CN107955800B

CN107955800B - Genetic engineering bacterium for producing ascomycin FK520 and preparation method thereof

Info

Publication number: CN107955800B
Application number: CN201610905199.1A
Authority: CN
Inventors: 陈少欣; 余志拓
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2016-10-17
Filing date: 2016-10-17
Publication date: 2021-05-28
Anticipated expiration: 2036-10-17
Also published as: CN107955800A

Abstract

The invention discloses a genetic engineering bacterium for producing ascomycin FK520 and a preparation method thereof. The gene engineering bacteria are the engineering bacteria integrating VGB genes in the genome of streptomyces hygroscopicus naturally producing FK 520. Fermentation experiments prove that when the engineering bacterium is used for preparing the ascomycin FK520, the fermentation unit of the obtained FK520 is improved by 1.38 times compared with the fermentation unit of the FK520 obtained by fermenting streptomyces hygroscopicus naturally producing the FK 520.

Description

Genetic engineering bacterium for producing ascomycin FK520 and preparation method thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a genetic engineering bacterium for producing ascomycin FK520 and a preparation method thereof.

Background

Ascomycin FK520 is a macrolide compound with 23-membered ring, has immunosuppressive and antifungal activities, and has molecular formula C₄₃H₆₇NO₁₂Molecular weight of 789.99, and is easily soluble in methanol, acetone, etc. FK520 is an ethyl analogue of Tacrolimus (FK 506), which inhibits the activity of T cells and the synthesis of related inflammatory factors, and its derivative, "pimecrolimus", has become an important immunosuppressive drug.

At present, FK520 is mainly produced in a large scale by a method of fermentation of Streptomyces hygroscopicus (Streptomyces hygroscopicus), but in the actual production process, the fermentation process is difficult to control, the thallus grows too fast, thick and dense, and the accumulation of a secondary metabolite FK520 is seriously restricted by the reduction of oxygen utilization. Controlling the FK520 fermentation process becomes a production problem.

Disclosure of Invention

The invention aims to solve the technical problem that the oxygen of the existing streptomyces hygroscopicus is limited in the fermentation process, and provides the streptomyces hygroscopicus capable of efficiently utilizing the oxygen in the FK520 fermentation process, a corresponding gene, a recombinant vector, a transformant and a method for preparing ascomycin FK520, so that the oxygen dissolution in the streptomyces hygroscopicus fermentation process can be improved, the respiration effect of bacteria is promoted, and the fermentation unit of the obtained FK520 is improved.

One of the technical solutions for solving the above technical problems of the present invention is: a genetically engineered bacterium for producing ascomycin FK520 is an engineered bacterium which integrates a hemoglobin gene (VGB) in the genome of streptomyces hygroscopicus naturally producing FK 520. The VGB gene can be a natural exogenous VGB gene or a VGB gene which is codon-optimized for streptomyces hygroscopicus naturally producing ascomycin FK 520. Preferably, the nucleotide sequence of the VGB gene is shown as SEQ ID No. 1.

The second technical scheme for solving the technical problems is as follows: the nucleotide sequence of the VGB gene is shown in SEQ ID No. 1.

The third technical scheme for solving the technical problems is as follows: the nucleotide sequence of the VGB complete sequence gene is shown in SEQ ID No. 3. The VGB complete sequence gene comprises an ermE promoter and a VGB gene.

The fourth technical scheme for solving the technical problems is as follows: a recombinant vector comprising the VGB gene of the invention. Wherein, the skeleton of the recombinant vector is a vector which is conventional in the field. Preferably, the skeleton of the recombinant vector is plasmid pSET-152. Preferably, the nucleotide sequence of the plasmid pSET-152 is shown as SEQ ID No. 2. It can be ligated to the vector by the nucleotide sequence of the VGB gene of the present invention by a method conventional in the art. Preferably, the target VGB complete sequence gene fragment with sticky ends obtained after double digestion is inserted into the multiple cloning site of the pSET-152 vector and is connected by ligase to form the recombinant vector pSET-VGB containing the VGB gene of the invention.

The fifth technical scheme for solving the technical problems is as follows: a transformant comprising the recombinant vector of the present invention. Wherein, the host cell of the transformant can be a conventional host in the field, preferably Escherichia coli (Escherichia coli) ET12567, and the transformant can be obtained by transforming the recombinant vector pSET-VGB into the Escherichia coli ET 12567.

The sixth technical scheme for solving the technical problems of the invention is as follows: a method for preparing the genetic engineering bacteria for producing the ascomycin FK520 comprises the steps of jointing the transformant with streptomyces hygroscopicus naturally producing the FK520 and selecting a joint. Wherein, the natural FK 520-producing streptomyces hygroscopicus is a conventional natural FK 520-producing streptomyces hygroscopicus, and is preferably streptomyces hygroscopicus ATCC 14891. The host cell of the transformant may be a conventional host in the art, and a preferred host is E.coli ET12567(pUZ 8002). The backbone of the recombinant vector contained in the transformant is preferably the plasmid pSET-152.

The seventh technical scheme for solving the technical problems of the invention is as follows: a method for preparing ascomycin FK520 comprises the step of fermenting the genetically engineered bacterium for producing ascomycin FK520 to obtain FK520 from fermentation liquor.

Wherein, the seed solution required by the fermentation is the seed solution which is conventional in the field and is obtained after 7-8 days of producing ascomycin FK520 by slant culture at 28 ℃, spores of about 1/4 are transferred to a primary seed culture medium and culture for 48 hours at 28 ℃.

The fermentation is conventional in the art. Wherein the culture medium for fermentation is a conventional culture medium. Preferably, the medium of the fermentation comprises the following components: 6.0% of glycerol, 2.0% of yeast extract, 2.0% of soybean cake powder, 0.02% of monopotassium phosphate and trace elements; the percentage is a mass-volume percentage of the medium (e.g., 1% is 1g/100ml, and all "mass-volume percentages" in the present invention have the same meaning). Preferably, the trace elements consist of 0.001% of ferrous sulfate, 0.001% of copper sulfate, 0.001% of zinc sulfate and 0.000015% of cobalt chloride, wherein the percentage is the mass volume percentage of the culture medium. Preferably, the fermentation is also stirred; more preferably, the rotation speed of the stirring is 600-800 rpm; preferably 780 rpm. The temperature of the fermentation is the conventional temperature required by the fermentation of the streptomyces hygroscopicus naturally producing FK520 in the field, and is preferably 28 ℃. The fermentation period is preferably 5 to 8 days; more preferably 7 days. The dissolved oxygen amount of the fermentation is preferably 5-40%; more preferably 15-40%, the percentage is volume percent.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The invention has the following beneficial effects:

the VGB gene is introduced into streptomyces hygroscopicus naturally producing FK520, so that the respiration of thalli is promoted, enough ATP and NADPH are generated, the accumulation of FK520 is further promoted, the production efficiency of FK520 is improved, and the production cost of FK520 is reduced.

The fermentation of the streptomyces hygroscopicus for naturally producing FK520 is an aerobic fermentation process, but the dissolved oxygen in the fermentation liquid is still low even if the aeration and stirring conditions reach the highest parameters during the fermentation. However, the exogenous VGB gene is introduced into the streptomyces hygroscopicus naturally producing FK520, so that the respiration of the thallus is promoted, the dissolved oxygen in the fermentation process of the streptomyces hygroscopicus is improved, the inhibition of oxygen in the fermentation process of the thallus is removed, the positive effect on the growth of the thallus, particularly on the accumulation of secondary metabolites is achieved, and the method has certain universality.

On-tank studies found that FK520 fermented by the VGB-modified strain had 1.38 times as many units of FK520 fermented by the non-modified natural FK 520-producing strain under the same culture conditions. And the fermentation unit of rapamycin obtained by fermenting the naturally rapamycin-producing streptomyces hygroscopicus with the VGB gene is 0.917 times of that of rapamycin obtained by fermenting unmodified streptomyces hygroscopicus. Indicating that the insertion of the VGB gene does not promote the respiratory action of all S.hygroscopicus.

Drawings

FIG. 1 is a map of the pSET-VGB plasmid.

FIG. 2 shows the fermentation process of VGB modified FK520 producing strain.

FIG. 3 is a FK520 liquid phase detection spectrum.

FIG. 4 shows the fermentation process of an unmodified FK 520-producing strain.

FIG. 5 is a fermentation process of VGB engineered rapamycin producing bacteria.

FIG. 6 shows the fermentation process of an unmodified rapamycin producing strain.

Detailed Description

The inventor of the present invention has found that the utilization rate of oxygen by Streptomyces hygroscopicus can be improved by introducing the VGB gene into the Streptomyces hygroscopicus ATCC 14891 naturally producing FK520 through extensive research and repeated experiments. Therefore, the invention constructs a streptomyces hygroscopicus gene engineering strain containing VGB gene through gene engineering, and improves the problem of low dissolved oxygen in the FK520 fermentation process.

Firstly, cloning is carried out on the exogenous VGB gene, and the exogenous VGB gene can be obtained by cloning by a conventional method.

Then, a recombinant vector for site integration is constructed, and the VGB gene is connected to the plasmid pSET-152 by a conventional method in the field, such as double-enzyme digestion, so that the gene engineering plasmid pSET-VGB containing the VGB gene is constructed.

Then, the host cell is transformed with the recombinant vector to obtain a transformant. The transformant strain and the streptomyces hygroscopicus wild strain are co-cultured and are jointed to obtain a mutant strain containing VGB gene in a genome, so that the utilization rate of the streptomyces hygroscopicus on oxygen can be improved, and the problem of low dissolved oxygen in the FK520 fermentation process is solved. The cultivation of the obtained strains is a routine procedure in the art. In the present invention, in-tank culture is employed.

The sampling treatment method of the fermentation tank sample comprises the following steps:

adding acetone with the volume of 4 times into 300 mu l of fermentation liquor, performing ultrasonic treatment for 20min, centrifuging at 12000rpm for 3min, and taking supernatant for HPLC analysis.

The detection method of the fermentation product is an HPLC analysis method (isocratic system):

column: hypersil BDS C18,5 μm,4.6mm × 150 mm;

mobile phase: 35% water (ph4.8) + 65% acetonitrile;

column temperature: 55 ℃;

wavelength: 210 nm;

flow rate: 1.0 ml/min;

time: and 15 min.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The used tool enzyme and DNA molecular weight Marker are purchased from TaKaRa company, and the specific reaction conditions and the used method refer to the commercial specification. The gel recovery kit used was purchased from a manufacturer, and the method of use was referred to the commercial instructions. pSET-152 was purchased from TaKaRa, and its nucleotide sequence is shown in SEQ ID NO. 2. ET12567 competent cells were purchased from TaKaRa. Streptomyces hygroscopicus ATCC 14891 is purchased from American type culture Collection and rapamycin producing strain NRRL 5491 is purchased from American type culture Collection.

Example 1

Construction of exogenous VGB Gene-introduced engineering Strain

1. Construction of VGB-containing genetic engineering plasmid pSET-VGB

According to the preference of a codon of streptomyces hygroscopicus, the original exogenous VGB gene sequence from Vitreoscilla sp is modified, enzyme cutting sites of NdeI and AscI are added to two ends of the optimized VGB gene, namely the SEQ ID No.1 sequence consists of the optimized VGB gene and the enzyme cutting sites of NdeI and AscI at two ends; adding XbaI restriction enzyme cutting site at the front end of ermE promoter; the rear end of the terminator is integrated with a BamHI restriction site, and the whole gene sequence integrated with the restriction sites XbaI and BamHI is shown in SEQ ID No. 3. The designed sequence is completely synthesized, a target gene fragment is obtained by double digestion of XbaI and BamHI, and the target gene fragment is inserted into XbaI and BamHI sites of the pSET-152 vector, so that the recombinant vector pSET-VGB shown in figure 1 can be obtained. The sequence of pSET-152 is shown in SEQ ID No. 2.

2. Transformation of the recombinant vector into ET12567 competent cells

Taking 50 ul of competent cells ET12567, adding the recombinant vector pSET-VGB into the competent cells, uniformly blowing the competent cells, and standing the competent cells on ice for about 30 min; after heat shock is carried out for 45s at 42 ℃, standing on ice for 2 min; adding 450 μ l of fresh LB culture medium (without antibiotics) into the centrifuge tube, mixing, shaking at 37 deg.C, culturing at 150rpm for 45min, and recovering; centrifuging at 8000rpm for 1min, decanting the supernatant, adding 100 μ l LB medium, blowing, mixing, diluting, and spreading on antibiotic-containing LB plate (chloramphenicol, kanamycin, and apramycin concentrations of 25 μ g/ml, and 50 μ g/ml, respectively); the recombinants can be obtained by culturing at 37 ℃ overnight.

3. Introduction of VGB Gene into naturally FK 520-producing S.hygroscopicus by conjugation transfer

Inoculating streptomyces hygroscopicus ATCC 14891 naturally producing FK520 to a fresh slant, and culturing at 28 ℃ for 8 days until black spores are generated on the surface of white lawn. Spores (1ml) were collected by centrifugation in 1.5ml centrifuge tubes at 4000rpm for 5 min. The black spores precipitated at the bottom were resuspended in 500. mu.l TES buffer (0.05M, pH8.0), sealed with a sealing membrane, and heat-shocked in a water bath at 50 ℃ for 10 min. Naturally cooling for 5min, adding 500 μ l of 2 × spore pre-germination liquid (1% yeast extract, 1% casein, 0.01M CaCl)₂pH7.0, said percentages being mass volume percentages), at 37 ℃ and for 3h at 200 rpm. Then, the pregerminated spores were collected by centrifugation at 4000rpm for 5min and resuspended in 500. mu.l of LB or ddH₂O, the spore is a spore pre-germination liquid.

Coli ET12567 carrying the recombinant vector pSET-VGB was inoculated into 5ml of LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH7.0, said percentages being percentages by mass of the medium) containing chloramphenicol, kanamycin and apramycin at concentrations of 25. mu.g/ml, 25. mu.g/ml and 50. mu.g/ml, respectively, and cultured at 37 ℃ and 200rpm overnight. Inoculating 1% by volume (all the inoculum percentages are volume percentages) into 5ml of the same LB culture medium (with three antibiotics added), culturing at 37 deg.C to OD 0.5, centrifuging at 4000rpm for 5min, collecting thallus, washing with LB liquid culture medium for 2 times, and suspending in 500. mu.l of LB liquid culture medium as Escherichia coli solution.

Mixing the prepared spore pre-germination solution and an escherichia coli solution in a volume ratio of 1: 1 in a centrifuge tube, fully mixing, centrifuging at 4000rpm for 3min, pouring out supernatant, adding 100 microliter LB for re-suspension, blowing and uniformly mixing, and diluting and coating an MS agar culture medium (2% mannitol, 2% soybean cake powder, 2% agar, pH7.0, wherein the percentages are mass volume percentages of the culture medium). After culturing at 28 ℃ for 18 hours, the plates were covered with 1ml of sterile water each having an apramycin and nalidixic acid concentration of 50. mu.g/ml, and cultured at 28 ℃ for one week. Several single colonies were randomly picked from the MS plates and plated on MS plates containing 50. mu.g/ml apramycin for streaking, and after 7 days the zygotes were picked from the plates.

4. Detecting expression and activity of VGB gene in modified strain

(1) Colony PCR is utilized to detect whether the VGB gene is successfully introduced into the resistant strain grown on the apramycin resistant plate.

Primers were synthesized as follows:

an upstream primer VGB-5': 5'-ATGCTCGACCAGCAAACCAT-3', respectively;

the downstream primer VGB-3': 5'-TCATTCAACCGCTTGAGCGT-3' are provided.

The PCR conditions were as follows: the genomic DNA of the apramycin-resistant strain growing on the plate was used as a template for PCR amplification using two primers. The PCR system was a 50. mu.l system: part of a single colony picked by the aseptic gun head is a template, 10 XBuffer 5 mul, dNTP Mix 4 mul, an upstream primer VGB-5 '1 mul, a downstream primer VGB-3' 1 mul, a template 1 mul, rTaq enzyme 1 mul and ddH₂037 μ l. The PCR amplification step is as follows: (1) pre-denaturation at 94 ℃ for 10 min; (2) denaturation at 94 ℃ for 30 s; (3) annealing at 52 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the step 2-4 for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃.

The PCR product was purified by agarose gel electrophoresis, and a DNA fragment of about 450bp (the band could not be amplified by an unmodified control strain) was recovered by an agarose gel DNA recovery kit, and the nucleotide sequence of the DNA fragment was analyzed, whereby it was confirmed that the DNA fragment was VGB gene. It is shown that the VGB gene has been successfully integrated into the genome of S.hygroscopicus by the method of conjugative transfer, thus obtaining the genetically engineered strain producing ascomycin FK 520.

Example 2

On-tank studies of VGB-engineered strains

The deposited genetically engineered strain producing ascomycin FK520 as described in example 1 was removed from the glycerol tubes. After 8 days of slant culture at 28 ℃, spores of about 1/4 are transferred to a first-class seed culture medium, after 48 hours of culture at 28 ℃, the spores are inoculated into a 2L fermentation tank (6.0 percent of glycerol, 2.0 percent of yeast extract, 2.0 percent of soybean cake powder, 0.02 percent of monopotassium phosphate and trace elements, wherein the trace elements comprise 0.001 percent of ferrous sulfate, 0.001 percent of copper sulfate, 0.001 percent of zinc sulfate and 0.000015 percent of cobalt chloride, and the percentages are mass volume percentages of the culture medium), the inoculation amount is 5 percent, the fermentation is carried out at 28 ℃, and the fermentation period is 7 days. As shown in FIG. 2, the fermentation process increases oxygen demand in the biosynthesis process along with the growth of the bacteria, more oxygen is consumed, and the dissolved oxygen is reduced; along with the expression of VGB gene in the bacteria, the hemoglobin content is increased, the oxygen utilization rate is correspondingly improved, the oxygen consumption is reduced, and the dissolved oxygen content is increased. During the fermentation process, a dissolved oxygen electrode is inserted into the fermentation tank to detect dissolved oxygen, and the dissolved oxygen is maintained at more than 15%. Fermentation units of FK520 were assayed, with a maximum of 1269.7mg/L and a fermentation time of 161h (liquid phase assay profile, see FIG. 3).

Example 3

On-tank studies of unmodified natural FK520 producer

Preserved original streptomyces hygroscopicus ATCC 14891 which naturally produces FK520 is subjected to slant culture at 28 ℃ for 8 days from a glycerol tube, spores of about 1/4 are transferred to a seed culture medium, the culture medium is cultured at 28 ℃ for 48 hours, and then inoculated into a 2L fermentation tank (6.0% of glycerol, 2.0% of yeast extract, 2.0% of soybean cake powder, 0.02% of monopotassium phosphate and trace elements, wherein the trace elements consist of 0.001% of ferrous sulfate, 0.001% of copper sulfate, 0.001% of zinc sulfate and 0.000015% of cobalt chloride, the percentages are mass volume percentages of the culture medium), the inoculation amount is 5%, the fermentation is carried out at 28 ℃, and the fermentation period is 7 days. The fermentation process is shown in FIG. 4, after the original strain is fermented for 47h, the stirring speed reaches the upper limit of 780rpm, but the dissolved oxygen is less than 10%. Along with the growth of the thalli, the biomass of the thalli is gradually increased, the oxygen consumption is increased, the dissolved oxygen in the tank is gradually reduced to be less than 5 percent, even under the conditions of maximum rotating speed and maximum ventilation, the dissolved oxygen is still not improved, the metabolic process of the control bacteria is inhibited, the low dissolved oxygen has obvious influence on the fermentation yield, the maximum yield of FK520 is 921.8mg/L, and the fermentation time is 114 h.

Example 4

VGB gene was introduced into rapamycin-producing bacteria for on-tank studies

The VGB gene is integrated into a streptomyces hygroscopicus NRRL 5491 naturally producing rapamycin by the same method to obtain a VGB gene modified strain. Culturing rapamycin-producing bacteria modified by VGB gene on a slope at 30 ℃ for 30 days, transferring the bacteria to a seed culture medium after spores are mature, culturing for 72 hours at 30 ℃, and then inoculating the bacteria to a 2L fermentation tank (wherein the trace elements comprise 0.0125% of magnesium sulfate, 0.005% of zinc sulfate, 0.001% of molybdenum sulfate, 0.002% of ferrous sulfate and 0.00024% of cobalt chloride, the percentages are mass volume percentages of the culture medium), the inoculation amount is 10%, the bacteria are fermented at 30 ℃, and the fermentation period is 10 days. The fermentation process is shown in FIG. 5, the dissolved oxygen in the fermentation tank is maintained above 20% (the percentage is volume percent), and the maximum rapamycin yield is 782.5 mg/L.

Example 5

On-tank studies of unmodified natural rapamycin producing bacteria

The preserved original rapamycin producing bacteria NRRL 5491 are cultured from a glycerol tube at a temperature of 30 ℃ for 30 days by a slant, after spores are mature, the spores are transferred to a seed culture medium, and are cultured at a temperature of 30 ℃ for 72 hours and then inoculated into a 2L fermentation tank (2.0 percent of glucose, 2.0 percent of yeast extract, 8.0 percent of glycerol, 0.1 percent of potassium dihydrogen phosphate, 0.1 percent of dipotassium hydrogen phosphate and trace elements, wherein the trace elements consist of 0.0125 percent of magnesium sulfate, 0.005 percent of zinc sulfate, 0.001 percent of molybdenum sulfate, 0.002 percent of ferrous sulfate and 0.00024 percent of cobalt chloride, the percentages are mass volume percentages of the culture medium), the inoculation amount is 10 percent, the fermentation period is 10 days at a temperature of 30 ℃. The fermentation process is shown in FIG. 6, the dissolved oxygen content is maintained above 22.5% (the percentage is volume percentage) during the fermentation process, and the maximum rapamycin yield is 853.3 mg/L. Compared with the modified strain introduced with the VGB gene, the yield and the dissolved oxygen amount of the control strain are higher.

This indicates that the same VGB gene introduced into a similar S.hygroscopicus by the same method resulted in no increase, or even a slight decrease, in rapamycin production compared to the unmodified control strain. It was shown that the introduction of VGB gene did not promote the accumulation of rapamycin in this strain.

It will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above disclosure, and equivalents may fall within the scope of the invention as defined by the number of claims appended hereto.

Claims

1. ProducerThe genetically engineered bacterium of the cystomycin FK520 is characterized in that the genetically engineered bacterium is streptomyces hygroscopicus (Streptomyces hygroscopicus) (FK 520 produced naturallyStreptomyces hygroscopicus) ATCC 14891 has integrated into its genomeVGBGenetically engineered bacteria, saidVGBThe nucleotide sequence of the gene is shown as SEQ ID No. 1.

2. A method for preparing the genetically engineered bacterium of claim 1, comprising conjugating the transformant with Streptomyces hygroscopicus ATCC 14891 which naturally produces FK520, and selecting the conjugant; the transformant comprises a recombinant vector, and the recombinant vector comprises a nucleotide sequence shown as SEQ ID No.1VGBA gene.

3. The method of claim 2, wherein the recombinant vector comprises a backbone of plasmid pSET-152.

4. The method of claim 3, wherein the nucleotide sequence of the plasmid pSET-152 is set forth in SEQ ID No: 2.

5. A method for preparing ascomycin FK520, which comprises fermenting the genetically engineered bacterium of claim 1 to obtain FK520 from the fermentation broth.

6. The method of claim 5, wherein the fermentation medium comprises the following components: 6.0% of glycerol, 2.0% of yeast extract, 2.0% of soybean cake powder, 0.02% of monopotassium phosphate and trace elements; the trace elements consist of 0.001 percent of ferrous sulfate, 0.001 percent of copper sulfate, 0.001 percent of zinc sulfate and 0.000015 percent of cobalt chloride; the percentage is the mass volume percentage of the culture medium.

7. The method of preparing ascomycin FK520 of claim 6, where the fermentation is further stirred;

alternatively, the temperature of the fermentation is 28 ℃;

or the fermentation period is 5-8 days;

or the dissolved oxygen of the fermentation is 5-40%, and the percentage is volume percentage.

8. The method of claim 7, wherein the stirring is at a speed of 600 to 800 rpm;

or the fermentation period is 7 days;

or the dissolved oxygen amount of the fermentation is 15-40%.

9. The method of claim 8 for the preparation of ascomycin FK520, whereby the stirring is performed at 780 rpm.