Biosynthesis method of 1-bit C hydroxylated steroid compound

Abstract

The invention relates to the technical field of biology, in particular to a biosynthesis method of a 1-bit C hydroxylated steroid compound. According to the present invention, the 1-site C hydroxylation of the steroid compounds (including androstenedione, 17 alpha-hydroxyprogesterone, dinordinol 4-BA and the like) is achieved by using the Nigrospora oxyspora ATCC12772, such that the 1-site C hydroxylation of the steroid compounds (including androstenedione, 17 alpha-hydroxyprogesterone, dinordinol 4-BA and the like) is achieved; compared with the existing chemical method and biosynthesis method, the biosynthesis method provided by the invention has the characteristics of few synthesis routes, high conversion rate, high reaction product specificity and high safety.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for biosynthesizing 1-C hydroxylated steroids. Background Art

[0002] In the field of steroid drug synthesis, hydroxylation of steroids is the most crucial and valuable step, which has always attracted the attention of researchers. By introducing a hydroxyl group at a specific position of a steroid, active groups can be effectively added or specific carbon atoms can be activated, thereby endowing steroid drugs with unique physiological activities. However, traditional chemical synthesis methods have many deficiencies in achieving steroid hydroxylation. For example, in the synthesis of important steroid drugs such as calcitriol, eldecalcitol, and maxacalcitol, the methods for introducing a 1-hydroxyl group in the currently commonly used photochemical ring-opening method, total synthesis method, and structure modification method are all chemical synthesis methods. These methods often require multiple steps of reaction, and the process is cumbersome. For example, the method for introducing a 1-hydroxyl group on vitamin D3 disclosed in Patent CN103073468A requires four steps of chemical reactions, and the finally obtained product is a mixture of optical isomers, which requires complex separation and purification steps.

[0003] With the progress of biotechnology, the biosynthetic method has become a popular research direction for preparing hydroxylated steroid products. This method mainly relies on the catalytic action of microbial cells or enzymes to convert steroid substrates into hydroxylated products. If a suitable microorganism or enzyme can be screened, the reaction steps can be significantly reduced, and it is even expected to obtain the target product through a one-step reaction. For example, in 1952, Murry and Petersons of the Upjohn Company in the United States specifically introduced a hydroxyl group at the 11-carbon position of progesterone using Rhizopus nigricans, successfully shortening the production steps of cortisone from more than 30 steps of the synthesis method to 11 steps, and reducing the cost to one-thirtieth of the original. This fully demonstrates the great advantages of the biosynthetic method in terms of specificity and cost control.

[0004] However, there are numerous types, site coverages, and combinations of biotransformation reactions of steroids by microorganisms. Especially in the hydroxylation process, there are even stereoisomerism phenomena in the transformation products. Cases where specific biotransformations can be screened and developed into commercial processes are rare. Currently, the 1-hydroxylation of steroid compounds still faces severe challenges. The 1-hydroxylation of steroids in natural animals is completed by 1-hydroxylase in kidney cells. However, it is extremely difficult to utilize this enzyme on a large scale. Existing microbial transformation methods also have obvious defects. For example, in "Optimization of the Transformation Medium for 1α-Hydroxylation of Dehydroepiandrosterone by Response Surface Methodology", when using Penicillium decumbens to perform 1-hydroxylation of dehydroepiandrosterone, the feeding concentration is only 0.6‰, and the conversion rate is only 18.83%. It cannot achieve large-scale production and does not have industrial value. Thus, it can be seen that the introduction of the 1-hydroxy group of steroids currently faces the problem of low conversion rate, far from achieving the advantages of high efficiency, high selectivity, and low cost when introducing hydroxy groups at other positions.

[0005] In summary, the existing methods for 1-C hydroxylation of steroids have obvious deficiencies and are difficult to meet the demands of large-scale production and the market for steroid drugs. Therefore, developing an efficient microorganism and transformation method capable of completing 1-C hydroxylation of steroid compounds has important practical significance and application value. Summary of the Invention

[0006] In view of this, the present invention provides a biosynthetic method for 1-C hydroxylated steroid compounds. This method uses Nigrospora oryzae to achieve the 1-C hydroxylation of steroid compounds, and has a relatively high conversion rate.

[0007] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0008] Use of Nigrospora oryzae in the biosynthesis of 1-C hydroxylated steroid compounds.

[0009] The present invention successfully uses Nigrospora oryzae to achieve the 1-C hydroxylation of steroid compounds, with a high product yield, simple operation, and being safe and environmentally friendly. It effectively solves the problems existing in traditional chemical synthesis methods, such as long steps, poor selectivity, and high environmental and safety risks.

[0010] Experiments show that Nigrospora oryzae ATCC12772 can efficiently perform 1-C hydroxylation on steroid compounds, with a conversion rate of over 65%, providing an effective approach for the 1-C modification of steroid compounds.

[0011] The present invention also studied the transformation effect of Nigrospora sphaerica ATCC12772 on different steroid compounds, and the results showed that its conversion rates for different steroid compounds (such as androstenedione, 17α-hydroxyprogesterone, bisnorcholestane 4-BA) were also significantly different, and the conversion rate for androstenedione was the highest.

[0012] The present invention also provides a method for the biosynthesis of C1-hydroxylated steroid compounds, which comprises: fermenting and transforming a substrate with Nigrospora sphaerica.

[0013] In some embodiments, the Nigrospora sphaerica is Nigrospora oryzae ATCC12772.

[0014] In some embodiments, the substrates for the C1-hydroxylated steroid compounds include sex hormones or their derivatives, ring-opened steroids or their derivatives.

[0015] In some preferred embodiments, the sex hormones include androstenedione or 17α-hydroxyprogesterone; and the side-chain degraded steroids include at least one of bisnorcholestane 4-BA.

[0016] In the biosynthesis method of the present invention, before the fermentation transformation, it further includes the steps of seed culture and / or transformation culture of Nigrospora sphaerica.

[0017] The medium for the seed culture comprises the following components in mass percentages: carbon source 3%-5.5%, organic nitrogen source 1%-3%, inorganic nitrogen source 0.3%-0.7%, pH 4-4.5;

[0018] The carbon source includes one of glucose, maltose, sucrose, dextrin, glycerol;

[0019] The organic nitrogen source includes at least one of yeast extract, yeast powder, peptone, corn steep liquor, urea;

[0020] The inorganic nitrogen source includes at least one of ammonium sulfate, ammonium chloride, ammonium nitrate;

[0021] The conditions for the seed culture include: temperature 24-32°C, rotation speed 160-200 rpm , and culturing for 16-48 hours;

[0022] The medium for the transformation culture is the same as the seed medium or further includes at least one of phosphate, potassium salt, magnesium salt on the basis of the seed medium;

[0023] The conditions for the transformation culture are: temperature 24-32°C, rotation speed 160-200 rpm, and culturing for 15-20.

[0024] In the present invention, after the Nigrospora sphaerica undergoes the seed culture and / or transformation culture, it can directly ferment and transform the steroid compound, or be made into a resting cell transformation system and then ferment and transform the steroid compound.

[0025] In some embodiments, the method for preparing the resting cell transformation system includes: centrifuging Nigrospora sphaerica, collecting the mycelium, and washing and resuspending the mycelium with a phosphate buffer solution.

[0026] In some specific embodiments, the phosphate buffer solution is a 0.1 M PBS buffer solution with a pH of 6.0 - 7.0.

[0027] In the present invention, the fermentation transformation includes: reacting for 30 - 72 hours under the conditions of a temperature of 24 - 32 °C and a rotation speed of 160 - 200 revolutions per minute.

[0028] In the present invention, after obtaining the crude product through the fermentation transformation, it further includes the steps of inactivating the strains, filtering, and refining and purifying the crude product.

[0029] In some embodiments, the refining and purification includes: dissolving the crude product with an organic solvent, adding a recrystallization solvent for recrystallization to obtain a high-quality product.

[0030] In some embodiments, the organic solvent includes at least one of acetone and methanol, including but not limited to this.

[0031] In some embodiments, the recrystallization solvent includes ethyl acetate.

[0032] The present invention uses Nigrospora oryzae ATCC12772 to achieve the hydroxylation at the 1C position of steroid compounds (including androstenedione, 17α-hydroxyprogesterone, bisnorcholestanol, etc.). Compared with the existing chemical methods and biosynthesis methods, the biosynthesis method provided by the present invention has fewer synthesis routes, higher conversion rates, and the characteristics of high reaction product specificity and high safety. Description of the Drawings

[0033] Figure 1 It is the HPLC chromatogram of the conversion of androstenedione in Example 1.

[0034] Figure 2 It is the mass spectrum of the conversion product of androstenedione in Example 1.

[0035] Figure 3 It is the HPLC chromatogram of the conversion of androstenedione in Example 2.

[0036] Figure 4 It is the NMR spectrum of the conversion product of androstenedione in Example 1.

[0037] Figure 5HPLC chromatogram of 17-hydroxyprogesterone conversion in Example 3.

[0038] Figure 6 HPLC chromatogram of dihydroxy alcohol (4-BA) conversion in Example 4. Detailed implementation mode

[0039] The present invention provides a method for the biosynthesis of C1-hydroxylated steroids. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] In the present invention, all equipment, raw materials, etc. can be purchased from the market or are commonly used in this industry. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0041] The present invention uses a specific microorganism, Nigrospora oryzae, to specifically introduce a hydroxyl group at the C-1 position of multiple steroid compounds, efficiently achieving the introduction of a hydroxyl group at a specific carbon position of the steroid.

[0042] The specific conversion route of the present invention is as follows:

[0043]

[0044]

[0045] In the specific embodiments of the present invention, the Nigrospora oryzae used is Nigrospora oryzae ATCC12772. Experiments have shown that this strain has achieved hydroxylation at the C-1 position of steroid compounds and has a high conversion rate. Other microorganisms reported so far cannot achieve hydroxylation at the C-1 position or the conversion rate is significantly lower than that of the strain of the present invention.

[0046] Specifically, the solution provided by the present invention includes the following steps:

[0047] (1) Prepare the culture medium

[0048] Prepare the seed culture medium and the conversion culture medium. Perform moist heat steam sterilization and cool to room temperature for standby.

[0049] Seed culture medium: carbon source 3%-5.5%, organic nitrogen source 1%-3%, inorganic nitrogen source 0.3%-0.7%, pH 4-4.5.

[0050] In the above-mentioned culture medium, the mass concentration of the carbon source is 3%-5.5%, specifically it can be 3%, 3.5%, 4%, 4.5%, 5% or 5.5%; the carbon source is selected from one of glucose, maltose, sucrose, dextrin, glycerol, and further preferably glucose.

[0051] In the above-mentioned culture medium, the mass concentration of the organic nitrogen source is 1%-3%, specifically it can be 1%, 1.5%, 2%, 2.5%, 3%; the organic nitrogen source includes at least one of yeast extract, yeast powder, peptone, corn steep liquor, urea.

[0052] In the above-mentioned culture medium, the mass concentration of the inorganic nitrogen source is 0.3%-0.7%, specifically it can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7%; the inorganic nitrogen source can be at least one of substances such as ammonium sulfate, ammonium chloride, ammonium nitrate, and further preferably ammonium sulfate.

[0053] The conversion culture medium can have the same formula as the above-mentioned seed culture medium, or appropriate inorganic salt components such as phosphates, potassium salts, and magnesium salts can be added on the basis of the above-mentioned seed culture medium.

[0054] In the specific embodiments of the present invention, the compositions of the seed culture medium and the conversion culture medium are: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and the pH value is adjusted to 4-4.5.

[0055] (2) Seed culture

[0056] Under sterile conditions, inoculate the strain into the seed culture medium, set the culture temperature and the shaker speed, and then culture for 16-48 hours;

[0057] In the present invention, the strain Nigrospora oryzae ATCC12772 is a common commercially available product and is obtained through commercial channels.

[0058] The above-mentioned culture temperature range is 24-32°C, specifically it can be 24°C, 26°C, 28°C, 30°C, 32°C, and further preferably 28°C.

[0059] The above-mentioned culture conditions include the shaker speed, and the speed is 160-200 revolutions per minute, specifically it can be 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm.

[0060] The above-mentioned culture time is 16-48 hours, specifically it can be 16 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours.

[0061] (3) Conversion culture

[0062] After the seed culture is completed, an appropriate amount of seed liquid is inoculated into the transformation medium, and the inoculation amount is 5%-20%, and it is cultured for 15-20 hours under the conditions that the culture temperature is between 24-32°C and the shaking speed of the shaker is 160-200 revolutions per minute.

[0063] The above inoculation amount is 5%-20% (V / V), specifically it can be 5%, 10%, 15%, 20%, and the most preferred is 20%.

[0064] The above culture temperature range is 24-32°C, specifically it can be 24°C, 26°C, 28°C, 30°C, 32°C, and further preferably 28°C.

[0065] (4) Feeding for fermentation transformation

[0066] After the transformation strain is cultured well, it is directly fed or a resting cell transformation system is constructed for feeding, and then fermentation transformation is carried out under appropriate conditions.

[0067] The construction of the resting cell transformation system mentioned above refers to collecting mycelia of the transformation strain by centrifugation, washing with phosphate buffer, and then resuspending it in an equal volume of phosphate buffer of the fermentation centrifugate in a transformation flask.

[0068] The above phosphate buffer is preferably 0.1M PBS buffer with a pH of 6.0-7.0.

[0069] After being cultured well or the resting cell transformation system is constructed, the substrate is directly added for transformation, and the fermentation transformation conditions are that the transformation temperature is between 24-32°C and the shaking speed of the shaker is 160-200 revolutions per minute.

[0070] Among them, the transformation time is 30-72 hours.

[0071] Preferably, the fermentation transformation temperature is 28°C and the rotation speed is 180 revolutions per minute.

[0072] (5) Raising the temperature to end fermentation and separating and purifying.

[0073] After the fermentation transformation is completed, the strain is inactivated by raising the temperature, and the mixed filter cake of the thallus and the material is harvested by filtration, and the crude product of the transformation is obtained by dissolving and extracting with acetone, methanol, etc., and the fine product is obtained by recrystallization with ethyl acetate. The mother liquor is returned for fermentation transformation.

[0074] Description of the main raw materials involved in the present invention:

[0075] The present invention takes three steroid compounds with a hydroxyl group at position 1, including androstenedione, 17-hydroxyprogesterone, and bisnorcholestanol (4-BA) as examples to illustrate the biosynthesis method described in the present invention. The strain used in the examples is Nigrospora oryzae ATCC12772, which is purchased from the Guangdong Microbial Culture Collection Center.

[0076] Microbial transformation utilizes the cytochrome P450 enzyme system of microorganisms, and the utilization methods of this enzyme include growth cell transformation and resting cell transformation.

[0077] The input method of the substrate is solid powder feeding.

[0078] In the present invention, after the strain ATCC12772 is successively subjected to slant culture, seed culture and transformation culture, the above-mentioned microbial transformation is carried out.

[0079] Among them, for the slant culture of the strain, potato dextrose agar (PDA) medium is used and cultured at 26 °C for 5 days and then stored at 4 °C.

[0080] The formula of the PDA medium: 20% potato, 2% glucose, and 2% agar powder are added. The sterilization conditions are 121 °C for 30 minutes.

[0081] The formulas of the seed medium and the transformation medium are: 4.5% glucose, 2.0% corn steep liquor, 0.5% ammonium sulfate, and the pH value is adjusted to 4 - 4.5. The sterilization conditions of the medium are 121 °C for 30 minutes.

[0082] The following further elaborates the present invention in conjunction with embodiments:

[0083] Example 1

[0084] Prepare a seed liquid shake flask. The composition of the seed medium is: 4.5% glucose, 2.0% corn steep liquor, 0.5% ammonium sulfate, and the pH value is adjusted to 4 - 4.5. Use a 500 - milliliter Erlenmeyer flask, with a liquid loading of 100 milliliters, sterilize at 121 °C for 30 minutes. After cooling to room temperature, inoculate with a slant culture of Aspergillus niger, and culture on a shaker at 26 °C and 180 revolutions per minute for 22 hours (seed culture of the strain) to obtain the seed liquid;

[0085] Prepare a fermentation liquid shake flask. The composition of the fermentation medium is: 4.5% glucose, 2.0% corn steep liquor, 0.5% ammonium sulfate, and the pH value is adjusted to 4 - 4.5. Use a 500 - milliliter Erlenmeyer flask, with a liquid loading of 100 milliliters, sterilize at 121 °C for 30 minutes. Cool to room temperature.

[0086] Transfer 5 milliliters of the seed liquid into each fermentation liquid shake flask, and culture on a shaker at 26 °C and 180 revolutions per minute for 12 hours (transformation culture of the strain). Add 0.5 grams of androstenedione to each flask, and carry out transformation on a shaker at 26 °C and 180 revolutions per minute for 72 hours (fermentation transformation), and inactivate at 80 °C for 30 minutes. Combine the fermentation liquids and filter with filter paper. Extract the filter cake twice with 10 - fold volume of acetone and then combine. After concentration, centrifuge to obtain the crude sample. Dissolve the crude sample with methanol and send it for testing. After calculation, the conversion rate is 65.61%. The HPLC chromatogram of the crude sample is shown in Figure 1 , and the mass spectrometry results are shown in Figure 2。

[0087] Example 2

[0088] Prepare a seed liquid shake flask. The composition of the seed medium is: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and adjust the pH value to 4 - 4.5. Use a 500 - milliliter Erlenmeyer flask with a liquid volume of 100 milliliters. Sterilize at 121°C for 30 minutes. After cooling to room temperature, inoculate with a slant culture of Globisporangium eumorphum, and culture on a shaker at 26°C and 180 rpm for 22 hours to obtain the seed liquid;

[0089] Prepare a fermentation liquid shake flask. The composition of the fermentation medium is: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and adjust the pH value to 4 - 4.5. Use a 500 - milliliter Erlenmeyer flask with a liquid volume of 100 milliliters. Sterilize at 121°C for 30 minutes. Cool to room temperature.

[0090] Transfer 5 milliliters of the seed liquid into each fermentation liquid shake flask, and culture on a shaker at 26°C and 180 rpm for 16 hours. Centrifuge at 3000 rpm for 5 minutes, pour off the supernatant, resuspend with an equal volume of PBS buffer at pH 7.0 to prepare a resting cell transformation system. Prepare 1 bottle of the resting cell transformation system with an equal volume for each bottle of the fermentation liquid. Add 0.5 grams of androstenedione to each bottle, and carry out the transformation at 26°C and 180 rpm for more than 60 hours, and inactivate at 80°C for 30 minutes. After merging, filter with filter paper. Extract the filter cake with 10 times the volume of acetone twice and then merge. Concentrate and centrifuge to obtain the crude sample. Dissolve with methanol and send for detection. The HPLC chromatogram is shown in Figure 3 。After calculation, the conversion rate is 67.42%.

[0091] Example 3

[0092] Prepare a seed liquid shake flask. The composition of the seed medium is: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and adjust the pH value to 4 - 4.5. Use a 500 - milliliter Erlenmeyer flask with a liquid volume of 100 milliliters. Sterilize at 121°C for 30 minutes. After cooling to room temperature, inoculate with a slant culture of Globisporangium eumorphum, and culture on a shaker at 26°C and 180 rpm for 22 hours; Prepare a fermentation liquid shake flask. The composition of the fermentation medium is: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and adjust the pH value to 4 - 4.5. Use a 500 - milliliter Erlenmeyer flask with a liquid volume of 100 milliliters. Sterilize at 121°C for 30 minutes. Cool to room temperature.

[0093] Transfer 5 ml of the seed liquid into each fermentation liquid shake flask, culture it on a shaker at 26°C and 180 rpm for 12 hours, add 0.5 g of 17-hydroxyprogesterone to each flask, carry out transformation on a shaker at 26°C and 180 rpm for 72 hours, and inactivate at 80°C for 30 minutes. Combine the fermentation liquids and filter them with filter paper. Extract the filter cake twice with 10 times the volume of acetone and then combine them. After concentration, centrifuge to obtain the crude sample, dissolve it with methanol and send it for testing. The HPLC chromatogram is shown in Figure 5 . After calculation, the conversion rate is 10.40%.

[0094] Example 4

[0095] Prepare a seed liquid shake flask. The composition of the seed medium is: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and adjust the pH value to 4 - 4.5. Use a 500-ml Erlenmeyer flask, with a liquid loading of 100 ml, sterilize at 121°C for 30 minutes. After cooling to room temperature, inoculate with a slant culture of Globisporangium echinulatum, and culture it on a shaker at 26°C and 180 rpm for 22 hours; prepare a fermentation liquid shake flask. The composition of the fermentation medium is: glucose 4.5%, corn steep liquor 2.0%, ammonium sulfate 0.5%, and adjust the pH value to 4 - 4.5. Use a 500-ml Erlenmeyer flask, with a liquid loading of 100 ml, sterilize at 121°C for 30 minutes. Cool to room temperature.

[0096] Transfer 5 ml of the seed liquid into each fermentation liquid shake flask, culture it on a shaker at 26°C and 180 rpm for 12 hours, add 0.5 g of dihydroxyergocalciferol (4-BA) to each flask, carry out transformation on a shaker at 26°C and 180 rpm for 72 hours, and inactivate at 80°C for 30 minutes. Combine the fermentation liquids and filter them with filter paper. Extract the filter cake twice with 10 times the volume of acetone and then combine them. After concentration, centrifuge to obtain the crude sample, dissolve it with methanol and send it for testing. The HPLC chromatogram is shown in Figure 6 . After calculation, the conversion rate is 20.87%.

[0097] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of Nigrospora oryzae in the biosynthesis of 1-C hydroxylated steroidal compounds.

2. The use according to claim 1, characterized in that: The Nigrospora sphaerocephala is a strain with a preservation number of ATCC12772. 3.1 A biosynthetic method for a C-hydroxylated steroid compound, characterized in that: The substrate was fermented and converted using Nigrospora sphaerocephala.

4. The biosynthesis method according to claim 3, characterized in that The Nigrospora oryzae is Nigrospora oryzae ATCC12772; and / or The substrates of the 1α-hydroxylated steroid compounds include sex hormones or their derivatives, side chain degradable steroids or their derivatives.

5. The biosynthesis method according to claim 4, characterized in that The sex hormone includes androstenedione or 17α-hydroxyprogesterone; the side chain degrading steroid includes at least one of bile esters and 4-BA.

6. The biosynthesis method according to claim 3, characterized in that: Before the fermentation conversion is carried out, the method further comprises the step of carrying out seed culture and / or conversion culture on Aspergillus sphaeroides.

7. The biosynthesis method according to claim 6, characterized in that The medium for seed culture comprises the following components in percentage by mass: 3%-5.5% carbon source, 1%-3% organic nitrogen source, 0.3%-0.7% inorganic nitrogen source, pH 4-4.5; The carbon source includes one of glucose, maltose, sucrose, dextrin and glycerol; The organic nitrogen source includes at least one of yeast extract, yeast powder, peptone, corn steep liquor and urea; The inorganic nitrogen source includes at least one of ammonium sulfate, ammonium chloride and ammonium nitrate; The seed culture conditions include: temperature of 24-32°C, rotation speed of 160-200 rpm , culture for 16-48 hours; The transformation culture medium has the same composition as the seed culture medium or further comprises at least one of phosphate, potassium salt and magnesium salt on the basis of the seed culture medium; The transformation culture conditions are: temperature of 24-32° C., rotation speed of 160-200 rpm, and culture for 15-20 hours.

8. The biosynthesis method according to claim 3, characterized in that: After the seed culture and / or conversion culture fermentation conversion, the step of preparing a resting cell conversion system of Aspergillus sphaeroides is also included; The preparation method of the resting cell transformation system comprises: centrifuging the Nigrospora glomerata, collecting the mycelium, washing with a phosphate buffer, and resuspending the mycelium; The phosphate buffer is a 0.1 M PBS buffer with a pH of 6.0-7.

0.

9. The biosynthesis method according to claim 3, characterized in that: The fermentation conversion comprises: reacting for 30-72 hours at a temperature of 24-32° C. and a rotation speed of 160-200 revolutions per minute.

10. The biosynthesis method according to any one of claims 3 to 9, characterized in that: After the fermentation conversion obtains the crude product, the steps of inactivating bacteria, filtering and refining the crude product are also included; The refining and purification includes recrystallization purification.

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