrin, 12g/L of glucose, 20g/L of yeast powder, 3g/L of peptone, (NH)4）2SO4 2 g/L，MgSO2 1 g/L，CaCO38g/L, lysine2g/L histidine 2g/L, solvent water (distilled water or tap water), pH7.0, 115 deg.C sterilizing for 30 min.
Table 1: tacrolimus production and biological dry weight at different fermentation times
Claims (7)
1. Streptomyces tsukubaensis (B)Streptomyces tsuknbaenis) SCLX0001, preserved in China Center for Type Culture Collection (CCTCC), with a preservation date of 2021, 10 months and 21 days, and a preservation number of: CCTCC NO: M20211303, address: wuhan, Wuhan university, post 430072, China.
2. Use of the Streptomyces tsukubaensis SCLX0001 of claim 1 in the fermentative production of tacrolimus.
3. The method for producing tacrolimus by fermenting streptomyces tsukubaensis SCLX0001 as claimed in claim 1, comprising the following steps:
inoculating streptomyces tsukubaensis SCLX0001 to a fermentation culture medium, fermenting and culturing at the temperature of 28-32 ℃ and the rpm of 500-700 for 120-250 h to obtain a fermentation liquid containing tacrolimus, and then separating and purifying the fermentation liquid to obtain the tacrolimus.
4. The method of claim 3, wherein: the aeration quantity is 4-8L/min, the tank pressure is 0.04-0.06 MPa, and the dissolved oxygen in the tank is kept at 20-30% by controlling the stirring rotating speed and the aeration quantity in the fermentation process.
5. The method of claim 3, wherein: the initial fermentation medium consists of: 50-70 g/L of starch, 25-50 g/L of dextrin, 5-15 g/L of glucose, 15-20 g/L of yeast powder, 1-3 g/L of peptone, (NH)4）2SO4 1~2 g/L，MgSO2 0.5~1 g/L，CaCO34-8 g/L, water as solvent, and pH 6.8-7.2.
6. The method of claim 3, wherein: the initial fermentation medium consists of: 50-60 g/L of starch, 30-50 g/L of dextrin, 7-15 g/L of glucose, 15-20 g/L of yeast powder, 1-2 g/L of peptone, (NH)4）2SO4 1~1.5g/L，MgSO2 0.5~0.8g/L，CaCO35-8 g/L, water as solvent, and pH 6.8-7.2; and sterilizing at 115 deg.C for 30 min.
7. The method of claim 5, wherein: the initial medium consists of: 60g/L of starch, 40g/L of dextrin, 12g/L of glucose, 20g/L of yeast powder, 3g/L of peptone, (NH)4）2SO4 2g/L，MgSO2 1g/L，CaCO38g/L, water as solvent, pH 7.0.
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