CN1322112C - Mycobacterium fortuitum and its use in production of testosterone by conversion of microbe - Google Patents

Abstract

The invention relates to a Mycobacterium fortuitum mutant strain CGMCC 1241 and its application in microorganism transformation to produce testosterone. The strain can realize the conversion of the substrate animal phytosterols to the product testosterone with a high conversion rate, and has industrial application value.

Description

A kind of mycobacterium fortuitous and its application in the production of testosterone by microbial transformation

technical field

The invention relates to the technical field of microbial transformation, in particular to a mycobacterium fortuitous and its application in the production of testosterone by microbial transformation.

Background technique

Steroid drugs are a large class of extremely important drugs that have special therapeutic effects in clinical practice and cannot be replaced by other drugs. Among them, testosterone (17β-hydroxyandrost-4-en-3-one, Testosterone, TS; has the following formula I structure)

As an androgen drug, it can promote the development of secondary sexual characteristics and the maturation of sex organs in male animals. In recent years, it has been used as an alternative therapy to treat primary or secondary hypogonadism in male patients. There have been a lot of research abroad on this. In addition, testosterone is an important precursor of anabolic hormones. The main functions of anabolic hormones are to improve protein metabolism, promote protein synthesis and reduce protein dysgenesis, reduce calcium and phosphorus excretion, relieve bone marrow suppression, restore and enhance physical strength, diuresis and lower blood pressure. Press and so on. Due to its remarkable anti-aging and anti-cancer effects, its application range is expanding day by day.

The traditional process of producing testosterone is to carry out a series of chemical synthesis using steroidal saponins from natural plant resources as raw materials. It not only involves the consumption of natural resources, but also causes serious environmental pollution due to the participation of a large number of chemical reaction steps.

The application of microbial transformation technology has made great progress in the research on the preparation process of steroid drugs. Especially since the 1980s, breakthroughs have been made in the application of microbial transformation technology to the preparation of key steroid intermediates. The world's pharmaceutical powers, especially the United States and Japan, use their own agricultural by-products—phytosterols as raw materials to obtain key intermediates through microbial transformation to synthesize steroidal drugs, which not only solves the raw material bottleneck problem caused by limited plant resources , and greatly reduce the manufacturing cost and reduce environmental pollution.

For testosterone, foreign countries have also carried out corresponding research on microbial transformation methods. The current reports mainly use androstenedione (androst-4-ene-3, 17-dione, 4AD; see the following formula II structure) as a substrate for one-step microbial hydrogenation conversion into testosterone; or use a single sterol such as gluten Sterols undergo microbial conversion to testosterone.

Since the former involves a series of reaction steps in the preparation of the 4AD substrate itself, the latter is superior in comparison. If microbial strains with high enough transformation ability and easy large-scale cultivation can be obtained, the industrial application value of this line can be greatly enhanced.

Contents of the invention

The purpose of the present invention is to obtain a mutant strain capable of realizing a high conversion rate of testosterone and having industrial application value by means of strain selection and optimization of transformation conditions.

The testosterone-producing bacterial strain with high transformation rate disclosed by the invention is a mutant strain of Mycobacterium fortuitum, and the microbial preservation number of the bacterial strain is CGMCC No.1241. The strain is obtained from Mycobacterium fortuitum (Mycobacterium fortuitum) DSMZ2966 through natural selection, chemical and physical mutagenesis and other treatments. Specific mutagenesis and screening steps include:

1. Starting strain

Mycobacterium fortuitum DSMZ 2966

2. Medium

Slant/plate medium: Contains appropriate amount of glucose, yeast powder, peptone and agar. This medium is used for the cultivation of starting strains, the natural isolation of strains, and the routine passage of strains.

Transformation medium: glucose 4.0%, yeast extract powder 1.0%, potassium dihydrogen phosphate 0.7% sitosterol 0.5%, Tween 80 0.1%.

3. Analysis method of conversion product-HPLC

Sample preparation: quantitatively add 8ml of acetone to 2ml of the transformation solution, shake and mix well, and place overnight. Take 1ml of the supernatant and centrifuge at 15000rpm for 10 minutes, and the supernatant is used for HPLC detection.

Chromatographic column: C ₁₈ , ODS, 4.6mm×250mm, 5μm

Detector: Agilent 1100

Detection wavelength: 242nm

Column temperature: 50°C

Mobile phase: methanol: water = 80: 20 (volume ratio)

Flow rate: 0.8ml/min

4. Natural separation

Take a fresh slant of the strain and add a small amount of sterile water, gently scrape off the bacterial lawn and pour it into a triangular bottle filled with sterile glass beads and sterile water, shake it fully for 10 minutes, and use a sterile sterile cotton bag filled with absorbent cotton. Filter through a funnel to obtain a bacterial suspension. The bacterial suspension was serially diluted and spread on the plate medium, and cultivated until a single colony grew.

5. UV mutagenesis

(1) Long-wave ultraviolet mutagenesis

The bacterial suspension was obtained by the same method as in the natural isolation above, and the bacterial species was subjected to mutagenesis treatment with a radiation wavelength of 380 nm. After treatment, it was serially diluted and spread on plate culture medium, and cultured at 28°C in the dark.

(2) short-wave ultraviolet mutagenesis

The bacterial suspension was obtained by the same method as in the natural isolation above, and the bacterial species was subjected to mutagenesis treatment with a radiation wavelength of 260 nm. After treatment, it was serially diluted and spread on plate culture medium, and cultured at 28°C in the dark.

6. γ-ray ( ⁶⁰ Co) mutagenesis

Obtain the bacterial suspension by the same method as in the above-mentioned natural separation, take out 2ml and place it in a sterile stoppered Erlenmeyer flask with a capacity of 10ml. The flask was irradiated by ⁶⁰ Co radiation source. After treatment, it was serially diluted and spread on a screening plate, and cultured at 28°C.

7. NTG mutagenesis

After adding NTG to the bacterial suspension system prepared by phosphate buffer and culturing on a shaker at 28°C, samples were taken and serially diluted to coat a screening plate and cultured at 28°C.

8. Shake flask screening

After the single colony obtained through the breeding treatment was inoculated on a slant and cultured at 28°C to mature, it was inoculated into a shake flask containing a transformation medium for liquid culture transformation, and the transformation product was detected by HPLC.

After repeated screening, a mutant strain with a conversion rate more than 10 times that of the starting strain was finally obtained. The genetic stability of the mutant strain was proved by passage investigation, and the ability to transform into testosterone could be maintained at the same level after a reasonable number of passages. The public preservation number of this microbial strain is CGMCC 1241. See Figure 1 for the strain selection pedigree; see Figures 2 and 3 for the HPLC profiles of the respective transformation products of the starting strain and the screened mutant strain.

When using the CGMCC 1241 bacterial strain obtained by mutagenesis and screening of the present invention to prepare testosterone, 4AD and phytosterols can be directly transformed as substrates to generate testosterone; wherein phytosterols are selected from cholesterol, sitosterol, campesterol or mixtures thereof. The added concentration of the substrate sterol is 0.1%-3.0%.

When using the bacterial strain of the present invention to prepare testosterone, the available carbon source in the culture medium is selected from dextrin, citrate, glycerin, sucrose, maltose, mannitol, glucose, and soluble starch, with a content of 1.0%-6.0%; nitrogen The source can be selected from yeast powder, compound amino acid, peptone, corn steep liquor, urea, nitrate, ammonium salt, with a content of 0.2%-5.0%; trace elements such as phosphate, magnesium salt, and sodium salt can be added during the transformation; spit can also be added Surfactants such as Wen 80 are used to promote the better dissolution of solid sterols and improve the conversion rate.

When the present invention adopts CGMCC 1241 strain to prepare testosterone, the preferred transformation medium is 1.0%-6.0% of glucose, 0.2%-2.0% of yeast powder, 0.5%-1.5% of potassium dihydrogen phosphate, and 0.1%-3.0% of animal and plant sterols , Tween 80 0-0.5%; transformation temperature is 20°C-40°C, preferably 25°C-30°C; transformation pH is 4-9, preferably pH 5-7; transformation time is 5-9 days.

The testosterone is prepared by adopting the above-mentioned optimized combination of conditions, and finally a conversion rate of more than 80% can be realized from a 250ml Erlenmeyer shaker flask to a 10-ton fermenter. The converted product is subjected to steps such as extraction and column chromatography to obtain a testosterone product with a purity higher than 90%.

The mutant strain obtained by the invention achieves the purpose of producing testosterone with a high conversion rate, and has industrial application value.

Description of drawings

Figure 1. Mycobacterium fortuitum (Mycobacterium fortuitum) CGMCC 1241 strain breeding pedigree

Among them, NS means natural separation, UV ₃₈₀ means ultraviolet treatment with a wavelength of 380nm, UV ₂₆₀ means ultraviolet treatment with a wavelength of 260nm, and NTG means nitrosoguanidine treatment.

Figure 2. HPLC profile of the transformation product of the starting strain

Figure 3. HPLC profile of the transformation product of strain CGMCC 1241

Detailed ways

The investigation of the stability of the strain of embodiment 1 --- the impact of subculture on transformation

Incline medium: glucose 1.0%, yeast extract powder 1.0%, polypeptone 0.8% agar powder 1.5%.

Seed medium: glucose 1.0%, yeast extract powder 1.0%, potassium dihydrogen phosphate 0.2%.

Transformation medium: 4.0% glucose, 1.0% yeast extract powder, 0.7% potassium dihydrogen phosphate, 0.5% sitosterol, 0.1% Tween 80; pH6.5 before elimination.

Culture conditions: slant culture temperature 28°C, culture time 5 days; seed solution culture temperature 28°C, culture time 2 days; transformation culture temperature 28°C, culture time 5 days. The rotation speed of the shake flask is 220rpm/min, and the culture is shaken on a rotary shaker.

Passage Algebra 1 2 3 4 5 Relative generation of TS (%) 100 97 95 94 96

Embodiment 2 Mutant strain and the comparison of starting bacterial strain transformation rate

Culture medium and culture condition are the same as embodiment 1.

The results of the control test between the mutant strain and the starting strain are as follows:

bacteria Starting strain DSMZ 2966 Strain CGMCC 1241 TS generation 265μg/ml 4100μg/ml Relative amount of TS 100% 1547% relative increase —— 14.5 times

Embodiment 3 shake flask conversion test

Slant culture medium, seed culture medium and culture conditions at each stage are the same as in Example 1.

Transformation medium: 5.0% dextrin, 1.2% peptone, 0.1% potassium nitrate, 0.5% potassium dihydrogen phosphate, 1.0% cholesterol, 0.2% Tween 80; pH5.8 before elimination.

The culture conditions are the same as in Example 1.

The conversion rate was 80.2%.

Embodiment 4 shakes flask transformation test

Slant culture medium, seed culture medium and culture conditions at each stage are the same as in Example 1.

Transformation medium: 3.5% soluble starch, 1.0% glucose, 2.0% corn steep liquor, 0.8% potassium dihydrogen phosphate, 2.0% mixed phytosterol (sitosterol: campesterol = 2:1), 0.1% Tween 80.

The transformation culture time was 7 days, and the rest of the culture conditions were the same as in Example 1.

The conversion rate was 83%.

Embodiment 57 tons fermenter transformation test

Slant medium, seed medium, transformation medium, slant and seed culture conditions are the same as in Example 4. The conditions of the 7-ton fermenter are controlled as follows: 25°C of temperature, 1:1 (vol:vol) ventilation, stirring speed 300rpm, cycle 7 days. The conversion rate of testosterone was 81.7%.

Example 6

The conversion solution obtained according to Example 5 was soaked in acetone, extracted with ethyl acetate, concentrated the extract, and obtained testosterone with a purity of 92% through silica gel column chromatography (eluent cyclohexane:ethyl acetate=7:1). product.

Claims (5)

1, a kind of mycobacterium fortutitum (Mycobacterium fortuitum) mutant strain, the microbial strains preserving number is CGMCC No.1241.

2, the application of mycobacterium fortutitum mutant strain CGMCC No.1241 according to claim 1 in the preparation testosterone.

3, a kind of method that adopts claim 1 mycobacterium fortutitum mutant strain CGMCC No.1241 to prepare testosterone, it is characterized in that the conversion culture medium carbon source that this method adopts is selected from dextrin, Citrate trianion, glycerine, sucrose, maltose, N.F,USP MANNITOL, glucose, Zulkovsky starch, content is 1.0%-6.0%;

Nitrogenous source is selected from yeast powder, aminoacids complex, peptone, corn steep liquor, urea, nitrate, ammonium salt, content 0.2%-5.0%;

The substrate sterol is selected from 4AD and animals and plants sterol, and interpolation concentration is 0.1%-3.0%;

Invert point is 20 ℃-40 ℃, and conversion pH is 4-9; Transformation time 5-9 days.

4, mycobacterium fortutitum mutant strain CGMCC No.1241 according to claim 3 prepares the method for testosterone, it is characterized in that wherein said conversion substratum is glucose 1.0%-6.0%, yeast powder 0.2%-2.0%, potassium primary phosphate 0.5%-1.5%, animals and plants sterol 0.1%-3.0%, tween 80 0-0.5%.

5, mycobacterium fortutitum mutant strain CGMCC No.1241 according to claim 3 prepares the method for testosterone, it is characterized in that wherein said invert point is 25 ℃-30 ℃, and conversion pH is 5-7.

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