CN116445298A - Fusarium solani strain for producing cyclosporin A by fermentation and application thereof - Google Patents
Fusarium solani strain for producing cyclosporin A by fermentation and application thereof Download PDFInfo
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- CN116445298A CN116445298A CN202310570938.6A CN202310570938A CN116445298A CN 116445298 A CN116445298 A CN 116445298A CN 202310570938 A CN202310570938 A CN 202310570938A CN 116445298 A CN116445298 A CN 116445298A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 89
- 230000004151 fermentation Effects 0.000 title claims abstract description 89
- 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 title claims abstract description 73
- 229930105110 Cyclosporin A Natural products 0.000 title claims abstract description 73
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- 239000000243 solution Substances 0.000 description 9
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- 229960000988 nystatin Drugs 0.000 description 8
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
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- RTGDFNSFWBGLEC-SYZQJQIISA-N mycophenolate mofetil Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(/C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-SYZQJQIISA-N 0.000 description 1
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- 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 description 1
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N1/14—Fungi; Culture media 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
- 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
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- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a fusarium strain for producing cyclosporin A by fermentation, and further discloses application of the fusarium strain for producing cyclosporin A by fermentation. The fusarium solani Fusarium solani FIM-CS-66-69 strain of high-yield cyclosporin A is obtained through screening by the normal pressure room temperature plasma mutagenesis technology, the high-yield cyclosporin A can be fermented, in a fermentation experiment, the titer of the cyclosporin A produced by the fusarium solani FIM-CS-66-69 fermentation is up to 16551 mug/mL, the yield of the cyclosporin A is greatly improved, the content of homologs is low, and the method is more suitable for industrial fermentation production.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fusarium strain for producing cyclosporin A by fermentation, and further discloses application of the fusarium strain for producing cyclosporin A by fermentation.
Background
Cyclosporin a (CsA) is a cyclic peptide immunosuppressant consisting of 11 amino acids produced by fungal fermentation. At present, the composition is widely applied to the field of organ transplantation rejection resistance in clinic, is mainly used for rejection reaction of liver, kidney and heart transplantation, can be used together with adrenocortical hormone, and can also be used for treating some immune diseases.
It has been reported that the CsA-producing bacteria are mainly Trichoderma stephanotium, fusarium glomeratum and Fusarium solani of the genus Zostertagia, beauveria snow and Alternaria, and the like. Fujian province microbiological institute in 1983 reported that Fusarium solani producing Fusarium (Fusarium solani 4-11) was screened from domestic soil and put into industrial production for the first time, and cyclosporin A was developed successfully in China for the first time, but the conditions of unstable fermentation level and relatively low component content were present, and the market competitiveness was not strong. Especially with the use of immunosuppressant drugs such as mycophenolate mofetil, tacrolimus and the like, the CsA market price is continuously reduced, and the market competition is more vigorous.
At present, the breeding technology aiming at the improvement of cyclosporin A fermentation strains mainly adopts the breeding means of combining ultraviolet mutagenesis with different mutagens, protoplast fusion technology and the like in the traditional breeding method, and achieves a certain effect. If a UV and NTG composite mutagenesis method is adopted by army and the like, obtaining a fusarium solani mutant strain FS-un26 for producing cyclosporin A, wherein the cyclosporin yield is 1931 mug/mL, which is improved by 20.6% compared with the original strain; deng Lixin and the like, protoplast fusion and laser mutagenesis are carried out on fusarium 421502 for producing cyclosporin A, and high-yield strains are screened, and the titer is about 950 mug/mL; chinese patent CN1570130 (CN 1219889C) discloses a method for producing cyclosporin a by fermentation using fusarium solani ATCC46829, the strain fermentation level of which is 2023 μg/mL; wu Hui and the like, through fusion and ultraviolet mutagenesis of fusarium solani S-4-H protoplast, the fermentation capacity of the strain after mutagenesis is only about 3000 mug/mL; zhou Li and the like, ultraviolet mutagenesis screening is carried out on the white muscardine fungus Cs A19-12 of the cyclosporin A to obtain a mutagenesis strain CsA-12 with relatively stable genetic characters, and the titer of 7d fermentation reaches 3300 mug/mL. In 2014, , the fermentation of cyclosporin A is researched by adopting a batch fermentation fructose fed-batch mode, and the yield of cyclosporin A is 6000 mug/mL when the fermentation period is 10 d; in 2021, liu Yu, the fermentation conditions of the high-yield cyclosporine A of the beauveria bassiana are optimized, and the tank fermentation titer in a 10d fermentation period reaches 15414 mug/mL through the addition of precursor amino acid and the fed-batch fermentation of glucose in the fermentation process, so that the titer of the product is effectively improved, but the cost of fructose, amino acid and the like is high, and the simple batch fermentation is changed into the fed-batch fermentation, so that the production operation is complex, the labor cost is high, and the industrial production is not facilitated. It can be seen that although UV mutagenesis has achieved a certain effect in combination with different mutagens and fermentation process optimisation, the cyclosporin production capacity of the strain is also improved to some extent, but is generally not ideal. This is mainly due to the complexity and multinode nature of the metabolic network within the microorganism, in complex systems for the production of secondary metabolites, excellent industrial strains are not readily available.
The normal pressure room temperature plasma breeding technology (ARTP) is a microorganism genome rapid mutation technology, plasma generated by the technology is rich in various chemical active particles, multiple effects of generating genetic material damage on bacterial strain cells, causing cell membrane permeability, protein structure change and the like are achieved, and cells start SOS repair mechanisms, so that mismatch sites with rich types are generated in the repair process, the mutation rate is high, and the method has been successfully applied to strain breeding of various industrial microorganisms. In view of this, it is highly desirable for practitioners to obtain a strain that is suitable for industrial production and that is capable of producing cyclosporin A with high efficiency.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a fusarium solani strain for producing cyclosporin A by fermentation, so as to solve the problem of non-ideal performance of the cyclosporin A fermentation strain in the prior art;
the second technical problem to be solved by the invention is to provide the application of the fusarium solani strain in the fermentation production of cyclosporin A.
In order to solve the technical problems, the Fusarium solani strain is classified and named as Fusarium solani (Fusarium solani) FIM-CS-66-69, and is preserved in the microorganism strain preservation center of Guangdong province, the preservation number is GDMCC No.63322, the preservation date is 2023 month 04, and the preservation address is No. 5 building of laboratory building of national institute of Mitsui, guangzhou, first of all.
The invention also discloses application of the fusarium solani strain in fermentation production of cyclosporin A.
The invention also discloses a method for producing cyclosporin A by fermentation, which comprises inoculating the fusarium solani strain into a proper fermentation medium for fermentation culture.
Specifically, the fermentation medium comprises the following components in percentage by mass: 3.0 to 6.5 weight percent of corn starch, 0.2 to 1.5 weight percent of glucose, 0.2 to 1.5 weight percent of casein, 0.2 to 0.8 weight percent of yeast powder, 0.02 to 0.2 weight percent of potassium chloride, 0.02 to 0.2 weight percent of magnesium sulfate, 0.01 to 0.03 weight percent of monopotassium phosphate, 0.1 to 0.8 weight percent of calcium carbonate and natural pH.
Preferably, the fermentation medium comprises the following components in mass content: 3.5wt% of corn starch, 0.5wt% of glucose, 0.5wt% of casein, 0.5wt% of yeast powder, 0.05wt% of potassium chloride, 0.05wt% of magnesium sulfate, 0.02wt% of monopotassium phosphate, 0.3wt% of calcium carbonate and natural pH.
In particular, the method comprises the steps of, the method for producing cyclosporin A by fermentation, the conditions of the fermentation culture include: controlling the rotating speed to be 100-250rpm, and carrying out fermentation culture at 25-29 ℃ for 68-146h.
Specifically, the method for producing cyclosporin A by fermentation further comprises inoculating the fusarium solani strain into a seed culture medium for seed liquid culture;
the seed culture medium comprises the following components in mass content: corn starch 1.0-4.0wt%, casein 0.1-0.5wt%, glucose 1.0-5.5wt%, potassium chloride 0.02-0.2wt%, sodium nitrate 0.02-0.2wt%, magnesium sulfate 0.001-0.05wt%, potassium dihydrogen phosphate 0.05-0.8wt% and pH5.4-5.8.
Preferably, the seed culture medium comprises the following components in percentage by mass: corn starch 2.0wt%, casein 0.3wt%, glucose 1.2wt%, potassium chloride 0.05wt%, sodium nitrate 0.05wt%, magnesium sulfate 0.005wt%, potassium dihydrogen phosphate 0.2wt%, and pH5.4-5.8.
Specifically, the method for producing cyclosporin A by fermentation comprises the following conditions of seed liquid culture: the rotation speed is controlled to be 100-250rpm, and the seed liquid culture is carried out for 32-60h at the temperature of 25-29 ℃.
Specifically, the method for producing cyclosporin A by fermentation further comprises the step of inoculating the fusarium solani strain into a slant culture medium for activation;
the inclined plane culture medium is a PDA culture medium and comprises the following components in mass content: 15-25wt% of potato, 1-3wt% of glucose, 1.5-2.5wt% of agar and natural pH.
Preferably, the slant culture medium is a PDA culture medium and comprises the following components in percentage by mass: 20wt% of potato, 2wt% of glucose, 2.0wt% of agar and natural pH.
Specifically, the conditions of the slant medium activation step include: culturing at 25-29 deg.C for 6-12d.
The fusarium solani Fusarium solani FIM-CS-66-69 with high cyclosporin A yield is obtained through screening by a normal pressure room temperature plasma mutagenesis technology, the high cyclosporin A yield can be fermented, in a fermentation experiment, the titer of the cyclosporin A produced by the fusarium solani FIM-CS-66-69 fermentation is up to 16551 mug/mL, the yield of the cyclosporin A is greatly improved, the content of homologs is low, and the method is more suitable for industrial fermentation production; the strain FIM-CS-66-69 screened by the invention has good stability, and the cyclosporin A titer of the strain is basically stable when the strain is continuously transmitted for four generations, and the strain is maintained at the same higher level, and can be used as a production strain for further research and development.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,
FIG. 1 is a graph of ARTP jet time versus mortality in mutagenesis experiments;
FIG. 2 is a tree of the strain FIM-CS-66-69 according to the invention.
Detailed Description
In the following examples of the invention, the medium involved comprises:
the separation plate culture medium and the slant culture medium comprise the following components in mass content: 20wt% of potato, 2wt% of glucose, 2.0wt% of agar and the balance of distilled water, and sterilizing for 20min at 121 ℃ by high-pressure steam.
The seed culture medium comprises the following components in mass content: corn starch 2.0wt%, casein 0.3wt%, glucose 1.2wt%, potassium chloride 0.05wt%, sodium nitrate 0.05wt%, magnesium sulfate 0.005wt%, potassium dihydrogen phosphate 0.2wt%, pH5.4-5.8, and sterilizing with high pressure steam at 121deg.C for 30min.
The fermentation medium comprises the following components in mass content: corn starch 3.5wt%, glucose 0.5wt%, casein 0.5wt%, yeast powder 0.5wt%, potassium chloride 0.05wt%, magnesium sulfate 0.05wt%, potassium dihydrogen phosphate 0.02wt%, calcium carbonate 0.3wt%, and sterilizing with high pressure steam at 121deg.C for 30min.
In the following embodiments of the present invention, the detection of cyclosporin a content adopts high performance liquid chromatography, and the method comprises: and (3) adding 3 times of methanol into a proper amount of fermentation liquor, carrying out ultrasonic vibration extraction for 2 times, merging supernatant filtering films to obtain fermentation primary extract, and carrying out HPLC detection and analysis. The chromatographic conditions include: chromatographic column C18 (4.6 mm. Times.250 mm,5 μm), detection wavelength 210nm, flow rate 1ml/min, column temperature 60 ℃, mobile phase methanol-ultrapure water (84:16), sample injection amount 5. Mu.L. Taking cyclosporine A standard as a reference substance, calculating the titer according to the peak area of a sample/the peak area of a standard solution, the concentration of the standard solution and the dilution multiple, and taking an average value of 3 times of fermentation titers.
EXAMPLE 1 obtaining of mutant Strain FIM-CS-66-69
Taking a fusarium solani FIM-CS-66 strain with relatively stable genetic characters as an initial strain, transferring the strain to a slant culture medium, and culturing in a constant-temperature incubator for 8-12d at a culture temperature of 26 ℃; washing spores on the slant culture medium with physiological saline, scattering glass beads, filtering with warp cloth, and making into 10 6 individual/mL spore suspension.
10 mu L of the spore suspension prepared above was sucked by a pipette onto a round iron plate with a diameter of 1cm, placed in a normal pressure room temperature plasma mutagenesis system with helium as working gas, a power of 110W and a working gas flow of 10L/min at a treatment distance of 2mm, respectively treated for 5s, 10s, 20s, 30s, 40s, 50s, 60s, 75s and 90s, and the treated spore suspension was subjected to gradient dilution plating to prepare a mortality curve, as shown in FIG. 1. As can be seen from FIG. 1, there is a clear dose-response relationship between the mutagenic treatment dose and the mortality of the strain FIM-CS-66, with a gradual increase in mortality with the treatment time.
According to the mortality curve, selecting irradiation time of 40s lethal dose, and performing plasma mutagenesis on spore suspension of the strain FIM-CS-66; and the treated spore suspension is filled into a test tube with normal saline, and the mixture is uniformly mixed to obtain the mutagenized spore suspension for standby.
Respectively coating the spore suspension in a plate culture medium containing nystatin, wherein the nystatin concentration in the plate culture medium is 12.5mg/L, 25mg/L, 50mg/L, 100mg/L and 150mg/L; after 10 days of incubation at 28℃the growth of colonies on the different plates was observed and the results are shown in Table 1, with the minimum inhibitory concentration of nystatin corresponding to the minimum inhibitory concentration of nystatin in the plate medium from which no colonies were grown, and the minimum inhibitory concentration of nystatin was 100mg/L as determined in Table 1.
TABLE 1 Effect of nystatin concentration on spore growth of Strain FIM-CS-66
| Nystatin (mg/L) | 0 | 12.5 | 25 | 50 | 100 | 150 |
| Colony growth | +++ | ++ | + | +- | - | - |
The++ colony grows well; the++ colony grows better; + colony growth is general; small amount of growth of the + -colony; colony non-growth
Subjecting the obtained mutagenized spore suspension to gradient dilution with dilution ratio of 10 respectively -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Selecting 10 -4 、10 -5 、10 -6 Three dilutions of spore suspension were plated onto 100mg/L resistant isolation plates containing nystatin and incubated at 26℃for 6-10d in the absence of light.
Inoculating single colony growing on the resistance plate into a slant culture medium, culturing for 6-10 days, inoculating 0.2-0.5% of the single colony into a seed culture medium, and culturing at 28deg.C under 230r/min for 36-60 hr to obtain seed solution; inoculating the seed solution into the fermentation medium with 10% of inoculation amount, and culturing at 26 deg.C and 230r/min for 4-7d to obtain fermentation broth.
And adding 3 times of methanol into the obtained fermentation liquor, carrying out ultrasonic oscillation extraction for 2 times, combining the supernatant and the extract, filtering to obtain a fermentation primary extract, measuring the yield of cyclosporin A by using high performance liquid chromatography, and determining that the structure of a product in the fermentation liquor is correct.
By this method, the strain producing cyclosporin A in the largest amount was selected, and for convenience of description, the selected strain was designated as strain FIM-CS-66-69, and strain FIM-CS-66-69 was stored in glycerol.
EXAMPLE 2 identification of Strain FIM-CS-66-69
Identification of physiological and biochemical characteristics
The obtained strain FIM-CS-66-69 was streaked on a plate of a separation medium and inserted into a cover slip, and cultured at 26℃for 5-15 days, and morphological characteristics of single colonies and hyphae thereof were observed with an optical microscope, a transmission and scanning electron microscope.
The main forms and the physiological and biochemical characteristics of the obtained strain FIM-CS-66-69 are as follows: the colony on the flat plate is round, the surface is short villous, the colony is produced into soluble light green pigment, the mature colony presents radioactive wrinkles, the center is raised, the back is earthy yellow, and the extended branch peduncles are found by observation under a mirror, and the two types of conidia with different sizes are produced. The large conidium sickle shape has 3 diaphragms, and grows in conidium seat or myxosporium, and the size is 22.5-37.5 μm×3-4 μm. The small conidia are spindle-shaped to oval shape, have 1 septum, and have a size of 4.5-24 μm×2.5-4 μm. The strain FIM-CS-66-69 is an oxygen consuming bacterium, and dissolved oxygen has remarkable effect on the generation of cyclosporin A in the fermentation process, the optimal growth temperature is 25-29 ℃, and the optimal growth pH is 5.5-7.5; the highest cyclosporin A yield is obtained when the shaking table rotates at 200-280r/min and is cultured for 4-7 d.
Molecular biological identification
Sequencing the ITS sequence of the strain FIM-CS-66-69, wherein the measured sequence is shown as SEQ ID NO. 1; comparing the ITS sequences of the detected strains with the existing sequences in the GenBank database, and carrying out homology analysis; after LPSN (http: the corresponding model strain ITS gene sequence is selected from the// www.bacterio.cict.fr) website, the system evolution analysis is performed by using CLUSTAL-X software, the generated comparison file is performed by using MEGA software adjacent method, and the topology analysis is the result of 1000 times of repeated sampling, as shown in figure 2. The ITS sequence analysis shows that the sequence homology of the strain FIM-CS-66-69 with Fusarium solani (Fusarium solani) is 99.79%.
SEQ ID NO.1：
TACCTAAAACGTTGCTTCGGCGGGAACAGACGGCCCTGTAACAACGGGCCGCCCCCGCCAGAGGACCCCTAACTCTGTTTTTATAATGTTTTTCTGAGTAAACAAGCAAATAAATTAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACAACCCTCAGGCCCCCGGGCCTGGCGTTGGGGATCGGCAGAAGCCCCCTGTGGGCACACGCCGTCCCTCAAATACAGTGGCGGTCCCGCCGCAGCTTCCATTGCGTAGTAGCTAACACCTCGCAACTGGAGAGCGGCGCGGCCACGCCGTAAAACACCCAACTTCTGAATGTTGACCTCGAATCAGGTAGGAAT。
The strain FIM-CS-66-69 is finally determined to be Fusarium solani (Fusarium solani) which is classified and named as Fusarium solani Fusarium solani FIM-CS-66-69 and is preserved in the Guangdong province microorganism strain preservation center, namely the 5 th floor of the laboratory building of the 100 th university of Mitsui in the View district of Guangzhou, guangdong province, with the preservation number of GDMCC No.63322 and the preservation date of 2023, 04 month and 04.
EXAMPLE 3 fermentation of cyclosporin A by the developing Strain FIM-CS-66
Activation of the starting Strain FIM-CS-66: the strain FIM-CS-66 preserved with glycerol was transferred onto a slant medium and cultured in a constant temperature incubator for 6-10d at 26 ℃.
Preparing FIM-CS-66 seed solution: the single colony obtained by activating the strain FIM-CS-66 is inoculated into a seed culture medium (100 mL of the seed culture medium is filled in a 500mL triangular flask), and is cultured for 46 hours at the temperature of 28 ℃ and under the condition of 230r/min to obtain seed liquid.
Fermentation culture: the seed solution thus obtained was inoculated into a fermentation medium (100 mL of fermentation medium in a 500mL Erlenmeyer flask) at an inoculum size of 10% (v/v), and subjected to fermentation culture at 26℃for 120 hours under 230r/min, followed by detection of the resulting fermentation broth.
The results showed that the yields of cyclosporin A from three shake flask fermentations were 3218. Mu.g/mL, 3132. Mu.g/mL and 3166. Mu.g/mL, respectively. Further, the purity of cyclosporin A in the fermentation broth was found to be 63.5%, 65.2% and 61.8%, respectively.
EXAMPLE 4 mutagenesis Strain FIM-CS-66-69 fermentation cyclosporin A
Activation of the Strain FIM-CS-66-69: the strain FIM-CS-66-69 preserved with glycerol was transferred onto a slant medium and cultured in a constant temperature incubator for 6-10d at 26 ℃.
Preparing FIM-CS-66-69 seed liquid: inoculating 0.5cm of lawn obtained by activating the strain FIM-CS-66-69 into a seed culture medium (100 mL of seed culture medium is filled in a 500mL triangular flask), and culturing at 28 ℃ under 220r/min for 46h to obtain a seed solution.
Fermentation culture: the seed solution thus obtained was inoculated into a fermentation medium (100 mL of fermentation medium in a 500mL Erlenmeyer flask) at an inoculum size of 10% (v/v), and subjected to fermentation culture at 26℃for 120 hours under 230r/min, followed by detection of the resulting fermentation broth.
The results showed that the yields of cyclosporin A from three shake flask fermentations were 16580. Mu.g/mL, 16660. Mu.g/mL and 16551. Mu.g/mL, respectively. Further, the purity of cyclosporin A in the fermentation broth was found to be 87.2%, 88.3% and 86.1%, respectively.
Therefore, the screened strain can not only ferment cyclosporin A efficiently, but also has better cyclosporin A purity in fermentation liquor and lower influence of byproducts.
EXAMPLE 5 production of cyclosporin A by fermentation of mutant Strain FIM-CS-66-69
Shake flask seed culture: inoculating the strain FIM-CS-66-69 lawn into seed culture medium (280 mL seed culture medium in 1000mL triangular flask), and culturing at 26deg.C under 230r/min for 46 hr to obtain shake flask seed solution.
Seed tank seed culture: shake flask seed solution was inoculated in an amount of 0.5% into a seed medium (70L seed medium was contained in a 100L tank) at a culture temperature of 28℃under a tank pressure of 0.05MPa at an air flow rate of 1: culturing for 43h under the condition of 1vvm and stirring speed of 100-200r/min, obtaining seed liquid in the seed tank.
Fermentation and culture in a fermentation tank: the prepared seed solution was inoculated into a fermentation medium (700L of fermentation medium in a 1-ton tank) at an inoculum size of 10% (v/v), and at a culture temperature of 26℃under a tank pressure of 0.05MPa at an air flow rate of 1:: 0.8-1.8vvm, stirring speed of 100-280r/min, controlling dissolved oxygen in the process to be not lower than 30%, fermenting and culturing for 120h, and placing the obtained fermentation liquor into a tank for detection.
The results of the assays showed that the cyclosporin A yields of the three batches of fermentation were 16633. Mu.g/mL, 16589. Mu.g/mL and 16609. Mu.g/mL, respectively. Further, the purity of cyclosporin A in the fermentation broth was 90.1%, 87.5% and 89.3%, respectively.
Further proved by the invention, the screened strain not only can ferment cyclosporin A efficiently, but also has better cyclosporin A purity in fermentation liquor.
EXAMPLE 6 genetic stability verification of Strain FIM-CS-66-69
The strain FIM-CS-66-69 of cyclosporin A which was selected and stored as described above was serially cultured and passaged (F1, F2, F3, F4, F5), and the fermentation titer was measured after 500mL shaking flask fermentation, and the results were shown in Table 2 below, with respect to the primary strain (F0) which had grown well.
TABLE 2 Effect of passage on cyclosporin A production by Strain FIM-CS-66-69
| Strain algebra | F0 | F1 | F2 | F3 | F4 | F5 |
| Relative potency (%) | 100 | 101.4 | 100.6 | 99.3 | 97.6 | 93.0 |
As shown by the results in the table 2, the strain FIM-CS-66-69 screened by the invention has no obvious influence on the four-generation fermentation level, and the cyclosporin A titer is basically stable and maintained at the same higher level, so that the strain FIM-CS-66-69 has better genetic stability; the highest cyclosporin A yield of target strain FIM-CS-66-69 is not lower than 16551 mug/mL, and the purity of cyclosporin A in fermentation broth is not lower than 86%, so that the impurity content can be effectively controlled, and the downstream purification of cyclosporin A is facilitated.
In summary, the mutant strain Fusarium solani FIM-CS-66-69 (Fusarium solani) screened in the present invention can be used as a production strain for further research and development.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. As will be apparent to one of ordinary skill in the art, other variations or modifications may be made in the various forms based on the above description. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. Fusarium solani strain, classified and named as Fusarium solani (Fusarium solani) FIM-CS-66-69, is deposited in the Guangdong province microorganism strain collection with the deposit number of GDMCCNo.63322 and the deposit date of 2023, 04 and 04.
2. Use of the fusarium solani strain of claim 1 for the fermentative production of cyclosporin a.
3. A method for fermentative production of cyclosporin a comprising inoculating the fusarium solani strain of claim 1 in a suitable fermentation medium for fermentative culture.
4. A method for fermentative production of cyclosporin a according to claim 3, wherein said fermentation medium comprises the following components in mass content: 3.0 to 6.5 weight percent of corn starch, 0.2 to 1.5 weight percent of glucose, 0.2 to 1.5 weight percent of casein, 0.2 to 0.8 weight percent of yeast powder, 0.02 to 0.2 weight percent of potassium chloride, 0.02 to 0.2 weight percent of magnesium sulfate, 0.01 to 0.03 weight percent of monopotassium phosphate, 0.1 to 0.8 weight percent of calcium carbonate and natural pH.
5. The method for producing cyclosporin a by fermentation according to claim 3 or 4, wherein the conditions of the fermentation culture comprise: controlling the rotating speed to be 100-250rpm, and carrying out fermentation culture at 25-29 ℃ for 68-146h.
6. The method for fermentative production of cyclosporin a according to any one of claims 3 to 5, further comprising inoculating the fusarium solani strain of claim 1 in a seed medium for seed liquid culture;
the seed culture medium comprises the following components in mass content: corn starch 1.0-4.0wt%, casein 0.1-0.5wt%, glucose 1.0-5.5wt%, potassium chloride 0.02-0.2wt%, sodium nitrate 0.02-0.2wt%, magnesium sulfate 0.001-0.05wt%, potassium dihydrogen phosphate 0.05-0.8wt% and pH5.4-5.8.
7. The method for fermentative preparation of cyclosporin A according to claim 6, wherein said seed liquid culture conditions comprise: the rotation speed is controlled to be 100-250rpm, and the seed liquid culture is carried out for 36-60h at the temperature of 25-29 ℃.
8. The method for fermentative production of cyclosporin a according to any one of claims 3 to 7, further comprising the step of inoculating the fusarium solani strain of claim 1 in a slant medium for activation;
the inclined plane culture medium is a PDA culture medium and comprises the following components in mass content: 15-25wt% of potato, 1-3wt% of glucose, 1.5-2.5wt% of agar and natural pH.
9. The method for fermentative production of cyclosporin a of claim 8, wherein said conditions of said slant medium activation step comprise: culturing at 25-29 deg.C for 6-10d.
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