Method for synthesizing 6 alpha, 11 alpha, 22-trihydroxy-23, 24-dinorchol-4-ene-3-ketone through microorganisms

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for synthesizing 6 alpha, 11 alpha, 22-trihydroxy-23, 24-dinorchol-4-ene-3-ketone through microorganisms. The invention provides absidia coerulea Syph-GBC-2021-005 which is preserved in the China Center for Type Culture Collection, and the preservation number of the absidia coerulea Syph-GBC-2021-005 is CCTCC (China Center for Type Culture Collection) NO: M 2021341. The strain can efficiently convert 4-HBC to synthesize 6 alpha, 11 alpha-dihydroxy-4-HBC, and has the advantages of high conversion rate, high selectivity and high product yield. The method for synthesizing the 6 alpha, 11 alpha-dihydroxy-4-HBC by utilizing the microorganisms, provided by the invention, is high in conversion rate and short in conversion period, provides an important precursor compound for synthesis of a steroid drug, provides a new method for synthesis and development of the steroid drug, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a method for microbial synthesis of 6α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one. Background Art

[0002] Currently, there are more than 400 steroid hormone drugs on the global market, ranking second only to antibiotic drugs. Hydroxylation is one of the important reactions for the functionalization of steroid compounds, which refers to the introduction of a hydroxyl group onto the group of a steroid compound. Hydroxylation can provide intermediates for the chemical synthesis of steroid drugs. By hydroxylation at different sites of steroid drugs, they can have different physiological functions, thereby obtaining potential steroid drug intermediates. In addition, hydroxylation can increase the polarity of steroid compounds, and further enhance the biological activity of the compounds. A large number of studies have shown that hydroxylated steroids generally exhibit higher biological activity compared to non-hydroxylated steroids with lower polarity.

[0003] The traditional production process of steroid compounds uses diosgenin as the raw material and undergoes multiple-step chemical synthesis. Some researchers have also directly extracted steroid intermediates or non-steroid compounds from animal and plant tissue fluids and then carried out multiple-step chemical synthesis. However, the process is complex, with low yield, high energy consumption, and serious environmental pollution, and it cannot meet the sustainable development of the steroid drug industry. Due to the increasing demand for steroid hormone drugs at present, the preparation of steroid compounds by microbial transformation has become a research hotspot. Microbial transformation has the advantages of high regioselectivity and stereoselectivity, strong specificity, mild reaction conditions, short reaction time, high yield, green and safe, etc., avoiding the adverse effects such as time-consuming, expensive, and environmentally unfriendly brought by the chemical synthesis method for preparing steroid drugs, and is suitable for popularization and application in the field of steroid drug preparation.

[0004] 22-Hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) is an important intermediate for the synthesis of progestins and corticosteroids such as progesterone. Although 6α / β, 7α / β, 8α / β, 9α / β, 11α / β, 12α / β, 14α / β, and 15α / β are all reported hydroxylation sites of steroid compounds, currently, the hydroxylated products of steroid hormone intermediates are commonly found at 7α, 7β, 9α, 14α, and 15α, while the hydroxylation at 6α and 11α has been rarely reported. Therefore, using microorganisms with high conversion efficiency to hydroxylate 4-HBC to form the novel bioactive steroid compound 6α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one (6α,11α-dihydroxy-4-HBC) not only provides an important precursor compound for steroid drug synthesis but also opens up a new route for steroid drug synthesis. Summary of the Invention

[0005] The present invention provides a microorganism for efficiently converting 22-hydroxy-23,24-bisnorchol-4-ene-3-one into 6α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one, and a method for synthesizing 6α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one using the microorganism.

[0006] The present invention has screened and obtained Absidia coerulea ( Absidia coerulea ), Syphu-GBC-2021-005, and found that this strain can efficiently and highly specifically hydroxylate 22-hydroxy-23,24-bisnorchol-4-ene-3-one at the 6α and 11α sites to synthesize 6α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one. The reaction formula is as Figure 1 shown. Based on this strain, the present invention further provides a method for synthesizing 6α,11α,22-trihydroxy-23,24-bisnorchol-4-ene-3-one using this strain.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] The present invention provides Absidia coerulea ( Absidia coerulea ), Syphu-GBC-2021-005, which was deposited at the China Center for Type Culture Collection (abbreviation: CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code 430072) on April 9, 2021. The taxonomic name is Absidia coerulea Absidia coerulea , and the deposit number is CCTCC NO: M2021341.

[0009] The present invention provides a microbial preparation, which contains the above-mentioned Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005.

[0010] Preferably, in the microbial preparation, Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005 exists in the form of viable bacteria.

[0011] The microbial preparation can be a liquid preparation or a solid preparation.

[0012] In addition to Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005, the microbial preparation may further contain excipients allowed in the field of microbial preparations, including but not limited to carriers, lyoprotectants, etc.

[0013] The above-mentioned microbial preparation can be prepared by conventional technical means, with or without excipients allowed in the field of microbial preparations.

[0014] The present invention also provides a preparation method of the above-mentioned microbial preparation, and the method includes: the step of culturing Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005. The culturing is preferably carried out at 25-30 °C.

[0015] Based on the function of Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005, the present invention provides the following applications of this strain: The present invention provides the above-mentioned Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005 or the microbial preparation for use in the biotransformation of 22-hydroxy-23,24-bisnorchol-4-en-3-one to prepare 6α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one.

[0016] The present invention provides the above-mentioned Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005 or the microbial preparation for use in the preparation of a biocatalyst for catalyzing the formation of 6α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one from 22-hydroxy-23,24-bisnorchol-4-en-3-one.

[0017] The present invention provides a method for synthesizing 6α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one using microorganisms, and the method includes: using 22-hydroxy-23,24-bisnorchol-4-en-3-one as a substrate, and through the above-mentioned Absidia coerulea (Absidia coerulea )Perform biotransformation on Syphu - GBC - 2021 - 005 to obtain 6α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one.

[0018] The above - mentioned method includes: inoculating the blue mucor ( Absidia coerulea )Syphu - GBC - 2021 - 005 into a transformation medium containing 22 - hydroxy - 23,24 - bisnorchola - 4 - en - 3 - one for biotransformation.

[0019] Preferably, in the transformation medium, the concentration of 22 - hydroxy - 23,24 - bisnorchola - 4 - en - 3 - one is 0.5 - 20 g / L.

[0020] More preferably, in the transformation medium, the concentration of 22 - hydroxy - 23,24 - bisnorchola - 4 - en - 3 - one is 0.5 - 10 g / L; even more preferably 0.5 - 5 g / L; even more preferably 1 - 3 g / L.

[0021] The 22 - hydroxy - 23,24 - bisnorchola - 4 - en - 3 - one can be dissolved with methanol as a cosolvent.

[0022] Preferably, the transformation medium further comprises the following components: sucrose 25 - 35 g / L, yeast extract 5 - 15 g / L, corn steep liquor 5 - 15 g / L, K2HPO4 1 - 3 g / L, KH2PO4 1 - 2 g / L, MgSO4 0.4 - 0.6 g / L, FeSO4 0.04 - 0.06 g / L.

[0023] The pH of the transformation medium is preferably 6.5 - 7.0.

[0024] Preferably, the temperature of the biotransformation is 25 - 30 °C, and / or the pH is 6.5 - 7.0.

[0025] Preferably, the transformation is carried out at 25 - 30 °C and a rotation speed of 180 - 250 r / min for 72 - 120 h.

[0026] Preferably, the inoculation amount of the blue mucor ( Absidia coerulea )Syphu - GBC - 2021 - 005 is 5% - 15%. Even more preferably 8% - 12%.

[0027] Preferably, inoculate the seed liquid of the blue mucor ( Absidia coerulea )Syphu - GBC - 2021 - 005 into a transformation medium containing 22 - hydroxy - 23,24 - bisnorchola - 4 - en - 3 - one for biotransformation.

[0028] The preparation of the seed liquid includes the following steps: Take a fresh PDA slant of Absidia coerulea ( Absidia coerulea ), Syphu-GBC-2021-005, scrape the spores and inoculate them into the seed medium, and culture for 45 - 52 h to obtain the seed liquid.

[0029] Among them, the seed medium includes the following components: potato starch 40 - 50 g / L, yeast extract 2 - 4 g / L, corn steep liquor 5 - 15 g / L, CaCO3 2 - 4 g / L, MgSO4 0.4 - 0.6 g / L, FeSO4 0.04 - 0.06 g / L.

[0030] The pH of the transformation medium is preferably 6.5 - 7.0.

[0031] The temperature of the seed culture is preferably 25 - 35 °C.

[0032] The above method further includes: after the transformation is completed, collecting the transformation liquid and extracting 6α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one therefrom.

[0033] The present invention also provides the application of 6α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one in the preparation of anti - inflammatory drugs.

[0034] The beneficial effects of the present invention at least include: Absidia coerulea ( Absidia coerulea ), CCTCC NO: M 2021341 provided by the present invention can efficiently transform 4 - HBC to synthesize 6α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one, having the advantages of high conversion rate, high selectivity, and high product yield. 6α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one has anti - inflammatory activity and can be used as an intermediate for synthesizing steroid drugs or for developing anti - inflammatory drugs.

[0035] The method provided by the present invention for hydroxylating 4 - HBC with Absidia coerulea ( Absidia coerulea ), CCTCC NO: M 2021341 to synthesize the novel steroid compound 6α,11α,22 - trihydroxy - 23,24 - bisnorchola - 4 - en - 3 - one with biological activity has a conversion rate (product yield) that can reach 72%, and the transformation period is short. This method has the advantages of simple operation, low cost, and high yield. It not only provides an important precursor compound for steroid drug synthesis, but also provides a new method for the synthesis and development of steroid drugs or anti - inflammatory drugs, and has good application prospects and economic value. Description of the Drawings

[0036] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 It is the reaction formula for the conversion of 4-HBC to synthesize 6α,11α-dihydroxy-4-HBC in the invention content.

[0038] Figure 2 It is the key HMBC correlation of the conversion product of Absidia coerulea CCTCC NO: M 2021341 in Example 2 of the present invention.

[0039] Figure 3 It is the HMBC spectrum of the conversion product of Absidia coerulea CCTCC NO: M 2021341 in Example 2 of the present invention.

[0040] Figure 4 It is the NOESY spectrum of the conversion product of Absidia coerulea CCTCC NO: M 2021341 in Example 2 of the present invention. Detailed implementation manners

[0041] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in combination with the drawings in the present invention. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0042] Unless otherwise specified, the chemical reagents used in the following examples are all conventional commercially available reagents, and the technical means used are conventional means well known to those skilled in the art.

[0043] The medium formulations used in the following examples are as follows: Potato medium (g / L): potato 200.00, glucose 20.00. After peeling and cutting the potato, weigh it, add distilled water and boil for 0.5 h, then filter the residue using double-layer gauze, add glucose to dissolve and make up the volume, and the pH is natural. Add agar (20.00 g / L) to the medium to obtain the PDA medium.

[0044] Seed medium (g / L): potato starch 45.00, yeast extract 3.00, corn steep liquor 10.00, CaCO3 3.00, MgSO4 0.50, FeSO4 0.05. Stir and dissolve potato starch with a small amount of distilled water, gelatinize it, then add other components in sequence and dissolve them. After volume fixation, adjust the pH to 6.5 and dispense into 50 mL / 250 mL conical flasks.

[0045] Transformation medium (g / L): sucrose 30.00, yeast extract 10.00, corn steep liquor 10.00, K2HPO4 2.00, KH2PO4 1.60, MgSO4 0.50, FeSO4 0.05. After volume fixation, adjust the pH to 6.5 and dispense into 50 mL / 250 mL conical flasks.

[0046] The above media were autoclaved at 68.95 kPa and 115.0 °C for 30 min.

[0047] Example 1 Obtaining of microorganisms for transforming 4-HBC to synthesize 6α,11α-dihydroxy-4-HBC In the present invention, a microorganism capable of transforming 4-HBC to synthesize 6α,11α-dihydroxy-4-HBC was obtained. After identification, it was Absidia coerulea ( Absidia coerulea ), and it was named Syphu-GBC-2021-005. Absidia coerulea Syphu-GBC-2021-005 was deposited at the China Center for Type Culture Collection (abbreviation: CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code 430072) on April 9, 2021. Its taxonomic name was Absidia coerulea, and the deposit number was CCTCC NO: M 2021341. In the present invention, Absidia coerulea Syphu-GBC-2021-005 is also referred to as Absidia coerulea CCTCC NO: M 2021341.

[0048] Example 2 Biocatalytic synthesis of 6α, 11α-dihydroxy-4-HBC by Absidia coerulea CCTCC NO: M 2021341 Using Absidia coerulea CCTCC NO: M 2021341 to biocatalytically synthesize 6α, 11α-dihydroxy-4-HBC from 4-HBC, the specific method is as follows: 1. Preparation of seed liquid Take a fresh PDA slant of Absidia coerulea CCTCC NO: M 2021341, scrape two loops of spores with a sterile inoculation loop, and inoculate them into the seed medium. Incubate at 28 °C and 220 r / min for 48 h to obtain the seed liquid.

[0049] 2. Biotransformation Accurately weigh 4-HBC and put it into a sterilized centrifuge tube. Add methanol to fully dissolve it and prepare a substrate stock solution with a concentration of 50 mg / mL. Using aseptic operation, pipette the stock solution into the transformation medium so that the final concentration of 4-HBC is 1 g / L. Inoculate the seed solution from Step 1 into the above transformation medium containing 4-HBC at an inoculation amount of 10% (v / v). Additionally, set up a substrate control group and a strain blank control group. Incubate at 28 °C and 220 r / min for 120 h to obtain the transformation solution.

[0050] 3. Treatment of Transformation Products Pour the transformation solution into a centrifuge tube, add ethyl acetate to make it balanced, and centrifuge at 3000 rpm for 10 min. After centrifugation, pour the supernatant into a separatory funnel. Add an equal volume of ethyl acetate to the centrifuge tube, tighten the tube cap and wash the bacterial cells thoroughly. Then pour the organic phase into the separatory funnel, shake well, and let it stand for 15 min. Repeat the above extraction process 3 times. Combine the upper organic phases and perform rotary evaporation under reduced pressure. After the liquid is completely evaporated, add 5 mL of methanol to redissolve it in an Eppendorf tube, and add an appropriate amount of anhydrous magnesium sulfate to absorb water and dry it, thus obtaining the sample of the transformation product to be detected.

[0051] 4. Identification of Microbial Transformation Products Dissolve the product sample prepared in Step 3 in deuterated chloroform and perform mass spectrometry (MS), nuclear magnetic resonance (NMR), HMBC, HMQC, and NOESY detections to analyze the structure of the product.

[0052] According to the analysis of the mass spectrometry results, MS (ESI) m / z 363.25, the relative molecular mass of the transformation product is calculated to be 362.25. Compared with the relative molecular mass of the substrate 4-HBC (C 22 H 34 O2) which is 330.50, it increases by 32, suggesting an increase of 2 oxygen atoms. The molecular formula is C 22 H 34 O4.

[0053] 1 In the 1H NMR (600 MHz, CDCl3) spectrum, two methyl groups attached to quaternary carbons are given δ H 0.80 (3H, s), 1.51 (3H, s), one methyl group attached to a tertiary carbon δ H 1.05 (3H, d, J J = 6.6 Hz), one proton signal on a double bond δ H 5.81 (1H, s), and one hydroxymethyl group attached to a tertiary carbon δH 3.37 (1H, dd, J J = 10.5, 2.9 Hz), 3.64 (1H, dd, J J = 10.4, 6.7 Hz), which are characteristic proton signals of 4-HBC. 13 The 13C NMR (150 MHz, CDCl3) spectrum shows a total of 22 carbon signals. Combining the analysis of the HMQC spectrum, it is inferred that the compound contains 3 methyl groups ( δ C 13.8, 17.2, 20.7), 7 methylene groups ( δ C 68.0, 39.5, 39.4, 34.7, 34.4, 28.9, 28.2), 7 tertiary carbons ( δ C 73.7, 69.4, 59.7, 55.5, 52.4, 38.8, 37.9) and 2 quaternary carbons ( δ C 39.0, 43.3), 1 carbonyl carbon ( δ C 200.8), 2 double bond carbons ( δ C 168.2, 127.4).

[0054] By comparing the 1 H and 13 13C NMR data (Table 1) of the transformation product and the spectrum of 4-HBC, it can be seen that the two compounds are extremely similar and have the same parent nucleus. The difference is that the product has two fewer methylene carbon signals than 4-HBC and has δ C 73.7 (C-6) and 69.4 (C-11) more. This indicates that the 6th and 11th positions of the product may be substituted by hydroxyl groups. Although there are no relevant carbon signals for H-6 and 11, the substitution at the 6th position can be confirmed by the correlation between δ H 5.81 (H-4) and δ C 73.3 (C-6) in the HMBC spectrum. Similarly, δ H 1.05 (H-9), δ H 2.36 (H-12) and δ H 1.29 (H-17) and δ C 69.4 (C-11) confirm the substitution of the 11th position by a hydroxyl group. The above spectra are shown in Figure 2 andFigure 3 。

[0055] Table 1 1 H (600 MHz) and 13 13C NMR (150 MHz) data (CDCl3)

[0056] The relative configuration of the transformation product was determined by the NOESY spectrum, see Figure 4 。There are NOE correlations between H-11 / H-18 and H-6 / H-8, indicating that the configuration is 6α, 11α.

[0057] Based on the above results, it can be concluded that the product of the transformation of 4-HBC by Absidia coerulea CCTCC NO: M 2021341 is 6α, 11α-dihydroxy-4-HBC.

[0058] The results of the biocatalysis experiment showed that the conversion yield (i.e., the product yield) of the biocatalytic synthesis of 6α,11α-dihydroxy-4-HBC using Absidia coerulea CCTCC NO: M 2021341 could reach 71.94%.

[0059] Example 3 Pharmacological effects of 6α, 11α-dihydroxy-4-HBC Using the conventional LPS-induced RAW264.7 cell model in the art, the in vitro anti-inflammatory activity of 6α, 11α-dihydroxy-4-HBC prepared in Example 2 above was evaluated. A blank control group, an LPS model group (treated with LPS), a positive control group (treated with LPS + 50 µM L-arginine), and an experimental group (treated with LPS + 50 µM 6α, 11α-dihydroxy-4-HBC) were set up respectively. The cell viability and NO production of each group were detected. After experimental determination, compared with the blank control group, at a concentration of 50 µM 6α, 11α-dihydroxy-4-HBC, the cell viability was 96.13%. The detection results of NO production showed that compared with the LPS model group, the NO production inhibition rate of the positive control group was 55.83%, and the NO production inhibition rate of the experimental group reached 48.63%, indicating that 6α, 11α-dihydroxy-4-HBC has a strong inhibitory effect on NO production in the LPS-induced RAW264.7 cell inflammation model and can be used as a candidate compound for the treatment of inflammation.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Absidia coerulea ( Absidia coerulea ), Syphu-GBC-2021-005, characterized in that It is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M 2021341.

2. A microbial preparation, characterized in that, The microbial preparation contains Absidia coerulea described in claim 1 ( Absidia coerulea ) Syphu-GBC-2021-005.

3. Use of Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005 or the microbial preparation according to claim 2 in the biotransformation of 22-hydroxy-23,24-bisnorchol-4-en-3-one as a substrate to prepare 6α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one.

4. Use of Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005 or the microbial preparation according to claim 2 in the preparation of a biocatalyst for catalyzing the formation of 6α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one from 22-hydroxy-23,24-bisnorchola-4-en-3-one.

5. A method for synthesizing 6α,11α,22-trihydroxy-23,24-dinorchola-4-en-3-one using microorganisms, characterized in that, The method includes: using 22-hydroxy-23,24-bisnorchol-4-en-3-one as a substrate, and performing biotransformation with the blue mold Absidia coerulea (Syphu-GBC-2021-005) described in claim 1 to obtain 6α,11α,22-trihydroxy-23,24-bisnorchol-4-en-3-one.

6. The method according to claim 5, characterized in that, The method includes: inoculating the Aspergillus fumigatus blue ( Absidia coerulea ) Syphu-GBC-2021-005 into a transformation medium containing 22-hydroxy-23,24-bisnorchol-4-en-3-one for biotransformation.

7. The method according to claim 6, characterized in that, In the conversion medium, the concentration of 22-hydroxy-23,24-bisnorchola-4-en-3-one is 0.5 - 20 g / L.

8. The method according to claim 6, characterized in that, The conversion medium further comprises the following components: sucrose 25 - 35 g / L, yeast extract 5 - 15 g / L, corn steep liquor 5 - 15 g / L, K2HPO4 1 - 3 g / L, KH2PO4 1 - 2 g / L, MgSO4 0.4 - 0.6 g / L, FeSO4 0.04 - 0.06 g / L; And / or, the temperature of the bioconversion is 25 - 30 °C, and / or, the pH is 6.5 - 7.

0.

9. The method according to any one of claims 6 to 8, characterized in that The inoculation amount of the said Absidia coerulea ( Absidia coerulea ) Syphu-GBC-2021-005 is 5%-15%. Use of 10.6α,11α,22-trihydroxy-23,24-bisnorchola-4-en-3-one in the preparation of anti-inflammatory drugs.

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