A method for converting progesterone into 11α,17α-diOH-progesterone by Gibberella fujikuroi

Abstract

The present invention discloses a method for converting progesterone into 11α, 17α-diOH-progesterone by utilizing Gibberella intermedia, belongs to the field of biotechnology, and is particularly related to a method for converting progesterone into 11α, 17α-diOH-progesterone by utilizing Gibberella intermedia CA3-1. The strain is deposited in the General Microbiological Center of China Committee for the Collection of Microorganisms, with a deposit number of CGMCC No.4903. Gibberella intermedia CA3-1 can convert progesterone into 11α, 17α-diOH-progesterone. When the substrate feed concentration is 3g / L, the concentration of 11α, 17α-diOH-progesterone is 0.626g / L, and the conversion rate of progesterone into 11α, 17α-diOH-progesterone is 18.9%. At present, the synthesis of 11α, 17α-diOH-progesterone provides a more diversified precursor for the research of steroidal drugs and their intermediates, with certain research value and market significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for converting progesterone into 11α,17α-diOH-progesterone by utilizing Gibberella intermedia CA3-1. Background Art

[0002] Progesterone, also known as progesterone or luteinizing hormone, is an important intermediate in the synthesis of steroidal corticosteroids and steroidal progestogens. It can be used to synthesize steroid hormones such as hydrocortisone, progesterone acetate, cortisone acetate, betamethasone, chlormadinone acetate, and megestrol acetate. In vivo, progesterone can be hydroxylated into different products. Among them, 11α-diOH-progesterone is widely reported to be used in the synthesis of progestogens such as chlormadinone acetate, while its 11α,17α-diOH-progesterone product is less reported.

[0003] Steroidal compounds are primarily prepared through traditional chemical synthesis and the currently more commonly used microbial transformation method. Compared to traditional chemical synthesis, microbial biotransformation offers numerous advantages, including fewer reaction steps, milder conditions, scalable production, and environmental friendliness. Microbial transformation technology can selectively introduce one or more hydroxyl groups into the progesterone nucleus, thereby synthesizing intermediates for various steroidal drugs, which are used in the production of various anti-inflammatory drugs. Due to the unique biological activity and medicinal value of steroidal compounds, China has identified the development of steroid hormone drugs as a key focus of recent technological development in the pharmaceutical industry.

[0004] In recent years, researchers have found several strains capable of converting progesterone, such as Glucophyllum rhizomyces, Absidia coerulea, and Rhizopus nigricans, all of which can efficiently convert 17α-progesterone into 11α, 17α-diOH-progesterone. However, there have been no reports of directly converting progesterone to 11α, 17α-diOH-progesterone. Summary of the Invention

[0005] Purpose of the Invention: To overcome the shortcomings of the prior art, the present invention provides a method for converting progesterone to 11α,17α-diOH-progesterone using Gibberella intermedia CA3-1. The present invention utilizes Gibberella intermedia CA3-1 to directly convert progesterone to 11α,17α-diOH-progesterone. The synthesis of 11α,17α-diOH-progesterone provides a more diverse precursor for the research of steroidal drugs and their intermediates. Furthermore, the C11α and C17α dihydroxylation reaction, as a new type of steroidal hydroxylation reaction, opens up a new pathway for steroidal drug synthesis and has considerable research value and market significance.

[0006] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] The present invention aims to provide a novel dihydroxylated progesterone product (11α,17α-diOH-progesterone) and a method for its conversion and synthesis. The substrate progesterone is converted to 11α,17α-diOH-progesterone using the fungus Gibberella intermedia CA3-1 via a one-step conversion process. This fungus is deposited at the General Microbiology Center of the Chinese Academy of Sciences, China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 4903 and the deposit date May 24, 2011. It is classified as Gibberella intermedia.

[0008] The first object of the present invention is to provide a method for converting progesterone into 11α,17α-diOH-progesterone using Gibberellin, comprising activating and fermenting Gibberellin CGMCC No. 4903 to obtain a strain fermentation broth, and using the strain fermentation broth to bioconvert the substrate progesterone into 11α,17α-diOH-progesterone.

[0009] In the present application, the gibberella is Gibberella intermedia CA3-1, which is deposited at the General Microbiology Center of the Chinese Culture Collection Committee of the Chinese Academy of Sciences, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 4903, a deposit date of May 24, 2011, and a classification name of Gibberella intermedia.

[0010] Optionally, in one embodiment of the present application, the concentration of the substrate progesterone is 1-5 g / L.

[0011] Optionally, in one embodiment of the present application, the concentration of the substrate progesterone is 3 g / L.

[0012] Optionally, in one embodiment of the present application, the conversion process includes adding the substrate progesterone to the fermentation liquid of Gibberella, and converting it at a conversion temperature of 28° C. and a rotation speed of 200-220 r / min for 48-72 hours to obtain a conversion liquid.

[0013] Optionally, in one embodiment of the present application, the substrate progesterone is added to the Gibberella fermentation broth, comprising washing the cultured bacteria in the fermentation broth with 0.1-0.5M PBS buffer, and then resuspending them in fresh 30mL / 250mL PBS buffer to prepare resting cells of Gibberella CA3-1 strain, and then adding the substrate progesterone into the resting cells of Gibberella CA3-1 strain.

[0014] Optionally, in one embodiment of the present application, the activation culture includes using Gibberellic acid CGMCC No. 4903 as the production strain, activating and culturing it at 25-35°C and 150-220 r / min to obtain a seed liquid, and then diluting the seed liquid and applying it to a PDA solid culture medium; picking a ring of the CA3-1 strain on the PDA solid culture medium, inoculating it into the seed culture medium, and culturing it at 25-35°C and 150-220 r / min for 12-20 hours to the middle and late stages of logarithmic growth, thereby obtaining a seed culture liquid of the CA3-l strain.

[0015] Optionally, in one embodiment of the present application, the components of the PDA solid culture medium include: 200-500 g / L potatoes, 20-50 g / L glucose, 2-10 g / L yeast powder, 10-20 g / L agar powder, the pH is adjusted to 6.5-7.5, and sterilized under high pressure steam at 121°C for 15-20 min.

[0016] Optionally, in one embodiment of the present application, the components of the seed culture medium include: 10-30 g / L glucose, 5-15 g / L yeast powder, 0.5-2 g / L NaCl, 1-5 g / L KH2PO4, pH adjusted to 6.5-7.5, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0017] Optionally, in one embodiment of the present application, the fermentation culture comprises inoculating the seed liquid into a fermentation medium at an inoculum amount of 4% to 8% by volume, the liquid volume being 25 to 50 mL of the fermentation medium in a 250 mL conical flask, culturing at a temperature of 25 to 35° C. and a rotation speed of 150 to 220 r / min for 20 to 24 hours to obtain a fermentation liquid;

[0018] Optionally, in one embodiment of the present application, the components of the fermentation medium include: 10-30 g / L glucose, 5-15 g / L yeast powder, 1-5 g / L corn steep liquor, 1-5 g / L NaCl, the pH is adjusted to 6.5-7.5, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0019] Optionally, in one embodiment of the present application, the method for converting progesterone into 11α,17α-diOH-progesterone using the above-mentioned microorganisms is carried out, and the specific steps are as follows:

[0020] (1) Using Gibberella CA3-1 as the production strain, activate and culture at 25-35°C and 150-220 r / min to obtain seed liquid, and then dilute the seed liquid and spread it on a PDA plate;

[0021] (2) Preparation of a cell liquid culture of the CA3-1 strain: Pick a loopful of the CA3-1 strain on the PDA solid medium obtained in step (1) and inoculate it into a sterilized 500 mL conical flask containing 50 to 100 mL of seed culture medium. The culture temperature is 25 to 35° C. and the culture is carried out on a shaker at a speed of 180 to 220 r / min for 12 to 20 h until the middle to late logarithmic growth period, thereby obtaining a seed culture of the CA3-1 strain.

[0022] (3) Fermentation culture: The cell liquid culture prepared in step (2) is inoculated into a fermentation medium at an inoculum size of 4% to 8% (v / v), and the volume of the fermentation medium is 25 to 50 mL in a 250 mL conical flask. The culture is carried out under the same conditions for 20 to 24 hours to obtain a bacterial fermentation liquid;

[0023] (4) Biotransformation: The cultured cells from step (3) were washed with 0.1-0.5 M PBS buffer and then resuspended in fresh 30 mL / 250 mL PBS buffer to prepare resting cells of Gibberella CA3-1. An appropriate amount of substrate progesterone was accurately weighed and added to the resting cells of Gibberella CA3-1 to a final concentration of 1-5 g / L. The transformation was carried out at a transformation temperature of 28°C and a rotation speed of 200-220 r / min for 48-72 h to obtain a transformation solution.

[0024] The second purpose of this application is to provide 11α,17α-diOH-progesterone prepared by the above method.

[0025] Optionally, in one embodiment of the present application, as Figure 1 , 11α,17α-diOH-progesterone has the following structural formula:

[0026] In this application, the reaction formula for converting progesterone into 11α,17α-diOH-progesterone is as follows: Figure 2 :

[0027]

[0028] Beneficial Effects: The present invention provides a method for converting progesterone to 11α,17α-diOH-progesterone using Gibberella intermedia CA3-1. The method utilizes Gibberella intermedia CA3-1 to directly convert progesterone to 11α,17α-diOH-progesterone. The synthesis of 11α,17α-diOH-progesterone provides a more diverse precursor for the research of steroidal drugs and their intermediates. Furthermore, the C11α and C17α dihydroxylation reaction, as a new type of steroidal hydroxylation reaction, opens up a new pathway for steroidal drug synthesis and has considerable research value and market significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the structural diagram of 11α,17α-diOH-progesterone.

[0030] Figure 2 This is the reaction formula for the conversion of progesterone to 11α,17α-diOH-progesterone.

[0031] Figure 3 、 4 This is the spectrum test result of 11α,17α-diOH-progesterone.

[0032] Figure 5 The purpose of this study was to study the process of converting progesterone (3 g / L) into 11α,17α-diOH-progesterone by Gibberella intermedia CA3-1 strain in fermentation medium. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings and examples. The present invention can be better understood based on the following examples. However, it is readily understood by those skilled in the art that the specific material proportions, process conditions, and results described in the examples are merely illustrative of the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0034] The present application provides a strain for converting progesterone to produce 11α,17α-diOH-progesterone. The strain is Gibberella intermedia CA3-1, which is deposited at the General Microbiology Center of the Chinese Culture Collection of Microorganisms, Chinese Academy of Sciences, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 4903 and a deposit date of May 24, 2011. The strain is classified as Gibberella intermedia.

[0035] In the embodiment of the present application, the method for converting progesterone into 11α,17α-diOH-progesterone using Gibberella sp. CGMCC No. 4903 comprises the following steps:

[0036] (1) Activation culture to prepare seed solution:

[0037] Gibberellic acid CGMCC No. 4903 was used as the production strain, and activated and cultured at 25-35°C and 150-220 r / min to obtain a seed solution, which was then diluted and spread onto a PDA plate (also known as a PDA solid culture medium);

[0038] In one example, the composition and proportion of the PDA solid culture medium are: 200-500 g / L of potatoes, 20-50 g / L of glucose, 2-10 g / L of yeast powder, 10-20 g / L of agar powder, pH 6.5-7.5, and sterilized under high pressure steam at 121°C for 20 minutes;

[0039] (2) Preparation of cell liquid culture of CA3-1 strain:

[0040] Pick a loop of the CA3-1 strain on the PDA solid medium in step (1) and inoculate it into a sterilized 500 mL conical flask containing 50-100 mL seed medium. The culture temperature is 25-35°C and the shaker is placed at a speed of 150-220 r / min for 12-20 h until the middle and late logarithmic growth stage, thereby obtaining a cell liquid culture (i.e., seed culture solution) of the CA3-1 strain.

[0041] In one example, the composition and ratio of the seed culture medium are: glucose 10-30 g / L, yeast powder 5-15 g / L, NaCl 0.5-2 g / L, KH2PO4 1-5 g / L, pH adjusted to 6.5-7.5, and sterilized by high-pressure steam at 121°C for 15-20 minutes;

[0042] (3) Fermentation and culture to prepare bacterial fermentation liquid:

[0043] The cell liquid culture (i.e., seed culture solution) prepared in step (2) is inoculated into the fermentation medium at an inoculum size of 4% to 8% (v / v), and the liquid volume is 25 to 50 mL of fermentation medium in a 250 mL conical flask, and the culture is continued at 25 to 35° C. and a rotation speed of 150 to 220 r / min for 20 to 24 h to obtain a bacterial fermentation liquid;

[0044] In one example, the composition and ratio of the fermentation medium are: glucose 10-30 g / L, yeast powder 5-15 g / L, corn steep liquor 1-5 g / L, NaCl 1-5 g / L, pH adjusted to 6.5-7.5, and sterilized by high-pressure steam at 121° C. for 15-20 min;

[0045] (4) Biotransformation:

[0046] The cultured cells from step (3) were washed with 0.1-0.5 M PBS buffer and then resuspended in fresh 30 mL / 250 mL PBS buffer to prepare resting cells of Gibberella CA3-1. An appropriate amount of the substrate progesterone was accurately weighed and added to the resting cells of Gibberella CA3-1 to a final concentration of 1-5 g / L. The transformation was carried out at a transformation temperature of 28°C and a rotation speed of 200-220 r / min for 48-72 hours to obtain a transformation solution.

[0047] Example 1 Preparation of Seed Liquid of Gibberella intermedia CA3-1 Strain

[0048] Gibberellic acid CGMCC No. 4903 was used as the production strain, and activated and cultured at 30°C and 220 r / min to obtain seed liquid, which was then diluted and coated on a PDA solid culture medium.

[0049] The composition and proportion of the PDA culture medium are as follows: 200 g / L potato, 20 g / L glucose, 5 g / L yeast powder, 20 g / L agar powder, natural pH, and sterilized under high pressure steam at 121°C for 20 min.

[0050] Example 2 Preparation of Cell Liquid Culture of Gibberella intermedia CA3-1 Strain

[0051] The Gibberellic acid CA3-1 strain on the PDA solid medium of Example 1 was picked and inoculated into a 500 mL conical flask containing 100 mL of seed culture medium. The culture was placed on a shaker at 30°C and cultured at a speed of 220 r / min for 24 h to the middle and late stages of logarithmic growth to obtain a cell liquid culture (i.e., seed culture solution) of Gibberellic acid CA3-1.

[0052] The composition and proportion of the seed culture medium are as follows: glucose 10-30 g / L, yeast powder 5-15 g / L, NaCl 0.5-2 g / L, KH2PO4 1-5 g / L; the pH of the culture medium is adjusted to 7.0 before sterilization, and sterilized under high-pressure steam at 121°C for 20 minutes.

[0053] Example 3 Preparation of Fermentation Broth of Gibberella intermedia CA3-1 Strain

[0054] The cell liquid culture of the Gibberellic acid CA3-1 strain prepared in Example 2 was inoculated at an inoculum size of 6% (v / v) into a 250 mL conical flask containing 30 mL of sterilized fermentation medium, and cultured on a shaker at 30°C and 220 r / min for 20 to 24 h to obtain a bacterial fermentation broth.

[0055] The composition and proportion of the fermentation medium are as follows: glucose 10-30 g / L, yeast powder 5-15 g / L, corn steep liquor 1-5 g / L, NaCl 1-5 g / L, pH 7.0, and sterilization under high-pressure steam at 121° C. for 20 minutes.

[0056] Example 4 Gibberella intermedia CA3-1 strain transforms progesterone

[0057] The cultured cells from Example 3 were washed three times with 0.2M PBS buffer and then resuspended in fresh 30 mL / 250 mL PBS buffer to prepare resting cells of Gibberella CA3-1. An appropriate amount of progesterone was accurately weighed and added to the resting cells of Gibberella CA3-1 to achieve a progesterone concentration of 1-5 g / L. The cells were incubated at a conversion temperature of 28°C and a rotation speed of 220 rpm for 48-72 hours to obtain a conversion solution.

[0058] Experimental example: product detection

[0059] The conversion solution obtained in Example 4 above was centrifuged at 8000-12000 r / min for 5-10 minutes, and the supernatant was extracted three times with an equal volume of ethyl acetate. The combined extracts were spun on a rotary evaporator until crystals appeared, and then redissolved with an equal volume of acetonitrile and filtered through a 0.22 μm organic membrane to remove impurities. The filtrate was determined to be 11α,17α-diOH-progesterone by LC-MS, and the content of 11α,17α-diOH-progesterone was analyzed by high performance liquid chromatography. Among them, the spectrum test results of 11α,17α-diOH-progesterone are as follows. Figure 3 As shown in Figure 2, the peak position of product 1 converted from progesterone by Gibberella is consistent with the peak time of 11α,17α-diOH-progesterone; Figure 4 As shown, by LC-MS analysis of the conversion product, an ion fragment with a mass-to-charge ratio of 347.2 was detected, which is consistent with [M+H] + Positive ion mode, that is, Gibberella converts progesterone to produce 11α,17α-diOH-progesterone.

[0060] like Figure 1 , 11α,17α-diOH-progesterone has the following structural formula:

[0061]

[0062] like Figure 5 The figure shows the conversion of progesterone by Gibberella intermedia CA3-1 strain in fermentation medium. When the substrate feed amount is 3g / L, the yield of the dihydroxy product 11α,17α-diOH-progesterone substrate conversion increases slowly from 0 to 30 hours, then increases significantly from 36 to 54 hours, reaching a maximum of 0.626g / L at 54 hours. The product molar yield is 18.9%. After 54 hours, the yield decreases. It is speculated that the decrease in yield may be caused by the metabolism of 11α,17α-diOH-progesterone by the strain into other products. In addition, the same phenomenon occurs when the feed amount is 1g / L and 5g / L.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for converting progesterone into 11α,17α-diOH-progesterone using Gibberella sp., characterized in that: Gibberella is subjected to activation culture and fermentation culture to obtain bacterial cells, which are washed to obtain resting cells. The resting cells are used to convert the substrate progesterone into 11α,17α-diOH-progesterone. The gibberella is Gibberella intermedia CA3-1, which is preserved in the General Microbiology Center of the Chinese Culture Collection of Microorganisms, Chinese Academy of Sciences, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No. 4903 and a preservation date of May 24, 2011. It is classified as Gibberella intermedia.

2. The method according to claim 1, characterized in that The concentration of the substrate progesterone is 1-5 g / L.

3. The method according to claim 2, characterized in that The concentration of the substrate progesterone is 3 g / L.

4. The method according to claim 1, wherein The conversion of the substrate progesterone to 11α,17α-diOH-progesterone includes adding the substrate progesterone to the resting cells of Gibberella sp., and converting at a conversion temperature of 28° C. and a rotation speed of 200-220 r / min for 48-72 hours to obtain a conversion solution.

5. The method according to claim 1, characterized in that The activation culture includes: using Gibberellic acid fungus CGMCC No. 4903 as a production strain, activating and culturing it at 25-35° C. and 150-220 r / min to obtain a seed liquid, and then diluting the seed liquid and applying it to a PDA solid culture medium; picking a ring of CA3-1 strain on the PDA solid culture medium, inoculating it into the seed culture medium, and culturing it at 25-35° C. and 150-220 r / min for 20-24 hours until the middle and late stages of logarithmic growth, thereby obtaining a seed culture liquid of the CA3-1 strain.

6. The method according to claim 5, characterized in that The seed culture medium comprises the following components: 10-30 g / L glucose, 5-15 g / L yeast powder, 0.5-2 g / L NaCl, and 1-5 g / L KH2PO4. The pH value is adjusted to 6.5-7.5, and the medium is sterilized by high-pressure steam at 121° C. for 15-20 minutes.

7. The method according to claim 1, characterized in that The fermentation culture comprises the following steps: inoculating the seed liquid into the fermentation medium at an inoculum amount of 4% to 8% by volume, culturing at a temperature of 25 to 35° C. and a rotation speed of 150 to 220 r / min for 20 to 24 hours to obtain a fermentation liquid.

8. The method according to claim 7, characterized in that The fermentation medium comprises the following components: 10-30 g / L glucose, 5-15 g / L yeast powder, 1-5 g / L corn steep liquor, and 1-5 g / L NaCl. The pH value is adjusted to 6.5-7.5, and the medium is sterilized by high-pressure steam at 121° C. for 15-20 minutes.

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