DE2746383A1 - Androst-4-ene-(3,17)-di:one prepn. from (17)-alkyl-steroid - by aerobic bacterial fermentation in aq. medium using Mycobacterium fortuitum, (DSM 1134) (NL 25.4.78) - Google Patents

Abstract

Androst-4-ene-3, 17-dione, (I), is prepd. by cultivating a specified microorganism mutant in an aq. nutrient medium, under aerobic conditions, in the presence of >=1 steroid, (II), contg. a 2-10C alkyl gp. in the 17-position. Other microorganism mutants which may be used and are capable of selectively degrading (II) and of collecting (I) in fermentation liquor, are derived from Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces. (I) is an intermediate for prepn. of steroid hormones, e.g. testosterone.

Description

New mutants of microorganisms and their use

the manufacture of androst-4-en-3.17-dione The conversion of steroids by microorganisms has already been studied quite extensively and in the literature released. The earliest relevant work appears to be from Mamoli and Vercellone of 1937 "Reports" 70, 470 and "Reports" 70, 2079. loc. cit is about the reduction of 17-ketosteroids to 17ß-hydroxysteroids with the help of Fermenting yeast reports. In 1952 (see US Pat. No. 2,602,769), Peterson and Murray report on the ile hydroxylation of progesterone with the help of the fungus Rhizopus nigricans. 1972 (see US Pat. No. 3,684,657), Kraychy et al. Report on the selective microbiological degradation of steroid 17-alkyl esters through fermentation of steroids having at least 8 carbon atoms in the 17-alkyl side chain with Mycobacterium sp. ITURL B-3683 with formation of androst-4-en-3,17-dione, androst-1, 4-diene-3, 17-dione and 20a-hydroxymethylpregna-1,4-dien-3-one. 1973 (cf.

US-PS 3,759,791) report Marsheck and coworkers about the selective microbiological representation of androst-4-en -3,17-dione by fermentation a steroid of the Cholestan or Stigmastan series with at least 8 carbon atoms in the 17-alkyl side chain with Mycobacterium characterized as Mycobacterium vaccae sp. NWIL B-3805.

Mutants by their ability to selectively break down steroids with 17-alkyl side chains of 2 up to and including 10 carbon atoms and for collection of androst-4-en-3,17-dione in the fermentation broth are obtained after the mutation measures or other mutation measures explained in more detail below from microorganisms of the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Mikrobacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces, preferably Mwcobacterium; Examples of species in these genera are M.

phlei, M. smegmatis, M. rhodochrous, M. mucosum, M. fortuitum and M. butyricum.

In the context of the present invention, in particular a new microorganism mutant, namely Mycobacterlum fortuium DSM 1134, for the selective breakdown of steroids with 17-alkyl side chains of 2 to 10 inclusive carbon atoms to androst-4-ene-3,17-dione used.

Examples of selectively degradable steroids according to the invention are sitosterols, Cholesterol, stigmasterol, campesterol and similar steroids with 17-alkyl side chains having 2 up to and including 10 carbon atoms. These steroid substrates can be either be used in pure form or in raw form.

As mentioned earlier, one gets through their ability to be selective Degradation of steroids with 17-alkyl side chains with 2 up to and including 10 carbon atoms marked and androst-4-en-3,17-dione in the fermentation broth accumulating mutants through mutation of microorganisms of the genus Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces.

For the purpose of the invention, the Species Mycobacter fortuitum ATCC 6842 to a new laboratory microorganism mutant mutated. The 1974 ATTC catalog explains in addition to listing ATCC 6842 the following: wJ.C. Cruz 2. Cold abscess. Acta Med.

Rio de Janeiro 1: 1 (1936). M. fortuitus ATTC 6842 sterols non-selective to low molecular weight compounds, for example C02 + H20. Thus is suitable this microorganism is not used for selective steroid degradation.

In the mutation of M. fortuitum ATTC 6842 by means of nitrosoguanidine A new mutant is obtained, which selectively uses steroid de with 17-alkyl side chains Able to degrade 2 up to and including 10 carbon atoms to androst-4-en-3,17-dione.

This microorganism mutant was found in the German Collection of Microorganisms of the Society for Radiation and Environmental Research m.b.H. Munich, Grisebachstraße 8, D-3400 Göttingen and was given the deposit number there DSM 1134.

The mutant microorganism according to the invention is different of the Mycobacterium species NRRL B-3805 used in US Pat. No. 3,759,791. the Mycobacterium species NRRL D-3805 has the general properties of Mycobacterium vaccae, which is a distinctly different species than the Mycobacterium according to the invention fortuitum acts (cf. for a comparison of these microorganisms "Bergey's Manual of Determinative Bacteriology, "8th Edition, The Williams and Wilkins Company, 1974, pages 695 and 696).

he morphology and the chemical or drug sensitivity of M. fortuitum DSM 1134 are of the corresponding properties of the output species M. fortuitum ATCC 6842 indistinguishable. Provide both M. fortuitum cultures acid-fast, immobile, non-spore-forming bacilli from the Mycobacteriaceae family of the order Actinomycetales. According to the Runyon'schon classification (cf. E.H. Runyon in ZMod. Clin. North Amorica ", 43, 273 (1959)) is a non-chromogenic, Mycobacterium belonging to group IV, i.e. it grows rapidly at low temperature with the formation of non-pigmented colonies on relatively simply composed ones Media.

M. fortuitum ATCC 6842 and M. fortuitum DSM 1134 can be compared with their effect on steroid molecules clearly differ from one another. As already carried out, the use of M. fortuitum ATCC 6842 is non-selective Breakdown of steroids when using M.

fortuitum DSM 1134, on the other hand, there is a selective breakdown of the steroids. This property of M. fortuitum DSM 1134 is a highly valuable one Property.

The mutation of M. fortuitum ATTC 6842 to a mutant that then is remuted to M. fortuitum DSM 1134, is done with the help of nitrosoguanidine. the Details of the mutation measures will be explained in more detail below. The mutation and conversion or transformation measures explained in more detail below are described in detail for a Mycobacterium, but it is straightforward possible similar or equivalent measures in the case of microorganisms of the others mentioned To apply genera.

The selective conversion according to the invention can be in a growing Culture of M. fortuitum DSM 1134 either by adding the respective steroid substrate to the culture during the incubation period or by adding the respective steroid substrate accomplish to the nutrient medium before inoculation. The steroid can either be used alone or in combination with another steroid. The preferred one however not mandatory concentration range for the steroid in the Culture is about 0.1 to about 100 g / l. The culture is in a nutrient medium using a source of carbon, for example an assimilable carbohydrate, and a nitrogen supplier such as an assimilable nitrogen compound or a proteinaceous material, grown or cultured. Preferred carbon suppliers are glucose, brown sugar, cane sugar, glizerin, starch, corn starch, lactose, Dextrin, molasses and the like. Preferred nitrogen suppliers are corn steep liquor, Yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, Corn meal, milk solids, pancreatic digestion products of casein, fish meal, distillery residues, Animal peptone fluids, Meat and bone scraps, ammonium salts and the same. Advantageously, combinations of these carbon and nitrogen suppliers are used. The fermentation or fermentation media do not spray trace metals, e.g. zinc, magnesium, manganese, cobalt, iron and the like, to be added, as tap water and unpurified before the sterilization of the medium Components are used as components of the medium.

The conversion process takes approximately 72 hours to 15 days. The incubation temperature during the conversion process ranges from about 25 ° to about 37 ° C for Mycobacterium fortuitui DSM 1134 preferably 300C. The contents of the conversion vessel is ventilated with sterilized air and for easy growth of the microorganism emotional. in this way the efficiency of the X-conversion process can be increased.

After completion of the conversion, determined by thin layer chromatography, using commercially available silica gel plates and a solvent system from 2 volumes of ethyl acetate and 3 volumes of cyclohexane, the desired -converted Steroid isolated in conventional manner. For example, the fermentation or Fermentation or conversion mixture including the fermentation broth and the microorganism cells extracted with a water-immiscible organic solvent for steroids will. Suitable solvents are methylene chloride, chloroform, carbon tetrachloride, Ethylene chloride, trichlorethylene, ether, amyl acetate, benzene and preferably dichloromethane.

On the other hand, the fermentation broth and the cells initially in the usual known manner, for example by filtering or centrifuging, separated from each other and then extracted separately with suitable solvents will. The cells can either be mixed with a water or with a extracted with water immiscible solvents. The cell-free fermentation or fermentation broth can be mixed with water-immiscible solvents extract.

The extracts can be filtered through diatomaceous earth, whereupon the The filtrate is distilled to dryness in vacuo.

The distillation residue obtained in this way with the desired converted Steroid can be made in a minimum amount of a 20:80 mixture of ethyl acetate and Dissolve cyclohexane. This solution can then be chromatographed on silica gel. The androst-4-ene-3,17-dione can be extracted from the silica gel with a 15:85 mixture Elute ethyl acetate and chloroform. By evaporation of the solvent and recrystallization the evaporation residue from hexane then gives the desired compound in pure form.

What is involved in carrying out the method according to the invention, i. The product obtained in the conversion of the present invention is the known steroid intermediate Androst-4-ene-3,17-dione. This compound is suitable as an intermediate in the Manufacture of usable steroid hormones. For example, androst-4-ene-3,17-dione such as those known from U.S. Patents 2,143,453, 2,253,798, 2,264,888 and 2,356,154 Process to convert into testosterone.

The following examples are intended to further illustrate the process according to the invention illustrate. unless otherwise stated, mean all Percentages "percent by weight". Frner means all quantities in the case of solvent mixtures, unless otherwise stated, "volumes".

Example 1 Production of the mutant M. fortuitum DSM 1134 from M.

fortuitum ATCC 6842: a) Nitrosoguanidine mutanogenesis: cell from M. fortuitum AS 6842 are grown at a temperature of 28 ° C in a sterile seed medium of the following composition: sewing broth 8 g / l yeast extract 1 g / l Sodium propionate 0.5 g / l made up to 1 l with distilled water. The pH value the medium is treated with 1N NaOH before being sterilized for 20 minutes at 121 ° C 7.0 set.

The cells are grown to a density of about 5x108 / ml, beaded by centrifugation and then with an equal volume washed sterile 0.1N sodium citrate, pH 5.6. The washed cells are resuspended in the same volume of citrate buffer, followed by an aliquot is separated for titer determination (i.e. for cell counting). Finally will Nitrosoguanidine added to a final concentration of 50 μg / ml. The cell suspension is incubated in a water bath for 30 minutes at a temperature of 37 ° C, whereupon again an aliquot is removed to determine the titer. The rest is centrifuged off and with an equal volume of sterile 0.1 M potassium phosphate with a pH of 7.0 washed. Eventually, the cells are turned into a. sterile minimal salt medium without carbon supplier of the following composition: NH4NO3 1.0 g / l K2HPO4 0.25 g / l MgSO4.7H2O 0.25 g / l NaCl 0.005 g / l FeSO4.7H2O 0.001 g / l with distilled water made up to 1l resuspended. The pH of this medium is a 20 minute ago Sterilize at 121 ° C with 1N HCl set to 7.0. Finally the cells spread out to select the mutants.

b) Selection and isolation of the mutant M. fortuitum DSM 1134 The in the mutated cells are diluted and spread on plates, which contain a medium of the following composition (modified from Fraser and Jerrel, 1963, J. Biol. Chem. 205, 291 to 295): glycerine 10.0 g / l K2PO4 0.5 g / l NH4Cl 1.0 g / l MgSO4.7H2O 0.5 g / l FeCl3.6H2O 0.05 g / l with distilled water filled up to 1l After adding 15 g / l agar, the medium becomes 30 min long in an autoclave to a temperature of 121 0C and then heated in poured sterile petri dishes. Growing resp.

culturing on this medium eliminates most of them by the mutation process formed nutritional auxotrophs. For example, cultures are made to grow chemically defined media require vitamins, growth factors and the like, eliminated. After about 7 days of incubation at a temperature of 280C the colonies formed on appropriate test plates for selection of mutants, and then replicated back to the control sheet containing glycerin-based medium. The test plates are made according to the publication by G.E. Peterson, H.L. Lewis and J.R.

Davis "Preparation of uniform dispersions of cholesterol and other water-insoluble carbon sources in agar media ", J. Lipid Research 3, 275-276 (1962). The minimum salt medium on these plates is under a) of Example 1 described. After adding 15 g / l agar and 1.0 g / l of a suitable carbon supplier steroids such as sitosterol or androstenedione (AD) are added, whereupon the obtained Suspension is heated in an autoclave at 121 ° C. for 30 minutes. The sterile, hot one The mixture is then poured into a sterile mixing vessel and mixed for several minutes and finally poured into sterile petri dishes.

Difficulties can arise with these measures due to foam formation come, but these can be done by mixing the hot mixture through Reduce flaming on the surface of the melted agar plates. In this way uniform dispersions of water-insoluble carbon suppliers are obtained, as a result of which the production of very homogeneous, but opaque agar plates is facilitated.

The colonies grown on the control plates, but not the grown on the test panels containing AD as the sole carbon supplier Colonies are purified by streaking on nutrient agar plates. After waxing at a temperature of 280C, single clones are made with the aid of sterile toothpicks plucked from the nutrient agar plates and inoculating net-like patterned plates retested with AD as a carbon supplier. Purified isolates that are still have a phenotype different from that of the mother culture, are then in Shake flasks evaluated.

c) Shake bottle evaluation: There are 500 ml shake bottles with 100 ml of a biotransformation agent with the following composition: glycerine 10.0 g / l K2HP04 0.5 g / l NH4Cl 1.0 g / l MgS04 7H20 0.5 g / l FeCl3 6H20 0.05 g / l with distilled water made up to 1 1 used. Thereupon be in the medium 1 g / l soy flour and then 10 g / l sitosterol mixed in. After heating for 30 minutes of the bottles in an autoclave to 121 0C they are cooled to 280C and then inoculated with 10 ml of a seed prepared as follows.

The purified isolates from stage b) are at a temperature of Grown at 28 ° C on inclined agar. To the Inoculating a 500 ml bottle with 100 ml of sterile seeds of the following composition: Nutrient broth 8 g / l yeast extract 1 g / l glycerine 5 g / l made up with distilled water a loop full of cells removed from a suitable agar is used for 1 liter. The pH of the seed medium is raised before heating the bottles for 20 minutes 121 ° C in an autoclave with 1N NaOH adjusted to 7.0. The seed bottles will be Incubated for 72 hours at a temperature of 28 ° C.

As mentioned earlier, 10 ml of grown kit are used to inoculate all 500 ml bottles with 100 ml of sterile transformation medium each are used. On that the bottles are kept at a temperature of 28 ° to 30 ° C on a rotary shaker incubated, taking aliquots from time to time. These samples each comprise 10 ml and are dispensed by shaking with 3 volumes Extracted methylene chloride. Portions of the extracts are analyzed by thin layer chromatography using silica gel and the solvent system described, i. 2 volumes of ethyl acetate and 3 volumes of cyclohexane, as well as by gas / liquid chromatography analyzed. Evidence of the presence of Add and AD confirms the selective Breakdown of sitosterol by the new mutant from the mother Mycobacterium M. fortuitum ATCC 6842.

d) Remutation to form the mutant M. fortuitum DSM 1134 the obtained mutant, the nocii grows very slowly on the AD-slaltigen agar plates, is subjected to the mutation measures described, whereby one mutagenized Cells. These cells are then the measures outlined in parts b) and c) subjected to the new mutant Mycobacterium fortuitum DSM 1134, which for the formation of AD and small amounts of ADD (androst-1,4-diene-3,17-dione), receives.

Example 2 Conversion of sitosterol to AD: The medium used corresponds to the medium of example 1 c). This medium is heated by heating for 30 minutes Sterilized in an autoclave to 121 ° C., then cooled to 300 ° C. and finally with it 10 parts of a seed culture of the mutant Mycobacterium fortuitum DSM 1134, the corresponding Example 1 d) was inoculated. The inoculated mixture is agitated 336 hours at a temperature of 30 ° C to encourage submerged growth incubated.

After the incubation, the mixture is extracted with dichloromethane. The extract obtained is dried over anhydrous sodium sulfate and then through Freed from solvent by vacuum distillation. The distillation residue obtained is then added dissolved in a minimum of a 20:80 mixture of ethyl acetate and cyclohexane. The solution obtained is chromatographed on silica gel. The presence of androst-4-ene-3,17-dione and a small amount of androsta-1,4-diene-3,17-dione (ratio: about 8: 1) from the thin-layer chromatogram. The compounds obtained are from the Silica gel by eluting with a 15:85 mixed solvent of ethyl acetate and Separated chloroform, whereupon the individual compounds by evaporation of the solvent and recrystallization of the evaporation residue from IIexane isolated will.

Example 3 By replacing the sitosterol of Example 2 with cholesterol enadit one also prominent AD and a low I; narrow ADD.

Example 4 By replacing the sitosterol of Example 2 with stigmasterin Nan also mainly receives AD and a small amount of ADD.

Example 5 By replacing the sitosterol of Example 2 with campesterol one also obtains mainly AD and a small amount of ADD.

Example 6 By adding a combination of any steroids of Examples 2 to 5 to the sitosterol in Example 2 or by replacing the sitosterol in Example 2 by a combination of any steroids from Examples 2 to 5 one also gets mainly AD and a small amount of ADD.

Example 7 By replacing Mycobacterium fortuitum ATCC 6842 in Example 1 by a microorganism of the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia or Streptomyces one obtains mutants of microorganisms, which are distinguished by their ability to be selective Degradation of steroids with 17-alkyl side chains of 2 up to and including 10 carbon atoms and mark for the accumulation of mainly AD in the fermentation broth.

Example 8 By replacing the X. fortuitum DSM 1134 in Examples 2 to 6 by the mutants obtained according to Example 7 are also mainly obtained AD and a small amount of ADD.

Claims (13)

Claims 1. A process for the production of androst-4-en-3,17-dione, characterized in that Mycobacterium fortuitum DSM 1134 in an aqueous Nutrient medium under aerobic conditions in the presence of a steroid with 2 up to and including Grows 10 carbon atoms in the 17-alkyl side chain. 2. The method according to claim 1, characterized in that as Uses steroid sitosterol. 3. The method according to claim 1, characterized in that as Used steroid cholesterol. 4. The method according to claim 1, characterized in that as Used steroid stigmasterine. 5. The method according to claim 1, characterized in that as Uses steroid campesterine. 6. A process for the production of androst-4-e-3,17-dione, characterized in that that Mycobacterium fortuitum DSM 1134 in an aqueous nutrient medium under aerobic Conditions in the presence of a mixture of two or more steroids, of which each has 2 to 10 inclusive carbon atoms in the 17-alkyl side chain, breeds. 7. The method according to claim 6, characterized in that one Mixture of sitosterol and / or cholesterol and / or stigmasterol and / or campesterol used. 8. New laboratory mutant Mycobacterium fortuitum DSM 1134 9. Procedure for the preparation of androst-4-en-3,17-dione, characterized in that one Microorganism mutant from the group Arthrobacter, Bacillus, Brevibacterium, Corynobacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces, distinguished by their ability to selectively break down steroids with 17-alkyl side chains with 2 up to and including 10 carbon atoms and for the accumulation of androst-4-ene-3,17-dione excel in the fermentation booth, in an aqueous nutrient medium under aerobic conditions in the presence of a steroid having 2 to 10 carbon atoms inclusive in the 17-alkyl side chain grows. 10. The method according to claim 9, characterized in that the Mutant microorganism in an aqueous nutrient medium under aerobic conditions in Presence of a mixture of two or more steroids, each of which 2 has up to and including 10 carbon atoms in the 17-alkyl side chain. 11. The method according to claim 9, characterized in that as Steroid uses sitosterol, cholesterol, stigmasterol or campesterol. 12. The method according to claim 10, characterized in that one is a Mixture of sitosterol and / or cholesterol and / or stigmasterol and / or campesterol used. 13. Process for the preparation of androst-4-en-3,17-dione, thereby characterized in that a microorganism mutant of Mycobacterium fortuitum, distinguished by their ability to selectively break down steroids with 17-alkyl side chains with 2 up to and including 10 carbon atoms and for the accumulation of androst-4-ene-3,17-dione excels in the fermentation broth in an aqueous nutrient medium under aerobic conditions in the presence of a steroid having 2 to 10 carbon atoms inclusive in the 17-alkyl side chain grows.