Mycobacterium neogoldensis and its use in preparing steroid drug intermediates

Abstract

The present invention provides a new Mycobacterium aureum strain and its use in preparing steroid drug intermediates. The deposit number of the new Mycobacterium aureum strain is CGMCC No. 33600. The present invention also provides a method for preparing steroid drug intermediates based on the new Mycobacterium aureum strain. Compared with the prior art, the method of the present invention has mild reaction conditions, a short synthesis route, a wide range of raw material sources, minimal environmental pollution, a high yield of the target product of over 98%, and low impurity content, enabling efficient production of steroid drug intermediates.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to Mycobacterium neoaureum and its use in preparing steroid drug intermediates. Background Art

[0002] Steroidal compounds, also known as steroids, are a class of substances important for maintaining the vital functions of organisms and are widely present in all plants, animals, and most microorganisms. Steroidal compounds include cholesterol, bile acid, ergosterol, phytosterols, vitamin D, and various steroid hormones. Cholesterol is widely present in animals and is an essential substance for animal tissue cells. It affects cell membrane fluidity, cell proliferation, and differentiation, and is extremely important for maintaining the normal physiological state of cells. It is also the raw material for the synthesis of bile acid and vitamin D. Bile acid, as a sterol, is an important component of human digestive juices, emulsifying fat and promoting the digestive system's absorption of fat-soluble substances. Phytosterols and ergosterol are important components of plant and fungal cell membranes, respectively, and play an important role in ensuring membrane integrity, fluidity, cell viability, the activity of membrane-bound enzymes, and the transport of substances inside and outside cells.

[0003] Steroid drugs are widely used clinically, primarily for anti-toxic, anti-inflammatory, anti-shock, and anti-allergic effects. They can not only treat or alleviate anaphylactic shock, but are also used to treat other endocrine diseases such as Addison's disease. They have also become important adjunctive therapies for breast and prostate cancer. There is a huge global demand for steroid drugs each year, with over 300 approved for clinical use, and this number continues to grow, accounting for approximately 10% of the world's total pharmaceutical product output.

[0004] Currently, one method for producing steroidal drugs uses plant saponins as starting materials, chemically converting them into the key intermediate 16-pregnadienolone (16-DPA), which is then used for chemical synthesis to produce various steroidal drugs. This method is complex, costly, low-yield, and highly polluting. Another method uses plant sterols as starting materials and ferments them to produce side-chain degradation products such as androst-4-ene-3,17-dione (AD), 9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD), androst-1,4-diene-3,17-dione (ADD), or 10R,13S-20-hydroxymethylpregnadien-3-one (PHM). These side-chain degradation products are then used as key intermediates to prepare various steroidal hormone drugs through chemical and biological methods. Microbial conversion of plant sterols is a highly specific, efficient, low-cost, mild reaction conditions, and low-pollution method for producing steroidal drugs.

[0005] Early studies revealed that some microorganisms, such as Nocardia, Pseudomonas, Mycobacterium, and Arthrobacter, can utilize sterols as their sole carbon source for growth. The prerequisite for using microorganisms to convert plant sterols into steroidal drug intermediates is to alter the microbial metabolic pathways. As early as the 1980s and 1990s, researchers had obtained mutant strains that produced steroidal intermediates by altering the metabolic pathways of microorganisms that degrade steroids. The metabolites accumulated by different mutant strains were then used to identify possible metabolic pathways.

[0006] Steroid intermediate-producing bacteria are typically obtained through chemical or physical mutagenesis and screening, and the choice of screening method directly affects the screening efficiency and the properties of excellent mutants. Common starting strains include Mycobacterium, Nocardia, Rhodococcus, and Pseudomonas, with Mycobacterium being the most widely used. However, due to the lack of a complete understanding of the microbial metabolism and metabolic mechanisms of sterols, all genes involved in metabolism have not yet been fully identified. To date, engineered bacteria constructed entirely using molecular biology methods still suffer from metabolic imbalances and high impurity levels, which makes separation and purification difficult and leads to reduced product yields. Summary of the Invention

[0007] To address the above issues, the present invention aims to provide a new Mycobacterium auris and its use in the preparation of steroidal drug intermediates. The present invention also provides a method for preparing steroidal drug intermediates based on the provided new Mycobacterium auris. Compared with the prior art, the new Mycobacterium auris of the present invention can convert phytosterols into steroidal drug intermediates (PHMs) with a high conversion rate and low impurities. A qualified product can be obtained in a single extraction step, significantly simplifying the extraction and purification process.

[0008] The above object of the present invention is achieved by providing the following technical solutions:

[0009] In the first aspect, the present invention provides a new Mycobacterium aureus ( Mycobacterium neoaurum ), recorded as BT104-1, and its preservation number is CGMCC No. 33600.

[0010] In a second aspect, the present invention provides use of the new Mycobacterium aureus according to the first aspect of the present invention in preparing a steroid drug intermediate.

[0011] According to some embodiments of the present invention, the steroid drug intermediate is 10R,13S-20-hydroxymethylpregnane-4-ene-3-one (PHM).

[0012] The strain BT104-1 has the following properties:

[0013] 1. Colony morphological characteristics:

[0014] The strain of the present invention is cultured and grown on a nutrient solid culture medium at 30° C., and after 3-5 days, gray colonies with a diameter of about 3-10 mm are obtained. The colonies are irregularly round and have a dry surface.

[0015] 2. Morphological characteristics of strains:

[0016] like Figure 1 As shown, the strain of the present invention appears in a rod shape under a microscope, which is consistent with the microscopic morphology of Mycobacterium.

[0017] 3. Physiological and biochemical characteristics:

[0018] The bacterial strain of the present invention has a culture temperature of 28-35° C., an optimum growth temperature of 30° C., and grows better under the condition of a pH of 7.0-7.6.

[0019] 4. Nutritional characteristics:

[0020] The strain of the present invention does not require special nutrients, is cultured using a basic culture medium, and is obligately aerobic.

[0021] Wherein, the basal culture medium is a nutrient protein culture medium.

[0022] In a third aspect, the present invention provides a method for preparing a steroid drug intermediate, comprising: using the new Mycobacterium aureus according to the first aspect of the present invention to convert phytosterols into a steroid drug intermediate.

[0023] According to some embodiments of the invention, the method comprises the following steps:

[0024] (1) inoculating the Mycobacterium neoaureum into a seed culture medium for culturing to obtain a seed culture;

[0025] (2) inoculating the seed culture into a fermentation medium for culturing to obtain a fermentation liquid;

[0026] (3) Purifying and separating the fermentation broth.

[0027] According to some embodiments of the present invention, in step (1), the seed culture medium comprises: 5-15 g / L peptone, 1-5 g / L yeast extract, 1-10 g / L glycerol, 1-10 g / L Tween 80, and a pH of 7.0-7.6.

[0028] According to some embodiments of the present invention, in step (1), the culturing is carried out under the following conditions: temperature of 28-35°C, preferably 30-32°C; time of 2-4 days, preferably 2-3 days; rotation speed of 200-250 rpm, preferably 210-230 rpm.

[0029] According to some embodiments of the present invention, in step (2), the fermentation medium comprises: 5-20 g / L corn steep liquor powder, 5-20 g / L soybean cake powder, 1-5 g / L yeast extract, 2-10 g / L glycerol, 0.5-3.0 g / L glucose, 0.1-0.3 g / L citric acid, 0.005-0.015 g / L ammonium ferric citrate, 0.5-3.0 g / L K2HPO4, 0.01-0.10 g / L MgSO4·7H2O, 0.5-3 g / L (NH4)2SO4, 10-150 g / L phytosterols and 1-10 g / L Tween 80, with a pH of 7.0-7.6.

[0030] According to some embodiments of the present invention, in step (2), no vegetable oil is added.

[0031] According to some embodiments of the present invention, in step (2), the culture is shaking culture or aerobic submerged culture in a fermenter.

[0032] Preferably, the shaking culture is carried out under the following conditions: temperature of 28-35°C, preferably 30-32°C; rotation speed of 200-250 rpm, preferably 210-230 rpm; time of 4-10 days, preferably 6-10 days.

[0033] Preferably, the aerobic submerged culture in the fermentation tank is carried out under the following conditions: temperature of 28-35°C, preferably 30-32°C; time of 4-10 days, preferably 6-10 days; rotation speed of 400-500 rpm, preferably 440-460 rpm; fermentation tank pressure of 0.05-0.1 MPa, preferably 0.07-0.09 MPa, ventilation type of air, ventilation volume of 1-5 L / min, preferably 2-3 L / min; culture volume of 10-20 L, preferably 13-18 L.

[0034] According to some embodiments of the present invention, in step (3), the purification and separation comprises: centrifuging the fermentation broth, extracting the obtained precipitate with methanol, then centrifuging the extract, distilling the obtained supernatant under reduced pressure, and then adding water for crystallization.

[0035] Preferably, the purification and separation comprises: centrifuging the fermentation broth, adding methanol to the obtained precipitate, heating to reflux, then centrifuging the extract, filtering the obtained supernatant and performing vacuum distillation, then adding water and cooling to crystallize, and finally filtering with suction, rinsing the filter cake with a methanol-water solution with a volume ratio of 20-40%, and drying under reduced pressure.

[0036] Compared with the prior art, the present invention has at least the following beneficial effects:

[0037] The present invention provides a new Mycobacterium aureum BT104-1, which can be used to convert phytosterols into steroid drug intermediates PHM with a high conversion rate and few impurities. The yield of PHM can reach more than 98%, and a qualified product can be obtained through a one-step extraction, greatly simplifying the extraction and purification process.

[0038] The present invention also provides a method for producing a steroidal drug intermediate based on the novel Mycobacterium aureus. Compared to the prior art, the method provided by the present invention implements an oil-free fermentation process and uses a single solvent for separation and purification, resulting in a simple process and high yield. Furthermore, the method of the present invention has mild reaction conditions, a short synthesis route, a wide range of raw material sources, and minimal environmental pollution. The target product prepared by this method has a yield of over 98%, is low in impurities, and can effectively produce a steroidal drug intermediate. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0040] Figure 1 The bacterial morphology of strain BT104-1 under a microscope (1000 times);

[0041] Figure 2 is the HPLC spectrum of PHM standard;

[0042] Figure 3 The HPLC spectrum of the PHM fermentation broth prepared according to the method of Example 2 of the present invention.

[0043] Deposit of biological materials

[0044] The new Mycobacterium neoaurum BT104-1 provided by the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on March 17, 2025. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 33600 and the classification name: Mycobacterium neoaurum.

[0045] The Mycobacterium neoaurum MN HIL-4 used in the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC). The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain has a deposit number of CGMCC No. 17948 and a deposit date of June 17, 2019. The classification name is Mycobacterium neoaurum. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The strains, plasmids, kits, etc. used in the following examples are commercially available products unless otherwise specified.

[0048] Some of the materials used in the following examples are as follows:

[0049] NA medium: peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, agar 15 g / L, adjust the pH to 7.0.

[0050] Phytosterols were purchased from Spring Valley Biological Products Co., Ltd.

[0051] Phosphate buffer: NaH2PO4·2H2O concentration is 3.3 g / L, Na2HPO4·12H2O concentration is 10.3 g / L.

[0052] Example 1: Acquisition of new Mycobacterium aureus BT104-1

[0053] The new Mycobacterium aureus MN HIL-4 was used as the starting strain and the new Mycobacterium aureus of the present invention was prepared by the following method:

[0054] 1) Weigh 6 mg of nitrosoguanidine (NTG, purchased from Sigma) into a sterile centrifuge tube, add 0.05 mL of acetone to aid dissolution, and then add 1 mL of 0.2 mM phosphate buffer, pH 6.0, to completely dissolve the NTG to obtain a NTG solution.

[0055] 2) Prepare Mycobacterium neogoldensis MN HIL-4 at a concentration of 10 8-9 5 mL of bacterial suspension with a concentration of 10 cells / mL was mixed with the above-mentioned nitrosoguanidine solution to obtain a mixed solution;

[0056] 3) Immediately place the mixture obtained in step 2) in a 30°C water bath and shake for 10 minutes to 1 hour;

[0057] 4) Centrifuge the mixture treated in step 3) to collect the bacterial cells, then wash the cells twice with 5 mL of phosphate buffer to terminate the NTG mutagenesis. Finally, add 5 mL of sterile saline to the centrifuge tube and shake well.

[0058] 5) Diluting the mutagenized bacterial suspension obtained in step 4) 10-fold, spreading the solution on a NA medium plate, and culturing for 4-6 days to obtain single colonies;

[0059] 6) Pick a single colony to conduct a transformation test using phytosterol as a substrate (for specific steps, see Example 2), and screen out a mutant strain of Mycobacterium neoaureum BT104-1 with high PHM production.

[0060] The 16S RNA gene sequence of this strain (SEQ ID NO: 1) is shown below:

[0061]

[0062] The strain was identified as Mycobacterium neogoldensis.

[0063] Example 2: PHM fermenter conversion experiment

[0064] 1) The experimental strain BT104-1 was inoculated into 300 mL of seed culture medium in a 1000 mL Erlenmeyer flask and cultured with shaking at 220 rpm at 32°C for 48 h to obtain a seed culture;

[0065] The composition of the seed culture medium is as follows: 10 g / L peptone, 3 g / L yeast extract, 5 g / L glycerol, 5 g / L Tween 80, and 5 g / L, and the pH is adjusted to 7.0.

[0066] 2) inoculating the seed culture prepared in step 1) into 15 L of in-situ sterilized fermentation medium in a 30 L fermentor for aerobic submerged culture in the fermentor under the following conditions: temperature of 32° C., time of 6-10 days, incubation in the dark, rotation speed of 450 rpm, fermentor pressure of 0.08 MPa, aeration type of air at a rate of 2-3 L / min; sampling and observing experimental results daily, stopping fermentation when the residual phytosterol content was less than 1% (mass percentage), and replenishing water to a total volume of 15 L;

[0067] The fermentation medium was composed of 10 g / L corn steep liquor powder, 10 g / L soybean cake powder, 3 g / L yeast extract, 5 g / L glycerol, 1.0 g / L glucose, 0.2 g / L citric acid, 0.01 g / L ammonium ferric citrate, 1.0 g / L K2HPO4, 0.05 g / L MgSO4·7H2O, 1.5 g / L (NH4)2SO4, 100 g / L phytosterols, and 4 g / L Tween 80. The pH was adjusted to 7.0.

[0068] 3) taking the fermentation broth from step 2) and centrifuging at 4000 rpm for 10 min to obtain a precipitate;

[0069] 4) Weigh the precipitate obtained in step 3) and extract it with 5 volumes of methanol. Heat and reflux for 10 minutes. Centrifuge the resulting extract at 4000 rpm for 10 minutes. Filter the supernatant through a 0.22 µm organic filter membrane and transfer it to a distillation flask.

[0070] The filtrate in the distillation flask is distilled under reduced pressure to crystallize, and the distillation is continued until the methanol content in the distillate is less than 3% (mass percentage). Deionized water is added in an amount three times the weight of the residue in the distillation flask at this time, and the temperature is lowered to 5°C for crystallization, and maintained for 1-2 hours; then, the filter cake is filtered and rinsed with a 30% (volume ratio) methanol aqueous solution at -5°C, and then dried under reduced pressure at 45°C for 2 hours to obtain the PHM product.

[0071] Fermentation broth product analysis: Use a wide-mouth pipette to take 1 mL of the fermentation broth from step 2) and add 10 mL of ethyl acetate. Vortex intermittently for 10 minutes, then centrifuge at 6000 rpm. Place 1 mL of the supernatant in a 20 mL scintillation vial to evaporate the solvent. Reconstitute with 10 mL of acetonitrile and filter through a 0.22 µm organic membrane to remove impurities. The filtrate is analyzed by HPLC for PHM content. Calculate the PHM yield using the following formula. Calculate the conversion rate based on the PHM content in the fermentation broth and the phytosterol feed amount.

[0072] Product testing: Weigh the product obtained in step 4), dissolve it in acetonitrile, prepare a 1 mg / mL solution, filter it through a 0.22 µm organic membrane to remove impurities, and analyze the PHM content by high-performance liquid chromatography.

[0073] The HPLC column was Agilent ZORBAX SB C18 (5ORBAX, 4.6×150 mm), and the chromatographic conditions were as follows: gradient elution, water as phase A, methanol as phase B, a flow rate of 1.0 mL / min, 0-15.0 min: 30-95% volume fraction of phase B, the rest being phase A; 15.0-30.0 min: 95% volume fraction of phase B, the rest being phase A; equilibrium time 5 min.

[0074] The yield of PHM is calculated as follows:

[0075] PHM yield (%) = PHM peak area / total peak area × 100%;

[0076] The calculation formula for PHM concentration is as follows:

[0077]

[0078] Wherein, Ax is the peak area of PHM in the test sample; Ar is the peak area of PHM in the reference sample; Cr is the concentration of PHM in the reference sample (mg / mL).

[0079] like Figure 2 and Figure 3 As shown, Figure 2 is the liquid phase spectrum of PHM standard; Figure 3 HPLC spectrum of PHM fermentation broth.

[0080] from Figure 3 It can be seen that according to the method of the present invention, on the 7th day, the PHM yield of the fermentation broth reached 99.47%; the PHM concentration of the fermentation broth reached 62.01 mg / ml, and the molar conversion rate of phytosterols to PHM reached 77.98% (the molecular weight of phytosterols was 415).

[0081] After testing, on the 7th day, the PHM content in the PHM product was 98.7%.

[0082] In phytosterol conversion experiments using the original strain MN HIL-4, the main product was δ-lactone (HIL), accounting for 99.29% of the total product, and no PHM was found in the product (see Example 3 of patent application CN111500498A). In contrast, the new Mycobacterium aureum BT104-1 of the present invention selectively converts phytosterols into steroidal drug intermediates, PHM, with high conversion rates and low impurities.

[0083] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art who, without departing from the scope of the present invention, may make slight changes or modifications to the above-disclosed technical contents to obtain equivalent or equivalent embodiments falls within the scope of the present invention.

Claims

1. A new Mycobacterium aureum ( Mycobacterium neoaurum ) BT104-1, the new Mycobacterium aureus BT104-1 was deposited in the General Microbiology Center of China Culture Collection Administration on March 17, 2025, with the deposit number CGMCC No. 33600.

2. Use of the new Mycobacterium aureus BT104-1 according to claim 1 in the preparation of steroid drug intermediates; wherein, The steroid drug intermediate is 10R, 13S-20-hydroxymethylpregnane-4-ene-3-one.

3. A method for preparing a steroid drug intermediate, comprising: The new Mycobacterium aureum BT104-1 according to claim 1 is used to convert phytosterols into steroid drug intermediates; wherein the steroid drug intermediate is 10R,13S-20-hydroxymethylpregnane-4-ene-3-one.

4. The method according to claim 3, characterized in that The method comprises the following steps: (1) inoculating the Mycobacterium neogoldensis BT104-1 into a seed culture medium for culturing to obtain a seed culture; (2) inoculating the seed culture into a fermentation medium for culturing to obtain a fermentation liquid; (3) Purifying and separating the fermentation broth.

5. The method according to claim 4, characterized in that In step (1), the seed culture medium comprises: 5-15 g / L peptone, 1-5 g / L yeast extract, 1-10 g / L glycerol, 1-10 g / L Tween 80, and a pH of 7.0-7.6; and / or In step (1), the culture is carried out under the following conditions: temperature of 28-35°C; time of 2-4 days; rotation speed of 200-250 rpm.

6. The method according to claim 4, characterized in that In step (2), the fermentation medium comprises: 5-20 g / L corn steep liquor powder, 5-20 g / L soybean cake powder, 1-5 g / L yeast extract, 2-10 g / L glycerol, 0.5-3.0 g / L glucose, 0.1-0.3 g / L citric acid, 0.005-0.015 g / L ammonium ferric citrate, 0.5-3.0 g / L K2HPO4, 0.01-0.10 g / L MgSO4·7H2O, 0.5-3 g / L (NH4)2SO4, 10-150 g / L phytosterols and 1-10 g / L Tween 80, with a pH of 7.0-7.6; and / or In step (2), no vegetable oil is added; and / or In step (2), the culture is shaking culture or aerobic submerged culture in a fermenter.

7. The method according to claim 6, characterized in that The shaking culture is carried out under the following conditions: temperature of 28-35°C; rotation speed of 200-250 rpm; and time of 4-10 days; The aerobic submerged culture in the fermenter is carried out under the following conditions: temperature of 28-35°C; time of 4-10 days; rotation speed of 400-500 rpm; fermenter pressure of 0.05-0.1 MPa; ventilation type of sterile air; ventilation volume of 1-5 L / min; and culture volume of 10-20 L.

8. The method according to claim 4, characterized in that In step (3), the purification and separation comprises: centrifuging the fermentation broth, extracting the obtained precipitate with methanol, then centrifuging the extract, distilling the obtained supernatant under reduced pressure, and then adding water for crystallization.

9. The method according to claim 8, characterized in that The purification and separation comprises: centrifuging the fermentation broth, adding methanol to the obtained precipitate, heating to reflux, then centrifuging the extract, filtering the obtained supernatant and performing vacuum distillation, then adding water and cooling to crystallize, and finally performing suction filtration, rinsing the filter cake with a methanol-water solution with a volume ratio of 20-40%, and drying under reduced pressure.

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