阅读说明：本技术 一种高效发酵产大环内酰胺化合物fw05328-1的方法 (Method for producing macrolide compound FW05328-1 through high-efficiency fermentation ) 是由 孙菲 周剑 赵薇 江红 于 2020-09-11 设计创作，主要内容包括：本发明属于微生物发酵技术领域,具体涉及一种高效发酵产大环内酰胺化合物FW05328-1的方法。本发明所述高效发酵产大环内酰胺化合物FW05328-1的方法,在现有报道的生物合成途径基础上,通过对发酵培养基的优化,并添加3-氨基丁酸进行产物诱导,建立了适应于菌株产目标产物的发酵培养基及培养工艺,在10L-500L发酵罐发酵实验中,发酵产FW05328-1能力由249.8mg/L提高至654.2mg/L,大幅提高目标产物的产生能力(提高261.9％)；并且,发酵液中FW05328-1化合物组分比例由22.3％提高至74.8％(HPLC方法),提高幅度达到335.4％,从而为FW05328-1的规模化制备提供基础。(The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for producing a macrolide compound FW05328-1 by high-efficiency fermentation. The method for efficiently producing the macrolide compound FW05328-1 by fermentation establishes a fermentation culture medium and a culture process suitable for producing a target product by a strain by optimizing the fermentation culture medium and adding 3-aminobutyric acid for product induction on the basis of the existing reported biosynthesis way, and in a fermentation experiment of a 10L-500L fermentation tank, the capacity of producing the FW05328-1 by fermentation is improved to 654.2mg/L from 249.8mg/L, and the production capacity of the target product is greatly improved (improved by 261.9%); in addition, the component proportion of the FW05328-1 compound in the fermentation liquor is increased from 22.3 percent to 74.8 percent (by an HPLC method), and the increase range reaches 335.4 percent, thereby providing a basis for the large-scale preparation of the FW 05328-1.)

1. A method for producing a large lactam compound FW05328-1 by high-efficiency fermentation is characterized by comprising the step of inoculating a marine micromonospora fermentation strain into a fermentation medium containing 3-aminobutyric acid for fermentation culture.

2. The method for producing macrolide compound FW05328-1 by high-efficiency fermentation according to claim 1, wherein the mass concentration of 3-aminobutyric acid in the fermentation medium is 0.005-0.01 wt%.

3. The method for producing the macrolide compound FW05328-1 through the high-efficiency fermentation according to claim 1 or 2, wherein the fermentation medium comprises the following components by mass: 1.6-2.0% of soluble starch, 0.3-0.6% of glucose, 0.4-0.6% of peptone, 0.20-0.30% of soybean cake powder, 0.15-0.25% of yeast powder and K₂HPO₄·3H₂O 0.03-0.06％，(NH₄)₂SO₄ 0.03-0.06％，CaCO₃0.08 to 0.12 percent of sea salt, 0.8 to 1.2 percent of sea salt, 0.005 to 0.01 percent of 3-aminobutyric acid and pH value adjustment of 6.2 to 6.8.

4. A method for producing a macrolide compound FW05328-1 by high-efficiency fermentation according to any one of claims 1 to 3, wherein the conditions of said fermentation culture step are: inoculating the seed liquid with 1.0-2.0% of inoculum size, controlling the pressure of the tank to be 0.02-0.04Mpa, the rotating speed to be 180-220r/min, ventilating to be 0.8-1.2vvm, and performing fermentation culture at 26-30 ℃ for 5-7 d.

5. The method for producing the macrolide compound FW05328-1 through the high-efficiency fermentation according to claim 4, wherein in the fermentation culture step, the rotation speed is controlled at 180r/min in the early stage of the fermentation, when the thallus concentration reaches 7%, the rotation speed is increased to 200r/min, and the rotation speed is gradually increased to 220r/min according to the DO parameter and the mycelium growth change in the fermentation process.

6. The method for producing the macrolide compound FW05328-1 through high-efficiency fermentation according to any one of claims 1 to 5, wherein said method further comprises the step of inoculating said fermentation strain in a seed culture medium for seed liquid culture, said seed culture medium comprising the following components by mass: 1.0-2.0% of soluble starch, 0.4-0.6% of glucose, 0.4-0.8% of peptone, 0.4-0.6% of yeast powder and (NH)₄)₂SO₄0.04-0.06％，K₂HPO₄·3H₂O 0.03-0.06％，CaCO₃0.15-0.25 percent, 1.4-2.0 percent of sea salt and tap water, and adjusting the pH value to 7.0-7.4.

7. The method for producing the macrolide compound FW05328-1 by the high-efficiency fermentation according to claim 6, wherein said seed liquid culture step comprises a shake flask seed liquid culture and a seed tank culture step;

the conditions of the shake flask seed liquid culture step are as follows: inoculating the fresh slant digging block into the seed culture medium, and culturing at 27-29 ℃ and a controlled rotation speed of 200-;

the seed tank seed liquid culture conditions are as follows: inoculating the cultured shake flask seed culture solution into a seed tank containing the seed culture medium in an inoculation amount of 3.0-5.0%, controlling the temperature at 27-29 ℃, the tank pressure at 0.02-0.04Mpa and the rotation speed at 200-.

8. The method for producing the macrolide compound FW05328-1 through high-efficiency fermentation according to any one of claims 1 to 7, wherein said method further comprises the step of inoculating said strain to a solid slant culture medium of aspartyl having a high degree of activity, said solid slant culture medium comprising the following components in mass: soluble starch 1.5-3.5%, L-aspartyl 0.04-0.06%, KNO₃ 0.08-0.12％，K₂HPO₄·3H₂O 0.04-0.06％，NaCl 0.04-0.06％，MgSO₄·7H₂O 0.04-0.06％，CaCO₃0.08-0.12% of agar and 1-2% of agar, and adjusting pH value to 7.0-7.4.

9. The method for producing the macrolide compound FW05328-1 according to any one of claims 1 to 8, wherein said marine Micromonospora strain, classified and named as Micromonospora sp.FIM-MA181224, has been deposited in the China general microbiological culture Collection center with the collection number of CGMCC No. 17139.

10. An application of 3-aminobutyric acid in preparing a fermentation culture medium for efficiently producing a macrolide compound FW05328-1 by fermentation.

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for producing a macrocyclic lactam compound FW05328-1 through high-efficiency fermentation, and more particularly relates to a method for improving the fermentation yield of the macrocyclic lactam compound FW05328-1 based on amino acid induction.

Background

With the continuous progress of medical technology, more and more medical problems are overcome, and the service life of human beings is prolonged to a greater extent. However, cancer remains one of the major medical problems that people need to overcome, and cancer is the disease with the highest mortality rate in developed countries and developing countries, and the incidence rate and mortality rate of cancer are still increasing. At present, the main treatment means of cancer include surgery, radiotherapy and antitumor drug treatment, especially antitumor drug treatment, and the rapid development of the antitumor drug market is promoted along with the continuous increase of the global cancer incidence.

The FW05328-1 compound is a 26-membered polyene macrocyclic lactam compound with a new structure separated from a microbial secondary metabolite of micromonospora marinum FIM05-328 in 2017 by the institute of microorganisms in Fujian province (see Chinese patent CN107287131A for details). Pharmacodynamic experiments show that the compound has excellent inhibitory activity on human esophageal squamous carcinoma cell strains KYSE30, KYSE180 and EC109 in vitro, the inhibitory activity on human esophageal squamous carcinoma cell strains EC109 is particularly outstanding, and compared with structural analogues Salinilacticam A and medicinal cisplatin, the inhibitory activity of the compound is 165 times that of Salinilacticam A and 95 times that of cisplatin respectively. Therefore, the FW05328-1 compound is an antitumor drug precursor with better research prospect and has greater development value.

However, the compound FW05328-1, which is a natural product source, has extremely low fermentation titer, and is difficult to realize large-scale fermentation production. Therefore, Micromonospora sp.FIM-MA181224 is obtained by the institute of microorganisms in Fujian province through mutation breeding and the fermentation process is optimized (see the Chinese patent CN 110283747B for details), in the fermentation experiment of a 10L-500L fermentation tank, the titer of the compound FW05328-1 produced by the Micromonospora sp.FIM-MA181224 through fermentation reaches 249.8mg/L, and the method lays a foundation for the industrial production of the compound FW 05328-1.

Nevertheless, the fermentation titer of the macrocyclic lactam compound FW05328-1 still cannot meet the requirement of industrial production, mainly because, in addition to the expected further improvement of the fermentation titer, a series of FW05328-1 homologues are produced during the fermentation of Micromonospora sp.fim-MA181224 to produce the compound FW05328-1, which is not beneficial to the subsequent extraction of the target product by a separator. Therefore, how to improve the content of the target product FW05328-1 in the fermentation broth through the optimization of the fermentation process and effectively improve the component proportion of the target compound in the fermentation broth has important significance for the industrial research of the FW05328-1 compound.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a method for producing a macrolide compound FW05328-1 by high-efficiency fermentation, so as to solve the problems of low fermentation titer and low product proportion of the FW05328-1 compound in the prior art.

In order to solve the technical problems, the method for efficiently producing the macrolide compound FW05328-1 by fermentation comprises the step of inoculating a micromonospora marindus fermentation strain into a fermentation medium containing 3-aminobutyric acid for fermentation culture.

Specifically, in the fermentation medium, the mass concentration of the 3-aminobutyric acid is 0.005-0.01 wt%.

Specifically, the fermentation medium comprises the following components in percentage by mass: 1.6-2.0% of soluble starch, 0.3-0.6% of glucose, 0.4-0.6% of peptone, 0.20-0.30% of soybean cake powder, 0.15-0.25% of yeast powder and K₂HPO₄·3H₂O 0.03-0.06％，(NH₄)₂SO₄ 0.03-0.06％，CaCO₃0.08 to 0.12 percent of sea salt, 0.8 to 1.2 percent of sea salt, 0.005 to 0.01 percent of 3-aminobutyric acid and pH value adjustment of 6.2 to 6.8.

Preferably, the fermentation medium comprises the following components in percentage by mass: 1.8 percent of soluble starch, 0.5 percent of glucose, 0.5 percent of peptone, 0.25 percent of soybean cake powder, 0.2 percent of yeast powder and K₂HPO₄·3H₂O 0.05％，(NH₄)₂SO₄0.05％，CaCO₃0.1 percent of sea salt, 1 percent of 3-aminobutyric acid and tap water, and the pH value is adjusted to 6.6.

Specifically, the conditions of the fermentation culture step are as follows: inoculating the seed liquid with 1.0-2.0% of inoculum size, controlling the pressure of the tank to be 0.02-0.04Mpa, the rotating speed to be 180-220r/min, ventilating to be 0.8-1.2vvm, and performing fermentation culture at 26-30 ℃ for 5-7 d.

Preferably, in the fermentation culture step, the rotation speed is controlled to be 180r/min at the early stage of fermentation, when the thallus concentration reaches 7%, the rotation speed is increased to be 200r/min, and the rotation speed is gradually increased to be 220r/min according to DO parameters and the change of mycelium growth in the fermentation process.

More preferably, the conditions of the fermentation culture step are: inoculating with 1.8% inoculum size, controlling tank pressure at 0.03Mpa, controlling rotation speed at 180r/min in early stage of fermentation, ventilating at 1.0vvm, increasing rotation speed to 200r/min when the concentration of mycelia reaches 7% with the growth of mycelia, and gradually increasing rotation speed to 220r/min according to DO parameters and the growth change of mycelia in the fermentation process.

Specifically, the method further comprises the step of inoculating the fermentation strain into a seed culture medium for seed liquid culture, wherein the seed culture medium comprises the following components in percentage by mass: 1.0-2.0% of soluble starch, 0.4-0.6% of glucose, 0.4-0.8% of peptone, 0.4-0.6% of yeast powder and (NH)₄)₂SO₄0.04-0.06％，K₂HPO₄·3H₂O 0.03-0.06％，CaCO₃0.15-0.25 percent, 1.4-2.0 percent of sea salt and tap water, and adjusting the pH value to 7.0-7.4.

Preferably, the seed culture medium comprises the following components in percentage by mass: soluble starch 1.5%, glucose 0.5%, peptone 0.5%, yeast powder 0.5%, (NH)₄)₂SO₄0.05％，K₂HPO₄·3H₂O 0.05％，CaCO₃0.20 percent, sea salt 1.6 percent and tap water, and the pH value is adjusted to be 7.2.

Specifically, the seed liquid culture step comprises the steps of seed liquid culture in a shake flask and seed tank culture.

Specifically, the conditions of the shake flask seed liquid culture step are as follows: and inoculating the fresh slant digging block into the seed culture medium, and culturing at 27-29 ℃ and the controlled rotation speed of 200-.

Preferably, the cultivation step of the shake flask seed solution is as follows: and (3) digging blocks of the cultured fresh inclined plane according to the size of 0.5cm multiplied by 0.5cm, inoculating the blocks into a seed culture medium, and culturing at the temperature of 28 ℃ and at the speed of 220r/min for 72 hours to obtain a shake flask seed culture solution.

Specifically, the conditions of the seed tank seed liquid culture step are as follows: inoculating the cultured shake flask seed culture solution into a seed tank containing the seed culture medium in an inoculation amount of 3.0-5.0%, controlling the temperature at 27-29 ℃, the tank pressure at 0.02-0.04Mpa and the rotation speed at 200-.

Preferably, the culture steps of the seeding tank are as follows: inoculating the cultured shake flask seeds into a seed tank at 4.0% of inoculation amount, fermenting at 28 deg.C under 0.03Mpa at a rotation speed of 220r/min for 48h, and culturing in the seed tank.

Specifically, the method further comprises the step of inoculating the strain to a solid slant culture medium of the aspartokinase for activation culture, wherein the solid slant culture medium comprises the following components in percentage by mass: soluble starch 1.5-3.5%, L-aspartyl 0.04-0.06%, KNO₃ 0.08-0.12％，K₂HPO₄·3H₂O 0.04-0.06％，NaCl 0.04-0.06％，MgSO₄·7H₂O 0.04-0.06％，CaCO₃0.08-0.12% of agar and 1-2% of agar, and adjusting pH value to 7.0-7.4.

Preferably, the solid slant culture medium comprises the following components by mass: 2.0 percent of soluble starch, 0.05 percent of L-aspartyl and KNO₃ 0.1％，K₂HPO₄·3H₂O 0.05％，NaCl 0.05％，MgSO₄·7H₂O 0.05％，CaCO₃0.1 percent of agar and 1.5 percent of agar, and the pH value is adjusted to 7.2.

Preferably, the marine Micromonospora strain is classified and named as Micromonospora sp.FIM-MA181224, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation number of CGMCC No. 17139.

The invention also discloses application of the 3-aminobutyric acid in preparation of a fermentation culture medium for efficiently producing the macrolide compound FW05328-1 through fermentation.

Specifically, the invention also discloses a fermentation medium for efficiently producing the macrolide compound FW05328-1 by fermentation, which specifically comprises 0.005-0.01 wt% of 3-aminobutyric acid by mass concentration.

According to the method for efficiently producing the macrocyclic lactam compound FW05328-1 through fermentation, on the basis of the existing reported biosynthesis pathway, a fermentation culture medium and a culture process suitable for a strain Micromonospora sp.FIM-MA181224 to produce a target product are established through optimizing the fermentation culture medium and adding 3-aminobutyric acid to perform product induction, so that the capacity of producing the FW05328-1 through fermentation is improved to 654.2mg/L from 249.8mg/L in a fermentation experiment of a 10L-500L fermentation tank, and the production capacity of the target product is greatly improved (improved by 261.9%); and the method can change the metabolic pathway of microbial biosynthesis while improving the yield of microbial drugs, so that the metabolic pathway of microbial drug homologues is prevented, the metabolic flow is more shifted to the production of target drugs, the component proportion of the target drugs in fermentation metabolites is improved, the component proportion of the FW05328-1 compound in fermentation broth is improved from 22.3% to 74.8% (HPLC method), the improvement amplitude reaches 335.4%, the target products are favorably separated from a series of homologues, and the separation and purification steps are simplified, thereby providing a basis for the large-scale preparation of the FW 05328-1.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is a graph showing the fermentation profile in a 500L fermentor of the strain Micromonospora sp.FIM-MA181224 in example 5 of the present invention.

FIG. 2 is an HPLC chart showing the FW05328-1 component ratio in the fermentation broth of the strain Micromonospora sp.FIM-MA181224 in example 5 of the present invention.

Detailed Description

In the following examples of the present invention, the structural formula of the target compound FW05328-1 fermented is as follows:

in the following embodiment of the invention, the strain related to the fermentation culture of the compound FW05328-1 is Micromonospora sp.FIM-MA181224, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of West Lu No.1 of Chaozhou, the area of Chaoyang, Beijing, and the preservation number is CGMCC NO.17139, and the preservation date is 07/01.2019.

In the following examples of the present invention, the content detection of the macrolactam compound FW05328-1 in the fermentation broth adopts the HPLC-DAD method known in the prior art, which specifically comprises: agilent 1260Infinity ii HPLC system, DAD detector, column: agilent ZORBAX SB-C18(4.6 x 250mm, 5 μm), column temperature: 40 ℃, detection wavelength 298nm, mobile phase: 33% acetonitrile, flow rate: 1.0 mL/min.

Example 1

Culturing the preserved Micromonospora sp.FIM-MA181224 of Micromonospora marinum on an aspartyl solid culture medium at 35 ℃ for 15d to obtain a fresh slant of the Micromonospora sp.FIM-MA181224 of Micromonospora marinum.

Inoculating a fresh inclined plane digging block (0.5cm multiplied by 0.5cm) of Micromonospora sp.FIM-MA181224 of marine Micromonospora into a shake flask seed culture medium, culturing at 28 ℃ and 220r/min for 72h to obtain shake flask seeds, inoculating the shake flask seeds into a shake flask fermentation culture medium according to the inoculation amount of 4 percent, culturing at 28 ℃ and 220r/min for 6d, putting the shake flask in a bottle, and measuring the FW05328-1 content and the component proportion in a fermentation liquid by an HPLC method.

The preparation method of the seed culture medium comprises the following steps: 6g of corn starch, 5g of peptone, 5g of soybean meal, 3g of yeast powder and K₂HPO₄·3H₂O 0.5g，CaCO₃1g of sea salt and 1L of tap water, and the pH value is adjusted to be 7.2.

The preparation method of the fermentation medium comprises the following steps:10g of soluble starch, 5g of peptone, 2.5g of soybean cake powder, 2g of yeast powder and K₂HPO₄·3H₂O 0.5g，CaCO₃1g of sea salt, 10g of 3-aminobutyric acid and 1L of tap water, and the pH value is adjusted to 6.6.

Comparative example 1

The fermentation process of compound FW05328-1 of this comparative example differs from example 1 only in that the 3-aminobutyric acid is not added to the shake flask fermentation medium.

Comparative example 2

The fermentation process of compound FW05328-1 of this comparative example is the same as that of example 1 except that 3-aminobutyric acid is not added to the shake flask fermentation medium and an equal amount of 3-aspartic acid is added.

Comparative example 3

The fermentation process of compound FW05328-1 of this comparative example is the same as that of example 1 except that 3-aminobutyric acid is not added to the shake flask fermentation medium and lysine is added in an equal amount.

Comparative example 4

The fermentation process of compound FW05328-1 of this comparative example is the same as that of example 1 except that the 3-aminobutyric acid is not added to the shake flask fermentation medium and an equal amount of glutamic acid is added.

Comparative example 5

The fermentation process of compound FW05328-1 of this comparative example was identical to that of example 1 except that the 3-aminobutyric acid was not added to the shake flask fermentation medium and an equal amount of glycine was added.

After the fermentation and the flask placing are performed in the flask shaking way in the example 1 and the comparative examples 1 to 5, the HPLC method is used for measuring the fermentation liquid obtained by performing fermentation on the Micromonospora sp.FIM-MA181224 by using the culture medium of each scheme, the fermentation liquid obtained in the scheme is proved to be fermented to obtain the target compound, the content and the proportion of the FW05328-1 compound in the fermentation liquid are further measured, and the test results are shown in the following table 1.

TABLE 1 Effect of different amino acids on FW05328-1 fermentation titer and composition

Example 1

Comparative example 1

Comparative example 2

Comparative example 3

Comparative example 4

Comparative example 5

Fermentation titer (μ g/ml)

238.5

125.8

120.3

117.3

127.6

128.7

Component ratio (%)

74.2％

22.3％

20.7％

18.6％

24.1％

25.3％

As shown in the results in Table 1, in the method for producing the compound FW05328-1 by fermentation, 3-aminobutyric acid is added into a fermentation medium for induction, so that the capacity of producing the FW05328-1 by fermentation of Micromonospora sp.FIM-MA181224 can be remarkably improved, the fermentation titer is improved by 189.6%, and the component proportion of the FW05328-1 in a fermentation broth is improved from 22.3% to 74.2%, thereby greatly facilitating the later separation and purification work.

Example 2

In this embodiment, the fermentation medium in the fermentation method is further optimized, and a more suitable seed liquid culture medium and fermentation medium are selected.

The fermentation process of the compound FW05328-1 described in this example is the same as that of example 1, except that:

the seed culture medium comprises the following components in percentage by weight: 18g of soluble starch, 6g of glucose, 4g of peptone, 5g of yeast powder, (NH)₄)₂SO₄0.5g，K₂HPO₄·3H₂O 0.5g，CaCO₃2.0g, 16g of sea salt and 1L of tap water, and adjusting the pH value to be 7.2;

the fermentation medium comprises the following components in percentage by weight: 16g of soluble starch, 4g of glucose, 6g of peptone, 2.5g of soybean cake powder, 2g of yeast powder and K₂HPO₄·3H₂O 0.5g，(NH₄)₂SO₄0.5g，CaCO₃1.0g, 10g of sea salt, 0.1g of 3-aminobutyric acid and 1L of tap water, and the pH value is adjusted to 6.6.

Comparative example 6

The fermentation process of compound FW05328-1 of this comparative example is the same as that of example 2, except that:

the seed culture medium comprises the following components in percentage by weight: 6g of corn starch, 5g of peptone, 5g of soybean meal, 3g of yeast powder and K₂HPO₄·3H₂O 0.5g，CaCO₃1g of sea salt and 1L of tap water, and adjusting the pH value to be 7.2;

the fermentation medium comprises the following components in percentage by weight: 10g of soluble starch, 5g of peptone, 2.5g of soybean cake powder, 2g of yeast powder and K₂HPO₄·3H₂O 0.5g，CaCO₃1g of sea salt, 10g of 3-aminobutyric acid and 1L of tap water, and the pH value is adjusted to 6.6.

After the flask is placed in the flask for the flask fermentation in the above example 2 and comparative example 6, the content and the component ratio of FW05328-1 in the fermentation liquid are measured by the HPLC method, and the results are shown in table 2.

TABLE 2 Effect of different media on FW05328-1 fermentation titers and fractions

	Example 2	Comparative example 6
			Fermentation potency (mg/L)	336.7	241.2
Component ratio (%)	73.2％	72.8％

The results in Table 2 show that the optimized seed liquid culture medium and fermentation culture medium can improve the capability of Micromonospora sp.FIM-MA181224 for producing the anti-tumor compound FW05328-1 by fermentation by 39.6 percent to 336.7mg/L, and the component proportion of the FW05328-1 in the fermentation liquid reaches 73.2 percent.

Example 310L fermentor fermentation culture

Culturing the preserved Micromonospora sp.FIM-MA181224 of Micromonospora marinum on an aspartyl solid culture medium at 35 ℃ for 15d to obtain a fresh slant of the Micromonospora sp.FIM-MA181224 of Micromonospora marinum.

Inoculating fresh slant cut (0.5cm × 0.5cm) of Micromonospora sp.FIM-MA181224 of Micromonospora marinensis to shake flask seed culture medium, and culturing at 28 deg.C and 220r/min for 72 hr to obtain shake flask seed.

Inoculating the cultured shake flask seeds into a 10L fermentation tank by 1.5% inoculation amount, wherein the culture medium filling amount of the fermentation tank is 7L, the fermentation temperature is 28 ℃, the tank pressure is 0.03Mpa, the rotation speed is controlled to be 180r/min after fermentation is carried out for 0-48h, and the ventilation volume is 1: 1.0vvm, increasing the rotating speed to 200r/min when the bacterial concentration reaches 7% after fermenting for about 48 hours along with the growth of hyphae, and adjusting the ventilation volume to 1: 1.2vvm, after fermenting for 90 hours, gradually increasing the rotating speed to 220r/min according to DO parameters and the change of hypha growth in the fermentation process, supplementing 2ml of foam when the culture medium in the fermentation tank foams within 48-72 hours of fermentation, and discharging after the fermentation is finished for 144 hours of fermentation.

The seed culture medium comprises the following components and a preparation method: 15g of soluble starch, 5g of glucose, 5g of peptone, 5g of yeast powder, (NH)₄)₂SO₄0.5g，K₂HPO₄·3H₂O 0.5g，CaCO₃2g of sea salt, 16.5g of sea salt and 1L of tap water, and the pH value is adjusted to be 7.2.

The fermentation medium comprises the following components and a preparation method: 18g of soluble starch, 5g of glucose, 5g of peptone, 2.5g of soybean cake powder, 2.0g of yeast powder and K₂HPO₄·3H₂O 0.5g，(NH₄)₂SO₄0.5g，CaCO₃1.0g, 10g of sea salt, 0.075g of 3-aminobutyric acid and 1L of tap water, and the pH value is adjusted to 6.6.

In the embodiment, the content and the component proportion of the compound FW05328-1 in the fermentation broth are detected by HPLC in the whole fermentation process, the fermentation titer is 383.2mg/L when the fermentation is ended, and the component proportion of the FW05328-1 in the fermentation broth is 73.3%.

Example 4100L fermenter fermentation culture

The fermentation process of this example is the same as that of example 3, except that the amount of the medium in the 100L fermenter was 70L, the inoculum size was 1.8%, the seed solution cultured in the seed tank was inoculated to the 100L fermenter in an amount of 1.8%, and 20ml of DIE was added to the fermenter when the medium was foamed in the fermenter for 48 to 72 hours of fermentation.

The seeding tank culture method comprises the following steps: the seed in the shake flask cultured according to the shake flask culture process in the embodiment 3 is inoculated into a 10L fermentation tank by the inoculation amount of 4.0%, the liquid loading amount of the seed tank is 7L, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the rotation speed is controlled at 220r/min during the fermentation period, and the seed tank is fermented for 48 hours to be cultured and matured.

In the whole fermentation process, the content and the component proportion of a compound FW05328-1 in the fermentation broth are detected by HPLC, the fermentation titer is 603.8mg/L when the fermentation is ended, and the component proportion of the FW05328-1 in the fermentation broth is 74.1%.

Example 5500L fermenter fermentation culture

The fermentation process of this example was the same as that of example 4 except that the amount of the seed culture solution charged into the 500L fermenter was 350L and the inoculum size was 1.8%, the seed cultured in the seed tank was inoculated into the 500L fermenter at an inoculum size of 1.8%, and 100ml of DIE was added to the fermenter when the medium was bubbled in the fermenter during 48 to 72 hours of fermentation.

The seeding tank culture method comprises the following steps: the seed in the shake flask cultured according to the shake flask culture process in the embodiment 3 is inoculated into a 20L fermentation tank by the inoculation amount of 4.0%, the liquid loading amount of the seed tank is 14L, the fermentation temperature is 28 ℃, the tank pressure is 0.03MPa, the rotation speed is controlled at 220r/min during the fermentation period, and the seed tank is fermented for 48 hours to be cultured and matured.

In the whole fermentation process, the content and component ratio of the compound FW05328-1 in the fermentation broth are detected by HPLC, the fermentation curve of the fermentation tank is shown in figure 1, and the HPLC curve of the component ratio of FW05328-1 in the fermentation broth is shown in figure 2. As can be seen, the fermentation titer is 654.2mg/L when the fermentation is terminated, and the proportion of the FW05328-1 component in the fermentation broth is 74.8%.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.