Mycobacterium neoaurum and application thereof in preparation of steroid drug intermediate

Abstract

The invention provides mycobacterium neoaurum and application of the mycobacterium neoaurum in preparation of a steroid drug intermediate. The preservation number of the mycobacterium neogold is CGMCC (China General Microbiological Culture Collection Center) No. 28030. The invention also provides a method for preparing a steroid drug intermediate based on the mycobacterium neoaurum. Compared with the prior art, the method provided by the invention has the advantages of mild reaction conditions, short synthesis route, wide raw material source, small environmental pollution, high target product yield (up to 98% or above) and low impurity content, and can be used for effectively preparing the steroid drug intermediate.

Description

Technical Field

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

[0002] Steroid compounds, also known as steroids, are a class of important substances that maintain the life functions of organisms and are widely present in all animals and plants and most microorganisms. Steroid compounds include cholesterol, bile acids, ergosterol, phytosterols, vitamin D, and various steroid hormones. Cholesterol is widely present in animals and is an essential substance indispensable to animal tissue cells. It can affect the fluidity of cell membranes, cell proliferation and differentiation, and is extremely important for maintaining the normal physiological state of cells. Moreover, it is also a raw material for the synthesis of bile acids and vitamin D. Bile acids, as a kind of sterol, are an important component of human digestive juices, can emulsify fats, and promote the absorption of fat-soluble substances by the digestive system. Phytosterols and ergosterols are important components of the cell membranes of plants and fungi, 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, mainly for anti-toxic, anti-inflammatory, anti-shock, and anti-allergic effects, etc. They can not only treat or relieve anaphylactic shock but also be used to treat other endocrine diseases such as Addison's disease, and have also become important adjuvant drugs for the treatment of breast cancer and prostate cancer. There is a huge demand for steroid drugs globally every year. More than about 300 steroid drugs have been approved for clinical use, and this number is still increasing, accounting for about 10% of the total global pharmaceutical products.

[0004] Currently, in the production process of steroid drugs, one method is to use plant saponins as the initial raw material and convert them into the key intermediate 16-pregnadienolone (16-DPA) through chemical methods, and then produce various steroid drugs through chemical synthesis methods. This method has complex processes, high costs, low yields, and large pollution. Another method is to use phytosterols as raw materials to obtain 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-hydroxymethylpregn-4-en-3-one (PHM) through biological fermentation, and then use the above side-chain degradation products as key intermediates and prepare various steroid hormones through chemical and biological methods. Using phytosterols through microbial transformation is a method for producing steroid drugs with high specificity, high efficiency, low cost, mild reaction conditions, and small pollution.

[0005] Early studies found that some microorganisms can utilize sterols as the sole carbon source for growth, such as Nocardia, Pseudomonas, Mycobacterium, and Arthrobacter. The premise of using microorganisms to transform plant sterols into steroid drug intermediates is to change the catabolic pathway in the microorganisms. As early as in the 1980s and 1990s, researchers obtained mutant strains that produce steroid intermediates by changing the metabolic pathway of steroid degradation in microorganisms, and deduced the possible metabolic pathways based on the metabolites accumulated by different mutant strains.

[0006] Steroid intermediate-producing bacteria are usually obtained by chemical or physical mutagenesis treatment and screening, and the choice of screening method directly affects the screening efficiency and the properties of excellent mutant strains. Commonly used starting strains include Mycobacterium, Nocardia, Rhodococcus, and Pseudomonas, etc., among which Mycobacterium is the most widely used. However, since people have not fully grasped the microbial metabolic process and metabolic mechanism of sterols, not all genes involved in metabolism have been fully identified. So far, there are still problems of unbalanced material metabolism and high impurity content in the engineered bacteria constructed by completely using molecular biology methods, which makes separation and purification difficult and leads to a decrease in product yield. Summary of the Invention

[0007] In view of the above problems, the object of the present invention is to provide Mycobacterium neoaurum and its use in the preparation of steroid drug intermediates. The present invention also provides a method for preparing steroid drug intermediates based on the provided Mycobacterium neoaurum. Compared with the prior art, the Mycobacterium neoaurum of the present invention can convert plant sterols into steroid drug intermediate 9-OH AD, with high conversion rate and few impurities, and a qualified product can be obtained through one-step extraction, greatly 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 Mycobacterium neoaurum ( Mycobacterium neoaurum ), denoted as BT102-1, and its preservation number is CGMCC No. 28030.

[0010] In the second aspect, the present invention provides the use of the Mycobacterium neoaurum according to the first aspect of the present invention in the preparation of steroid drug intermediates.

[0011] According to some embodiments of the present invention, the steroid drug intermediate is 9α-hydroxy-androst-4-ene-3,17-dione (9-OH AD).

[0012] The strain BT102-1 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 is in the shape of a round rod 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 pH 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 an obligate aerobe.

[0021] Wherein, the basic 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 aureum 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 new Mycobacterium aureum 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: dry corn steep liquor 5 - 20 g / L, soybean cake powder 5 - 20 g / L, yeast extract 1 - 5 g / L, glycerol 2 - 10 g / L, glucose 0.5 - 3.0 g / L, citric acid 0.1 - 0.3 g / L, ammonium ferric citrate 0.005 - 0.015 g / L, K2HPO4 0.5 - 3.0 g / L, MgSO4·7H2O 0.01 - 0.10 g / L, (NH4)2SO4 0.5 - 3 g / L, phytosterol 10 - 150 g / L, and Tween 80 1 - 10 g / L, 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 cultivation is shake cultivation or aerobic deep - layer cultivation in a fermenter.

[0032] Preferably, the shake cultivation is carried out under the following conditions: the temperature is 28 - 35 °C, preferably 30 - 32 °C; the rotation speed is 200 - 250 revolutions per minute, preferably 210 - 230 revolutions per minute; the time is 4 - 10 days, preferably 6 - 10 days.

[0033] Preferably, the aerobic deep - layer cultivation in a fermenter is carried out under the following conditions: the temperature is 28 - 35 °C, preferably 30 - 32 °C; the time is 4 - 10 days, preferably 6 - 10 days; the rotation speed is 400 - 500 revolutions per minute, preferably 440 - 460 revolutions per minute; the pressure in the fermenter is 0.05 - 0.1 MPa, preferably 0.07 - 0.09 MPa; the ventilation type is sterile air, the ventilation volume is 1 - 5 L / min, preferably 2 - 3 L / min; the cultivation volume is 10 - 20 L, preferably 13 - 18 L.

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

[0035] Preferably, the purification and separation include: centrifuging the fermentation broth, adding methanol to the obtained precipitate, heating to reflux, then centrifuging the extract, filtering the obtained supernatant and subjecting it to vacuum distillation, then adding water and cooling for crystallization, finally performing suction filtration, washing the filter cake with a methanol - aqueous 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 Mycobacterium novum BT102-1, which can convert phytosterols into the steroid drug intermediate 9-OH AD with high conversion rate and few impurities. The yield of 9-OH AD can reach over 98%, and a qualified product can be obtained through one-step extraction, greatly simplifying the extraction and purification process.

[0038] The present invention also provides a method for producing steroid drug intermediates based on this Mycobacterium novum. Compared with the prior art, the method provided by the present invention realizes an oil-free fermentation process and uses a single solvent for separation and purification, making the process simple and with high yield. In addition, the method of the present invention has mild reaction conditions, a short synthesis route, wide sources of raw materials, and little environmental pollution. The yield of the target product prepared by this method can reach over 98%, with few impurities, and can effectively manufacture steroid drug intermediates. Brief Description of the Drawings

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

[0040] Figure 1 is the cell morphology diagram of strain BT102-1 under the microscope (1000 times);

[0041] Figure 2 is the HPLC chromatogram of the 9-OH AD standard product;

[0042] Figure 3 is the HPLC chromatogram of the 9-OH AD fermentation broth prepared by the method of Example 2 of the present invention.

[0043] Depositing of Biological Materials

[0044] The Mycobacterium novum BT102-1 provided by the present invention has been deposited with the China General Microbiological Culture Collection Center (abbreviated as CGMCC) on July 27, 2023. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 28030.

[0045] The Mycobacterium novum MN HIL-4 used in the present invention is deposited with the China General Microbiological Culture Collection Center (abbreviated as CGMCC). The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number of the strain is CGMCC No. 17948, and the deposit date is June 17, 2019. Detailed Embodiments

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

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

[0048] Some of the materials used in the following examples are shown below:

[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] Phytosterol, purchased from Chunzhigu Bioproducts Co., Ltd.

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

[0052] Example 1: Obtaining Mycobacterium neoaurum BT102-1

[0053] Using Mycobacterium neoaurum MN HIL-4 as the starting strain, the Mycobacterium neoaurum of the present invention was prepared by the following method:

[0054] 1) Weigh 6 mg of nitrosoguanidine (abbreviated as NTG, purchased from Sigma) into a sterile centrifuge tube, add 0.05 mL of acetone for solubilization, and then add 1 mL of 0.2 mM phosphate buffer at pH 6.0 to completely dissolve it to obtain a nitrosoguanidine solution;

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

[0056] 3) Immediately place the mixture obtained in step 2) in a 30°C water bath and shake it for 10 min - 1 h;

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

[0058] 5) Dilute the mutagenized bacterial suspension obtained in step 4) by 10-fold and spread it on an NA medium plate, and culture for 4 - 6 days to obtain single colonies;

[0059] 6) Pick single colonies for transformation experiments using phytosterol as the substrate (see Example 2 for specific steps), and screen from them to obtain the mutant strain Mycobacterium neoaurum BT102-1 with high yield of 9-OH AD.

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

[0061]

[0062] After identification, this strain belongs to Mycobacterium novum.

[0063] Example 2: 9-OH AD Fermenter Conversion Experiment

[0064] 1) Inoculate the experimental strain BT102-1 into 300 mL of seed medium in a 1000 mL Erlenmeyer flask, and culture it with shaking at 220 rpm at 32 °C for 48 h to obtain a seed culture.

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

[0066] 2) Inoculate the seed culture prepared in step 1) into 15 L of in-situ sterilized fermentation medium in a 30 L fermenter, and perform aerobic deep-layer culture in the fermenter. The culture conditions are: temperature 32 °C, time 6 - 10 days, dark culture, rotation speed 450 rpm, fermenter pressure 0.08 MPa, aeration type air, aeration rate 2 - 3 L / min; sample every day to observe the experimental results, and stop fermentation when the phytosterol residue is less than 1% (mass percentage), and make up the water to a total volume of 15 L.

[0067] Among them, the composition of the fermentation medium is as follows: corn steep liquor dry powder 10 g / L, soybean cake powder 10 g / L, yeast extract 3 g / L, glycerol 5 g / L, glucose 1.0 g / L, citric acid 0.2 g / L, ammonium ferric citrate 0.01 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 0.05 g / L, (NH4)2SO4 1.5 g / L, phytosterol 100 g / L, Tween 80 4 g / L; adjust the pH to 7.0.

[0068] 3) Take the fermentation broth from step 2), centrifuge at 4000 rpm for 10 min to obtain a precipitate.

[0069] 4) Weigh the precipitate obtained in step 3), add 5 times the volume of methanol for extraction, heat under reflux for 10 min, centrifuge the obtained extract at 4000 rpm for 10 min, and then filter the supernatant through a 0.22 µm organic membrane and transfer it to a distillation flask.

[0070] Distill the filtrate in the distillation flask under reduced pressure to crystallize it, continue distilling until the methanol content in the distillate is less than 3% (mass percentage), add 3 times the weight of the remaining substance in the distillation flask at this time of deionized water, and cool down to 5 °C for crystallization, and maintain for 1 - 2 h; then filter by suction, wash the filter cake with 30% (volume ratio) methanol aqueous solution at -5 °C, and then dry under reduced pressure at 45 °C for 2 h to obtain 9-OH AD product.

[0071] Detection of fermentation broth products: Use a wide-mouth pipette to take 1 ml of the fermentation broth from step 2), add 10 ml of ethyl acetate, vortex-shake intermittently for 10 min, then centrifuge at 6000 rpm. Take 1 ml of the supernatant and place it in a 20 ml scintillation vial to evaporate the solvent to dryness. Re-dissolve it with 10 mL of acetonitrile, and filter to remove impurities through a 0.22 µm organic membrane. Analyze the content of 9-OH AD in the filtrate by high performance liquid chromatography, and calculate the yield of 9-OH AD according to the following calculation formula. Calculate the conversion rate based on the 9-OH AD content in the fermentation broth and the feeding amount of phytosterol.

[0072] Product detection: Weigh the product obtained in step 4), dissolve it with acetonitrile to prepare a solution with a concentration of 1 mg / mL, and filter to remove impurities through a 0.22 µm organic membrane. Analyze the content of 9-OH AD by high performance liquid chromatography.

[0073] The high performance liquid chromatography column is Agilent ZORBAX SB C18 (5ORBAX, 4.6×150 mm). The chromatographic conditions are as follows: gradient elution is adopted, water is phase A, methanol is phase B, the flow rate is 1.0 mL / min. 0 - 15.0 min: phase B with a volume fraction of 30 - 95%, and the rest is phase A; 15.0 - 30.0 min: phase B with a volume fraction of 95%, and the rest is phase A; the equilibration time is 5 min.

[0074] The yield calculation formula of 9-OH AD is as follows:

[0075] Yield of 9-OH AD (%) = Peak area of 9-OH AD / Total peak area × 100%;

[0076] The calculation formula of 9-OH AD concentration is as follows:

[0077]

[0078] Among them, Ax is the peak area of 9-OH AD in the test sample; Ar is the peak area of 9-OH AD in the reference substance; Cr is the concentration of 9-OH AD in the reference substance (mg / mL).

[0079] As Figure 2 and Figure 3 shown, where Figure 2 is the liquid phase chromatogram of the 9-OH AD reference standard; Figure 3 is the HPLC chromatogram of the 9-OH AD fermentation broth.

[0080] From Figure 3It can be seen that according to the method of the present invention, on the 7th day, the yield of 9-OH AD in the fermentation broth reached 99.84%; the concentration of 9-OH AD in the fermentation broth reached 67.88 mg / ml, and the molar conversion rate of phytosterol to 9-OH AD reached 93.28% (the molecular weight of phytosterol is 415).

[0081] After detection, on the 7th day, the content of 9-OH AD in the 9-OH AD product was 98.5%.

[0082] In the conversion experiment of phytosterol using the starting strain MN HIL-4, the main product was δ-lactone (HIL), and the proportion of HIL in all products reached 99.29%, and 9-OH AD was not found in the products (see Example 3 of Patent Application CN111500498A). In contrast, the Mycobacterium neoaurum BT102-1 of the present invention can selectively convert phytosterol into the steroid drug intermediate 9-OH AD, with a high conversion rate and few impurities.

[0083] The above are only several exemplary embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by making some changes or modifications using the disclosed technical content by those skilled in the art without departing from the technical solution of the present invention fall within the scope of the present invention.

Claims

1. A new Mycobacterium aureum ( Mycobacterium neoaurum ), the new Mycobacterium gold was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 27, 2023, with the deposit number CGMCC No. 28030.

2. Use of the new Mycobacterium aureus according to claim 1 in the preparation of steroid drug intermediates.

3. The use according to claim 2, characterized in that: The steroid drug intermediate is 9α-hydroxy-androst-4-ene-3,17-dione.

4. A method for preparing a steroid drug intermediate, the method comprising: The new Mycobacterium aureum according to claim 1 is used to convert phytosterols into steroidal drug intermediates.

5. The method according to claim 4, characterized in that The method comprises the following steps: (1) inoculating the new Mycobacterium aureum 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.

6. The method according to claim 5, 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.

7. The method according to claim 5, 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.

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

9. The method according to claim 5, 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.

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

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