GB2103620A - Microbial production of testosterone and 1-dehydrotestosterone - Google Patents

Abstract

Testosterone and 1- dehydrotestosterone are produced from androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione or steroids with C2-8 17-side chains (e.g. sitosterol, cholesterol, stigmasterol, campesterol) using mutant microorganisms of genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia or Streptomycos, especially the novel Mycobacterium sp. NRRL B-12472 (a mutant of NRRL B-3805).

Description

SPECIFICATION Microbial process for producing testosterone compounds The present invention relates to a process for producing 17ss-hydroxyandrost-4-en-3-one (testosterone) and 17ss-hydroxyandrost-1,4-diene-3-one (1,2-dehydrotestosterone). More particularly, it provides a method for producing testosterone from steroids or 4-androstene-3,1 7-dione (AD) by fermentation with novel microorganism Mycobacterium sp. NRRL B-12472. It also provides a method for producing 12,dehydrotestosterone from 1 ,4-androstadiene-3,1 7-dione (ADD) by fermentation with the novel microorganism Mycobacterium sp. NRRL B-12472.

The transformation of steriods by microorganisms has been widely studied and documented. Apparently, the earliest such work as by Mamoli and Vercellone in 1937, Ber. 70,470 and Ber. 70,2079. They disclose the reduction of 17-ketosteroids to 17-hydroxysteroids by fermenting yeast. More recently, Peterson and Murray disclose the 11-hydroxylation of progesterone with the fungus Rhizopus nigricans; see U.S. Patent No.

2,602,769 (1952). Also recently, Kraychy et al, in U.S. Patent No. 3,684,657 (1972) discloses the selective microbiological degradation of 17-alkyl steroids by fermenting a steroid containing at least 8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683to prepare androst-4-ene-3, 17-dione, androst-1,4diene-3,17-dione, and 20-a-hydroxymethyl-pregna-1,4-diene-3-one. Even more recently, Marsheck et al, in U.S. Patent No.3,759,791(1973) disclose the selective microbiological preparation of androst-4-ene-3,17dione in good yield and 20a-hydroxymethyl-pregna-4-ene-3-one in lesser yield by fermenting a steroid containing at least8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805.

Microorganisms and mutants thereof involved in selective steroid degradation are widely known in the art as indicated above.

U.S. Patent 3,759,791 describes a process for producing androst-4-ene-3,1 7-dione, a precursor to testosterone, using Mycobacterium sp. NRRL B-3805. Japanese Patent J5 5037-121 describes a process for producing 1,2-dehydrotestosterone using an oxidizing inhibitor and a species of Mycobacterium or Brevibacterium.

The present invention particularly provides a process for producing testosterone which comprises: a) cultivating a testosterone producing micro-organism in an aqueous nutrient medium under aerobic condition in the presence of AD or a steroid having from 2 to 10 carbon atoms, inclusive, in the 17-alkyl side chain to produce testosterone in the fermentation beer, and, b) isolating said testosterone from the fermentation beer; a process for producing 1,2dehydrotestosterone which comprises: a) cultivating a 1,2-dehydrotestosterone producing microorganism in an aqueous nutrient medium under aerobic conditions in the presence of ADD to produce 1 2-dehydrotestosterone in the fermentation beer, and, b) isolating said 1,2-dehydrotestosterone from the fermentation beer;; lastly the invention provides a biologically pure culture of the microorganism Mycobacterium sp. having the identifying characteristics of NRRL B-12472, said culture being capable of producing testosterone from androstenedione or a steroid having from 2 to 10 carbon atoms inclusive in the 17-alkyl side chain and of producing 1 ,2-dehydrotestosterone from androstadiencedione, in recoverable quantities upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.

The utility of such process derives from, among other things, the fact that the steroids produced are valuable pharmacological materials. See Goodman and Gilman 4th Ed pages 1566-1580 and Japanese patentJ 55037-121 abstracted as Derwent66629 C/38. In addition, the method of the present invention produces higher yields than previously known microbial methods. And finally, this method may be used without the need for an inhibitor such as a chelating agent.

The novel microorganism Mycobacterium sp. NRRL B-12472, was obtained by mutagenesis of known organism Mycobacterium sp. NRRL B-3805. The microorganism thus uniquely adapted for the selective microbial degradation of the present invention may be obtained from the A.R.S. Culture Collection Research, Fermentations Laboratory, Northern Regional Research Laboratory, 18 N. University Ave., Peoria, IL 61604.

Other mutants of Mycobacterium, as well as the following genera of microorganisms, can be used in the subject invention so long as they have the ability to selectively degrade steroids having 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, and accumulate appreciable quantities of testosterone or 1 ,2-dehydrotestosterone in the fermentation beer. The genera of microorganisms which are within the scope of the subject invention are Athrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. The preferred genus in Mycobacterium. Exemplary species of this genus are M.phlei, M.smegmatis, M.rhodochrous, M.mucosum, M.fortuitum, M.butyricum and M.vaccae.Procedures are disclosed herein for mutating microorganisms of the above genera to give the desired mutants. Examples of suitable steroid substrates are sitosterol, cholesterol, stigmasterol, campesterol, and other steroids with 1 7-alkyl side chains of from 2 to 10 carbon atoms, inclusive. These steroid substrates can be either the pure or crude form.

In general the conditions for producing testosterone or 1,2-dehydrotestosterone by selective microbial degradation for the purposes of the invention process are, except for inclusion of the steroid, AD or ADD to be converted, the same as those for culturing the organism for normal growth.

The instant invention can be effected in a growing culture of Mycobacterium sp. NRRI B-12472 or other microorganism as described above by either adding the selected substrate to the culture during the incubation period or incorporating it in the nutrient medium prior to inoculation. The steroid, AD or ADD can be added singly or in combination with another steroid, AD or ADD. The preferred, but not limiting, range of concentration of the steroid in the culture is about 0.1 to 100 grams per liter. The culture is gown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch fructose syrup, cornstarch, lactose, dextrin, molasses, and the like.Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybeam meal, urea, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, ammonium salts, nitrate salts and the like.

Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, manganese, cobalt, iron and the like, need not be added to the fermentation medium since tap water and other ingredients containing these metals are used as components of the medium.

The transformation process can range from about 3 hours to 4 days. The incubation temperature during the transformation process can range from about 25" to about 37"C, with 30"C being preferred. The contents of the transformation vessel are aerated and agitated to facilitate growth of the microorganism, and thus, enhance the effectiveness of the transformation process.

Upon completion of the transformation process, as evidenced by thin layer chromatography using silica gel plates and a suitable solvent system, the desired transformed steroid is recovered by means well known in the art. For example, the fermentation (transformation) reaction mixture, including the fermentation liquor and cells, can be extracted with a water-immiscible organic solvent for steroids. Suitable solvents are methylene chloride (preferred), chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, ether, amyl acetate, toluene and the like.

Alternatively, the fermentation liquor and cells can be first separated by conventional methods, e.g.

filtration or centrifugation, and then separately extracted with suitable solvents. The cells can be extracted with either water-immiscible solvents. The fermentation liquor, freed of cells, can be extracted with water immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtrate taken to dryness by vacuum or other means. The resulting residue containing the desired transformed steroid then can be dissolved in 10 percent chloroform in methanol and this then concentrated with nitrogen on a steam bath until crystals appear. The solution then can be cooled to room temperature and filtered to remove precipitated steroid.

The desired transformed steroid can also be obtained from the remaining supernatant upon evaporation of the solvent from the supernatant.

Yields are calculated according to the formula 100P Y = Q(S-R) where Y represents percent yield, P weight of recrystallized product, 0 molecular weight of product divided by molecular weight of substrate, S initial weight of substrate, and R weight of recovered substrate.

Conversions are calculated according to the formula 100P c wherein C represents percent conversion and P, 0, and S retain the meaning previously assigned.

The following examples illustrate the invention. In the examples hereinafter set forth, temperatures are given in degrees centrigrade and relative amounts of materials in weight perc unit volume, except as otherwide noted.

Description of the preferred embodiments Example 1 Preparation of MutantMicrobacterium sp. NRRL B-12472 from M. sp NRRL B-3805 (a) Nitrosoguanidine Mutagenesis A medium consisting of ammonium chloride, 1 g; potassium phosphate, dibasic, 0.5 g; magnesium sulfate heptahydrate, 0.5 g; ferrous sulfate heptahydrate; 0.001 g, glycerol, 20 g; Tween 80,0.01 percent; and 1 litre of tap water is adjuted to pH 7.7 with potassium hydroxide. The resultant medium (MGYB + Tween 80) is added as 50 ml portions to shake flasks (250 ml) and sterilized by heating 20 minutes at 121"C. This medium is inoculated with 2.5 ml of a fluid culture of Mycobacterium sp. NRRL B-3805 grown in the same medium.The culture is incubated at 30 + 1"C on a rotary shaker with a 2 inch circular orbit at 200 rpm for 2 days.

The culture is pelleted by centrifugation and the cells are suspended in an equal volume of 0.05 M Trismaleic buffer (pH 8.0) containing 0.01 percent Tween 80. Fifteen milliliters of Tris-maleic buffer containing 1 mg/ml of nitrosoguanidine (NTG) are added to 15 ml of the cell suspension to give a final NTG concentration of 500 mg/ml. The cell suspension is incubated in a water bath shaker for 15 minutes at 30"C, after which the suspension is centrifuged once more. The cells are washed three times with a solution of 0.85 percent NaCI and 0.01 percentTween 80 and resuspended in MGYB + Tween 80.

(b) Isolation of Mutant M sp. NRRL B-12472 Following incubation on the shaker for 1 day at 30 + 1"C to allow for segregation of putative mutants from cell clumps, the culture is diluted serially in Trismaleic buffer with Tween 80. Aliquots from appropriate dilutions are spread on the surface previously prepared plates containing 20 to 25 ml of CGYA medium which has the following formula: Trypticase Soy Broth (Baltimore Biological Laboratories), 20 g; yeast extract, 5 g; glycerol, 20 g; agar, 15 g; and 1 liter of tap water and which is sterilized for 20 minutes at 121"C.

The plates are then incubated at 30 + 1"for7 days.

(c) Shake Tube Evaluation Resultant colonies which appear on the agar plates are picked at random and transferred to test tubes containing 4 ml of CGYB medium (CGYA medium in which the agar is omitted). The cultures are incubated statically for 7 days at 30 + 1"C. One-half milliliter portions of the inoculum cultures are then transferred to test tubes (24 x 125 mm) containing 10 ml of MGYB to which ca. 150 mg of sitosterols, N.F. had been added before sterilizing. After 7 days incubation on the shaker at 30 + 1"C, the shake tube cultures are extracted with 10 ml of methylene chloride. The methylene chloride extracts are analyzed by thin layer chromatography using silica gel and a solvent system consisting of 90:10 (by volume) toluene-ethanol.

Evidence of the presence of significant amounts of testosterone confirms the selective degradation of sitosteroid by the novel mutant produced from the parent M sp. NRRL B-3805.

Example Transformation of AD to Testosterone Fifty milliliter portions of MGB medium are added to shake flasks (250 ml) containing 500 mg of AD. The MGB medium is prepared as for MGYB medium except that the glycerol is omitted and 2 ml of the sterile 50 percent dextrose solution are added to each flask after autoclaving. Five replicate flasks of MGB medium are inoculated with 2.5 ml of a fluid culture ofM. sp. NRRL B-12472 and incubated at 30 + 1"C for 7 days on the shaker.

The combined whole culture broth (250 ml) is extracted with methylene chloride three times. The combined methylene chloride extract is dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue is redissolved in ethyl acetate-toluene (1 :: v/v) and chromatographed on a column containing 80 g Woelm silica gel. The column is eluted with ethyl acetate-toluene (1:5) and the fractions monitored by TLC. Fractions containing testosterone are evaporated in vacuo to give crystalline material which assayed by GC [Quantitative GC assay: 3 percent OV-1 7/OV-21 0 on Chromosorb W/HP, 80-100 mesh; at 240"C; using a F.I.D. (Flame lonization Detector)] for greater than 80 percent purity. The conversion yield was 28 percent for AD.The physicochemical spectra, IR and NMR, are consistent for the compound testosterone.

Example 3 Transformation of Sitosterol to Testosterone A process according to Example 2 is employed except that 750 mg of Sitosterols, N.F. is substituted for AD.

The combined whole culture broth was also processed as for Example 2, 19 percent conversion yield.

Example 4 Transformation of ADD to Testosterone A process according to Example 2 is employed except that ADD is subsituted for AD. The combined whole culture broth from 5 shake flasks is extracted three times with an equal volume of methylene chloride. The combined organic extracts are dried over anhydrous sodium sulfate, filtered, then evaporated in dryness in vacuo. The residue was assayed by GC for testosterone, 69 percent conversion yield.

Example 5 A process according to Example 2 is employed except that 1.050 g of the dried methylene chloride extract (GC assay of 39 percent) is chromatographed and eluted successively from the Woelm silica column using the following proportions and volumes of ethyl acetate:toluene: 1) 15:85,96 ml; 2) 25:75,96 ml; 3) 35:65, 104 ml; 4) 50:50, 160 ml; and 5) 80:30, 152 ml. Fractions containing testosterone, as determined by TLC, are pooled and evaporated, yielding 0.3487 g of solids.Recrystallization of this from aqueous acetone yields 0.307 g of testosterone, m.p. 153.5 - 154.5"C, hmax 241 mm (E16,000), [a]20 + 120.7" (C, 1.007, CHCI3). The infrared (KBr) and NMR (CDC13) spectra are identical with those of a known testosterone sample.

Example 6 A process according to Example 2 is employed except that 500 mg of Sitosterols N.F. are substituted for AD. Once half of the dried extract residue, 0.445 g (GC assay of 25 percent testosterone and 5 percent AD), is processed as in example 4. Fractions containing testosterone, as determined in thin layer chromatography are combined and evaporated, yielding 0.1035 g of dry residue. This is recrystallized from aqueous acetate, yielding 0.074 g of testosterone, m.p. 152 - 153.5"C, hmax 241 mm (E16,600). The infrared (KBr) and NMR (CDC13) spectra are identical with those of a known testosterone sample.

Example 7 A process according to Example 2 is employed except that 500 mg of ADD are substituted for AD. Thin layer chromatography of the dried methylene chloride extract, 0.957 g, indicated that the preponderant constituent is 1 ,2-dehydrotestosterone with minior amounts of ADD and testosterone. The extract is chromatographed in the same manner as in example 4. Fractions containing 1,2-dehydrotestosterone, as indicated by TLC analysis, are pooled and evaporated. The residue, 0.719 g, is recrystallized from ethyl acetate, yielding 0.510 g of 1 ,2-dehydrotestosterone, m.p. 170 - 172", [a]D = 21.7 (C, 1.012, CHCl3) Xmax 244.5 mm (E14640). The IR (KBr) and NMR (CDC13) spectra are identical with those of known sample of 1,2-dehydro testosterone. No impurities are detected by GC assay of the isolated materials.

Mycobacterium sp NRRL P-l 2472 is a fast-growing, gram-positive aerobic organism in the form of non-motile rods, somewhat resemling M.fortuitum (page 697, Bergeys Manual of Determinative Bacteriology, Eighth Edition). The culture was deposited on 18th May 1981.

Claims (13)

1. A process for producing testosterone which comprises: a) cultivating a testosterone producing microorganism in an aqueous nutrient medium under aerobic conditions in the presence of AD or a steroid having from 2 to 10 carbon atoms, inclusive, in the 1 7-alkyl side chain to produce testosterone in the fermentation beer, and b) isolating said testosterone from the fermentation beer.

2. A process as claimed in Claim 1 in which the microorganism is cultivated in the presence of AD.

3. A process as claimed in Claim 1 in which the microorganism is cultivated in the presence of a steroid having from 2 to 10 carbon atoms, inclusive, in the 17-alkyl side chain.

4. A process as claimed in Claim 3 in which the steroid is selected from sitosterol, cholesterol, stigmasterol and campesterol.

5. A process as claimed in Claim 1 in which the testosterone producing microorganism is a microorganism mutant from the group consisting of Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Norcarida, Protaminobacter, Serratia, and Streptomyces, said micro-organism being characterized by its ability to selectively degrade AD or steroids having 17-alkyl side chains offrom 2to 10 carbon atoms, inclusive, and accumulate testosterone in the fermentation beer.

6. A process as claimed in Claim 1 in which the said testosterone producing microorganism is a Mycobacterium mutant which is characterized by its ability to selectively degrade AD or a steroid having from 2 to 10 carbon atoms, inclusive, in the 17-alkyl side chain, and accumulate testosterone in the fermentation beer.

7. A process as claimed in Claim 1 in which the Mycobacterium mutant is Mycobacterium sp. NRRL B-12472.

8. A process for producing 1 ,2-dehydrotestosterone which comprises: a) cultivating a 1,2-dehydrotestosterone producing microorganism in an aqueous nutrient medium under aerobic conditions in the presence of ADD to produce 1 ,2-dehydro-testosterone in the fermentation beer, and b) isolating said 1 ,2-dehydrotestosterone from the fermentation beer.

9. A process as claimed in Claim 8 in which the 1,2-dehydrotestosterone producing microorganism is selected from the group consisting of Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Norcardia, Protaminobacter, Serrata, and Streptomyces, said microorganism being characterized by its ability to selectively degrade ADD and accumulate 1 ,2-dehydrotesto-sterone in the fermentation beer.

10. A process as claimed in Claim 9 in which the testosterone producing microorganism is a Mycobacterium mutant which is characterized by its ability to selectively degrade ADD and accumulate 1,2-dehydrotestosterone in the fermentation beer.

11. A process as claimed in Claim 10 in which the Mycobacterium mutant is Mycobacterium sp. NRRL B-12472.

12. A process as claimed in Claim 1 or Claim 8 substantially as herein described with reference to the Examples.

13. A biologically pure culture of the microorganism Mycobacterium sp. having the identifying characteristics of NRRL B-1 2472, said culture being capable of producing testosterone from AD or a steroid having from 2 to 10 carbon atoms, inclusive, in the 17-alkyl side chain and producing 1,2dehydrotestosterone from ADD, in recoverable quantities upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.