Application of fusarium tripteroides in synthesis of 7alpha-OH-DHEA

Abstract

The application of the fusarium tripteroides in synthesis of 7alpha-OH-DHEA comprises the following steps: 1) preparing a spore suspension: inoculating the fusarium tripteroides onto a PDA plate, culturing for 2-5 days at 25-30 DEG C, adding sterile water in a sterile state, and collecting spores; and 2) seed culture: inoculating to a seed culture medium according to an inoculum size of 0.5-2%, and culturing for 2-5 days at the temperature of 25-30 DEG C and at the speed of 180-220 rpm. 3) fermentation culture: inoculating 5-10% of the mixture to a fermentation culture medium, and culturing for 18-30 hours at the temperature of 25-30 DEG C and the rotating speed of 180-220 rpm. And 3) a transformation experiment: culturing the fermentation broth for 18-30 h, adding a substrate DHEA, carrying out the transformation experiment under the conditions of 25-30 DEG C and a rotating speed of 180-220 rpm, and supplementing a carbon source in the transformation process.

Description

Technical Field

[0001] The present invention relates to a steroid compound biotransformation method, in particular to a hydroxylation method. Background Art

[0002] Fusarium spp. is a filamentous fungus widely distributed in plants and soil, commonly found in tropical and subtropical regions. Many of its species are important plant pathogens. Fusarium produces a variety of secondary metabolites, some of which can cause fungal keratitis and onychomycosis in humans. In subsequent studies, many scholars have isolated a series of naphthoquinones, mucormycins and polyketides from Fusarium oxysporum subspecies, and related subspecies have also been used for side chain cleavage, hydrogenation, oxidation and isomerization, and hydroxylation of steroids.

[0003] Dehydroepiandrosterone (DHEA) is a multifunctional steroid hormone that participates in a wide range of biological processes, such as diabetes, obesity, atherosclerosis, anti-tumor, bone metabolism, etc. Hydroxylated DHEA has different physiological activities, among which the hydroxylated product 7α-OH-DHEA has been shown to have important pharmacological effects such as antioxidant, anti-tumor and immunomodulatory. Therefore, the production of 7α-OH-DHEA has always been the focus of steroidal academic and industrial circles.

[0004] At present, there are two main methods for industrial production of 7α-OH-DHEA: chemical method and biological method. Compared with the chemical method, the biological method has the characteristics of mild reaction conditions, regional and stereospecificity, enantiomeric specificity, and green environmental protection. Currently, the strains reported for synthesizing 7α-OH-DHEA include Colletotrichum flaxensis, Gibberella, Absidia cyanobacteria, and Mucor racemosus, but there are problems such as low substrate input, many by-products, and high separation difficulty, which cannot meet the requirements of industrial production.

[0005] The prior art utilizes the selective hydroxylation ability of the blue Absidia strain to biotransform DHEA to synthesize the target product 7α-OH-DHEA. Although the conversion rate is high, it is found through experiments that it will generate a byproduct 7β-OH-DHEA with a similar content. The two substances are chiral isomers and are difficult to separate. Chinese patent CN102517222A discloses a method for converting Gibberella to produce 7α-OH-DHEA. The substrate feed amount is 5.2g / L, the substrate conversion rate is 90%, and the product yield is 80%, but the byproduct is 7α, 15α-diOH-DHEA, and the subsequent product separation is difficult. Therefore, it has become an urgent problem to be solved in the prior art to select a new method to improve the selectivity of the biosynthesis of 7α-OH-DHEA, and then improve the efficiency of the biological method of synthesizing 7α-OH-DHEA. Summary of the invention

[0006] In order to solve the above technical problems, we unexpectedly found in the research that DHEA can be highly selectively monohydroxylated to 7α-OH-DHEA under appropriate conditions by using Fusarium tricinctum, which solves the problems of low feed amount and many by-products that are difficult to separate in the prior art. There is no report on the use of Fusarium tricinctum for steroid transformation. The use of Fusarium tricinctum strains for steroid hydroxylation can produce 7α-OH-DHEA, which has the advantages of high substrate feed concentration, low by-product content, and separated products, which is more conducive to industrial production.

[0007] The technical solution provided by the present invention is

[0008] The application of Fusarium trilineatum in the synthesis of 7α-OH-DHEA comprises the following steps:

[0009] 1) Preparation of spore suspension: Inoculate Fusarium thaliana on a PDA plate, culture at 25-30°C for 2-5 days, and then add sterile water to collect spores under aseptic conditions.

[0010] 2) Seed culture: inoculate into seed culture medium (250 mL Erlenmeyer flask containing 30-50 mL seed culture medium) at 0.5-2% inoculum, and culture at 25-30° C. and 180-220 rpm for 2-5 days.

[0011] 3) Fermentation culture: inoculate 5-10% into fermentation medium (250 mL Erlenmeyer flask containing 30-50 mL fermentation liquid), culture at 25-30° C., rotation speed 180-220 rpm, and culture for 18-30 h to obtain fermentation liquid.

[0012] 4) Conversion experiment: After the fermentation broth is cultured in step 3), the substrate DHEA is added, and the conversion experiment is carried out at 25-30° C. and a rotation speed of 180-220 rpm, and a carbon source is added during the conversion process.

[0013] The application of three-line Fusarium in the synthesis of 7α-OH-DHEA, further, the culture temperature of the three-line Fusarium strain is 25-30°C, the components of the seed liquid culture medium are glucose 10-30g / L, glycerol 5-15mL / L, peptone 5-15g / L, yeast extract 5-15g / L, NaCl 1-3g / L, KH2PO43-10g / L; the components and proportions of the fermentation liquid culture medium are glucose 20-50g / L, corn steep liquor 10-30g / L, NaNO31-3g / L), KCl2-10g / L, K2HPO41-3g / L, KH2PO41-3g / L, MgSO40.2-1.0g / L, FeSO4 0.02-0.1g / L.

[0014] The application of Fusarium trilineatum in the synthesis of 7α-OH-DHEA is a preferred technical solution, and the culture and conversion rotation speed is 180-220 rpm.

[0015] The application of Fusarium trilineatum in the synthesis of 7α-OH-DHEA is, as a preferred technical solution, the substrate is fed in a 1-3% (v / v) dimethyl sulfoxide aqueous solution.

[0016] The application of Fusarium trilineatum in the synthesis of 7α-OH-DHEA is a preferred technical solution, and the carbon source is supplemented by adding 3 to 10 g / L of glucose solution.

[0017] The application of the three-line Fusarium in the synthesis of 7α-OH-DHEA is, as an optimal technical solution, the seed culture medium is composed of: glucose 10g / L, glycerol 10mL / L, peptone 5g / L, yeast extract 5g / L, NaCl2g / L, KH2PO4 (5g / L, the fermentation broth culture medium is as follows: glucose 30g / L, corn steep liquor 10g / L, NaNO32g / L, KCl (5g / L), K2HPO4 (2g / L), KH2PO4 (1g / L), MgSO4 (0.5g / L), FeSO4 (0.02g / L).

[0018] The application of the three-line Fusarium in the synthesis of 7α-OH-DHEA provided by the present invention discovers for the first time that the three-line Fusarium strain has a monohydroxylation effect, and by optimizing the process conditions of microbial culture and substrate conversion, the three-line Fusarium strain is used to achieve a high conversion rate of 7α-OH-DHEA under a high substrate feed amount, thereby achieving the purpose of reducing costs, energy consumption and three wastes. DETAILED DESCRIPTION

[0019] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] In the embodiment

[0021] The deposit number of Fusarium tricinctum strain is CCTCC AF 2020017

[0022] Example 1

[0023] Preparation of spore suspension: Fusarium trilineatum was inoculated on a PDA plate and cultured at 28°C for 72 h. Then, 500 μL of sterile water was added under sterile conditions to collect the spores.

[0024] Seed culture: 1% inoculum (0.5 mL) was inoculated into the seed culture medium (250 mL Erlenmeyer flask was filled with 50 mL seed culture medium), and cultured at 28°C, 200 rpm for 72 h to obtain the seed culture solution. The seed culture medium is as follows: glucose (10 g / L), glycerol (10 mL / L), peptone (5 g / L), yeast extract (5 g / L), NaCl (2 g / L), KH2PO4 (5 g / L)

[0025] Fermentation culture: 10% inoculation (5 mL seed culture solution) to fermentation medium (250 mL Erlenmeyer flask with 50 mL fermentation solution), 28 ° C, 200 rpm, culture for 24 hours to obtain fermentation solution. The fermentation medium is as follows: glucose (30 g / L), corn steep liquor (10 g / L), NaNO3 (2 g / L), KCl (5 g / L), K2HPO4 (2 g / L), KH2PO4 (1 g / L), MgSO4 (0.5 g / L), FeSO4 (0.02 g / L).

[0026] Conversion experiment: After the fermentation broth was cultured, steroid compounds were added in 4 parallel groups, divided into Example 1-1 to Example 1 to 4, respectively, dehydroepiandrosterone (Dehydroepiandrosterone, CAS: 53-43-0), pregnenolone (Pregnenolone, CAS: 145-13-1), Bisnoralcohol (Bisnoralcohol, CAS: 60966-36-1), estrone (EstroneCAS: 53-16-7), and the conversion experiment was carried out at 30 ° C and 200 rpm.

[0027] In this example, the substrate feed concentration is 5 g / L, the cosolvent is 2% (v / v) DMSO solution, and after 72 hours of conversion, HPLC detection found that DHEA had a new product generated, and its conversion rate was 97.05%, and no new product was found in the other substrates. Further nuclear magnetic resonance identification of the product generated by DHEA conversion was 7α-OH-DHEA. This shows that the strain has a monohydroxylation effect and can effectively convert DHEA to 7α-OH-DHEA.

[0028] Example 2

[0029] Preparation of spore suspension: Fusarium trilineatum was inoculated onto a PDA plate and cultured at 28°C for 72 h. Then, 20 mL of sterile water was added under aseptic conditions to collect the spores.

[0030] Seed culture: 1% inoculum (20 mL) was inoculated into the seed culture medium (5 L fermenter was filled with 2 L seed solution), and cultured at 30°C, 200 rpm for 48 h to obtain the seed culture solution. The seed culture medium is as follows: glucose (10 g / L), glycerol (10 mL / L), peptone (5 g / L), yeast extract (5 g / L), NaCl (2 g / L), KH2PO4 (5 g / L)

[0031] Fermentation culture: Shake flask culture method is to inoculate (5mL seed culture solution) with 10% to fermentation medium (250mL triangular flask is filled with 50mL fermentation solution), 28°C, rotation speed 200rpm, culture for 24h. Fermentation tank culture method is to inoculate (300mL seed culture solution) with 10% to fermentation medium (5L fermentation tank is filled with 3L fermentation medium), 28°C, rotation speed 200rpm, culture for 18h. The fermentation medium is as follows: glucose (30g / L), corn steep liquor (10g / L), NaNO3 (2g / L), KCl (5g / L), K2HPO4 (2g / L), KH2PO4 (1g / L), MgSO4 (0.5g / L), FeSO4 (0.02g / L).

[0032] Conversion experiment: After 24 hours of shake flask fermentation, the substrate was added and the conversion experiment was carried out at 28°C and 200 rpm. In a 5L fermenter, the substrate DHEA was added after 18 hours of fermentation, and 5g / L of glucose was added at 0h and 120h of conversion, respectively, and the conversion experiment was carried out at 25°C, 250rpm, and 2.5L / min of ventilation.

[0033] In this example, the substrate feed concentration was 8 g / L, the cosolvent was 2% (v / v) DMSO, and the conversion rate of the target product 7α-OH-DHEA was 87.76% after 96 hours of shake flask conversion. The conversion rate of the target product 7α-OH-DHEA was 79.21% after 72 hours of fermentation tank conversion.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of Fusarium trilineatum in the synthesis of 7α-OH-DHEA is characterized in that: The following steps are involved: 1) Preparation of spore suspension: Inoculate Fusarium thaliana on a PDA plate, culture at 25-30°C for 2-5 days, and then add sterile water to collect spores under aseptic conditions. 2) Seed culture: inoculate the seed culture medium with an inoculum of 0.5-2%, and culture at 25-30°C and 180-220 rpm for 2-5 days. 3) Fermentation culture: 5-10% is inoculated into the fermentation medium, cultured at 25-30° C., at a rotation speed of 180-220 rpm for 18-30 hours to obtain a fermentation liquid. 4) Conversion experiment: After the fermentation broth is cultured in step 3), the substrate DHEA is added, and the conversion experiment is carried out at 25-30° C. and a rotation speed of 180-220 rpm, and a carbon source is added during the conversion process.

2. The use of Fusarium trilineatum in the synthesis of 7α-OH-DHEA as claimed in claim 1, characterized in that The culture temperature of the three-line Fusarium strain is 25°C to 30°C, and the components of the seed liquid culture medium are glucose 10-30g / L, glycerol 5-15mL / L, peptone 5-15g / L, yeast extract 5-15g / L, NaCl 1-3g / L, KH2PO4 3-10g / L; the components and proportions of the fermentation liquid culture medium are glucose 20-50g / L, corn steep liquor 10-30g / L, NaNO3 1-3g / L, KCl 2-10g / L, K2HPO4 1-3g / L, KH2PO4 1-3g / L, MgSO4 0.2-1.0g / L, and FeSO4 0.02-0.1g / L.

3. The use of Fusarium trilineatum in the synthesis of 7α-OH-DHEA as claimed in claim 2, characterized in that: The culture and transformation rotation speed is 180-220 rpm.

4. The use of Fusarium trilineatum in the synthesis of 7α-OH-DHEA as claimed in claim 3, characterized in that: The substrate is fed in a dimethyl sulfoxide aqueous solution with a volume percentage concentration of 1 to 3%.

5. The use of Fusarium trilineatum in the synthesis of 7α-OH-DHEA as claimed in claim 4, characterized in that: The carbon source supplementation method is to add 3-10g / L of glucose solution.

6. The use of Fusarium trilineatum in the synthesis of 7α-OH-DHEA as claimed in claim 5, characterized in that: The seed culture medium is composed of: glucose 10 g / L, glycerol 10 mL / L, peptone 5 g / L, yeast extract 5 g / L, NaCl 2 g / L, KH2PO4 5 g / L, and the fermentation broth culture medium is as follows: glucose 30 g / L, corn steep liquor 10 g / L, NaNO3 2 g / L, KCl 5 g / L, K2HPO4 2 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, FeSO4 0.02 g / L.