RU2126837C1 - Strain of bacterium mycobacterium smegmatis used for oxidation of plant and animal sterols to androst-4-ene-3,17-dione - Google Patents

Abstract

FIELD: biotechnology, microbiology, steroids. SUBSTANCE: invention relates to a microbiological method of androst-4-ene-3,17-dione producing. The strain Mycobacterium smegmatis able to transform plant and animal sterols (for example, sitosterol, cholesterol) to androst-4-ene-3,71-dione. The strain Mycobacterium smegmatis is deposited in VKPM at number Ac-1552. Strain is resistant to streptomycin that ensures to use its without risk to infect medium by foreign microflora. Strain VKPM Ac-1552 provides producing the end product at concentration 7-9 g/l. EFFECT: increased yield of the end product. 1 ex

Description

 The present invention relates to the field of biotechnology, and in particular relates to the production of steroid drugs using microorganisms.

 The aim of the invention is to obtain a new effective mutant strain of bacteria capable of transforming sterols of plant (sitosterol) and animal (cholesterol) origin into androst-4-en-3,17-dione (AD) - a key intermediate in the synthesis of steroid medications. Currently, these sterols are the most promising type of steroid raw materials.

 The transformation of steroids by microorganisms, including some species of the genus Mycobacterium, is widely described in the world scientific and patent literature [A.A. Akhrem, Yu.A. Titov "Steroids and microorganisms", Nauka, M., 1970; S.B. Mahato, S. Banerjee, and S. Podder "Phytochemistry", 1989, 28, p. 7]. All the described transforming microorganisms are characterized by a slow growth rate both in surface and in deep conditions, and therefore the transformation can last more than 10 days. These microorganisms do not secrete antibiotic substances, which in large-scale production often leads to contamination by extraneous microflora. In this regard, it is promising to have processes of growing vegetative seeds and fermentation (transformation), protected from such negative points. This is achieved by adding a certain amount of exogenous antibiotic to the medium for growing and transforming and, accordingly, using a transforming microorganism resistant to this antibiotic. A bacterial strain with similar properties has an advantage over other microorganisms capable of oxidizing sterols.

 To select a strain that oxidizes sterols, the Mycobacterium smegmatis strain isolated from biological material obtained from a veterinary clinic in Moscow was used as the initial parent culture.

 The initial culture was subjected to alternating effects of physical (UV irradiation) and chemical (nitrosomethyl biuret and nitrosoguanidine) mutagenic factors. By stepwise selection of the induced mutants, a new, original, streptomycin-resistant strain of Mycobacterium smegmatis was obtained.

 The strain Mycobacterium smegmatis is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) under the number VKPM Ac-1552 and is characterized by the following features.

 Cultural and morphological characters.

 (The media were prepared according to the book "Methods for the selection of producers of antibiotics and enzymes", M., 1978. The color of the colonies was determined on the Bondartsev scale).

 Medium with yeast extract and glycerin.

 The colonies are round, flat with an uneven edge, the pigment does not stand out on Wednesday, the color of the colonies is Zh-2 (red), the diameter of the colonies is 5-6 mm. Medium with glucose and peptone.

 The colonies are flat with uneven edges, the center of the colonies is slightly raised, the pigment does not stand out on Wednesday, the color is close to Zh-2 (reddish), the diameter of the colonies is 4.5-5 mm.

 Medium with glucose and BVK.

 The colonies are rounded flat with an uneven edge, with radial folding emerging at the edges, in the center are finely wrinkled, no pigment is released into the medium, color G-2, colony diameter 4.5 - 7.5 mm.

 Wednesday 1.

 The colonies are flat matte, lighter at the edges, centric folding, the pigment does not stand out on Wednesday, the color of the colonies is 0-3 (orange), diameter 4.0 - 7.0 mm.

 Wednesday 6 (corn).

 The colonies are oval flat with a rough surface, the pigment does not stand out on Wednesday, the color is 0-5 (flesh-pink), the diameter of the colonies is 5.5 - 7 mm.

 Wednesday 12.

 Colonies are flat with a rough surface, uneven edges, color 0 - 5 (flesh-pink), the diameter of the colonies is 5.5 - 6.6 mm.

 Wednesday 22 with meat extract and peptone.

 The colonies are flat, uneven-rounded, with an uneven edge, finely wrinkled, the pigment does not stand out on Wednesday, the color is Zh-4 (pale terracotta), the diameter of the colonies is 4.5 - 5 mm.

 Wednesday 63.

 The colonies are flat, uneven-rounded, wrinkled with uneven edges, the pigment does not emit on Wednesday, color M-7 (golden yellow), the diameter of the colonies is 4-5 mm.

 Soya Agar.

 The colonies are flat, uneven-rounded, with uneven edges, finely wrinkled, no pigment is released on Wednesday, the color is close to Zh-2 (reddish), the diameter of the colonies is 5.5 - 6.5 mm.

 The environment is pea.

 The colonies are flat rounded with uneven edges, a rough surface, the pigment does not stand out on Wednesday, the color of the colonies is 0-3 (orange), diameter 7.0 - 8.5 mm.

 The medium is peptone-corn.

 The colonies are uneven-rounded with a rough surface, along the edges of the colonies are uneven, lighter and smoother than in the middle part, the pigment is not released into the medium, the color of the colonies is 0-3 (orange), diameter 7 - 11 mm.

 Medium with glucose-aspartic.

 The colonies are uneven-rounded with uneven edges, the surface of the colonies is radially wrinkled, the pigment does not stand out on Wednesday, the color of the colonies is D-2 (apricot yellow), the diameter of the colonies is 6.0 - 8.0 mm.

 The environment is glycerol-aspartic.

 The colonies are unevenly rounded radially wrinkled, with uneven edges, the pigment does not stand out on Wednesday, the color of the colonies is 0-3 (orange), diameter 3.0 - 5.0 mm.

 Wednesday Gause.

 Weak growth, pinpoint colonies, B-5 color (dark flesh).

 Waxman’s environment.

Film colonies with a rough surface, irregular in shape, diameter 4.5 - 6.0 mm. In sieving, there are two types of colonies:
1 - most of them are filmy, irregular in shape;
2 - rounded with an uneven edge; color close to Zh-2 (reddish).

 Wednesday Chapek.

 The colonies are raised with a dry surface, uneven-oval, diffusely wrinkled, the pigment does not stand out in the medium, the color of the colonies is 0-3 (orange), the diameter of the colonies is 4.5-8.0 mm.

 Chapek-Dox environment.

 Weak growth, pinpoint colonies, B-5 color (dark flesh).

 Physiological signs of a microorganism.

Strict aerobic, the optimal temperature for growing crops on agar media 28 - 35 ^o C.

 Biochemical signs.

 Gelatin does not dilute; milk peptones, but does not coagulate; starch does not hydrolyze.

 Attitude to carbohydrates: assimilates glucose, fructose, inositol, mannitol; does not absorb arabinose, xylose, raffinose, galactose, sucrose, lactose, rhamnose, sorbitol, maltose.

 The culture is gram-positive, represented by short straight or curved elongated sticks (depending on growing conditions); sizes vary: 0.2 - 0.4 x 0.5 - 4.0 microns, spore does not form.

 The strain is resistant to 100 μg / ml streptomycin, non-pathogenic.

 The strain Mycobacterium smegmatis VKPM As-1552 is able to transform sterols of plant and animal origin with a concentration of 10 - 20 g / l with the formation of the target product androst-4-en-3,17-dione in a concentration of up to 7 - 9 g / l, respectively.

 The use of the strain Mycobacterium smegmatis VKPM Ac-1552 can be illustrated by the following example.

Example 1. The culture of Mycobacterium smegmatis at 6 days of age, grown on agar medium of the following composition (in g / l): glucose - 10, soy flour - 3, citric acid - 2.2, urea - 0.5, potassium dihydrogen phosphate - 0.5, ammonium chloride - 1, magnesium sulfate - 0.5, ferrous sulfate - 0.05, agar-agar - 25, distilled water, pH before sterilization - 6.8 - 7.0, transferred to 30 ml of liquid seed medium in conical flat-bottomed flasks with a capacity of 250 ml. The composition of the inoculum (g / l): glucose - 10, soy flour - 3, urea - 0.5, citric acid - 2.2, ammonium chloride - 1, potassium dihydrogen phosphate - 0.5, iron sulfate - 0.05, magnesium sulfate 0.5, calcium carbonate - 1.5, distilled water, pH before sterilization of 7.0. Incubated for 48 hours on a rocking chair at a stirring intensity of 220 rpm (eccentricity 5 cm) and a temperature of 30 ^o C.

 The grown culture is added in an amount of 10 vol.% To the medium of the following composition (g / l): glucose - 10, soy flour - 10, urea - 0.5, ammonium hydrogen phosphate - 1.5, citric acid - 2.2, magnesium sulfate 0.2, iron sulfate 0.05, calcium carbonate 1.5, distilled water, pH before sterilization 7.0. The medium is poured into similar 30 ml flasks. Before sterilization, well-homogenized cholesterol is added to the medium in an amount of 1% (concentration 10 g / l). Fermentation is carried out on a rocking chair under the conditions described for obtaining vegetative inoculum for 96 + 4 hours.

After fermentation (concentration of blood pressure - 7.0 g / l according to HPLC analysis), the culture fluid is separated from the biomass in a centrifuge. The biomass is extracted with aqueous acetone (60 - 65% content). The solvent was evaporated in vacuo to stop the run. From the resulting aqueous suspension, the technical target product is filtered. After recrystallization from methylene chloride and hexane, a blood pressure is obtained with a yield of 70% and a melting point of 172 - 174 ^o C.

Claims (1)

 The bacterial strain Mycobacterium smegmatis VKPM As-1552 used to oxidize sterols of plant and animal origin to androst-4-en-3,17-dione.