FR2509323A1 - PROCESS FOR THE PRODUCTION OF TESTOSTERONE IN TWO STEPS BY FERMENTATION - Google Patents

Abstract

ACCORDING TO THE INVENTION, TESTOSTERONE IS PREPARED IN TWO STEPS: A TESTOSTERONE-PRODUCING MICRO-ORGANISM IS GROWN IN AN AQUEOUS NUTRITIONAL ENVIRONMENT IN AEROBIC CONDITIONS IN THE PRESENCE OF 4-ANDROSTENE-3,17-DIONE OR A STEROID BEFORE A 17-ALKYL CC SIDE CHAIN INCLUDED TO PRODUCE TESTOSTERONE IN FERMENTATION BROTH; AND B. THE SAID TESTOSTERONE IS ISOLATED FROM THE FERMENTATION BROTH.

Description

The present invention relates to a process for the production

  17-hydroxyandrost-4-en-3-one (testosterone) and 179-

  In particular, the invention relates to a process for the manufacture of testosterone from steroids or 4-androstene-3,17-dione (hereinafter referred to as "steroidal"). AD) by fermentation using a new microorganism of the genus Mycobacterium sp The strain used according to the invention was deposited by the applicant at the Agricultural Research Culture Collection, Northerm Regional Research Center (NRRL), Peoria, Illinois 61 604, USA, under B-12 472 The invention also relates to a method for

  production of 1,2-dehydrotestosterone from 1,4-androsta-

  diene-3,17-dione (hereinafter referred to as ADD) by fermentation with

  the new microorganism Mycobacterium sp NRRL B-12 472.

  The transformation of steroids by microorganisms

  has been widely studied and has been the subject of numerous publications.

  Apparently, the first of these works were done by Mamoli

  and Vercellone in 1937, in Ber 70, page 470 and Ber 70, page 2079.

  These authors describe the reduction of 17-cetosteroids or 17hydroxysteroids by fermentation yeast. More recently, Peterson and Murray describe the 11-hydroxylation of progesterone by the fungus Rhizopus nigricans; see U.S. Patent No. 2,602,769 (1952) Recently, also, Kraychy et al. describe in U.S. Patent No. 3,684,657

  (1972) the selective microbiological degradation of 17-alkyl-

  steroid Ides by fermentation of a steroid containing at least B atoms

  of carbon in the 17-alkyl side-warp by Mycobacterium sp.

  NRRL B-3683 to prepare androst-4-ene-3,17-dione, androst-1,4-

  diene-3,17-dione and oc-hydroxymethyl-pregna-1,4-diene-3-one.

  More recently, Marsheck et al describe in the patent

  United States of America No. 3,759,791 (1973)

  selective biologic of androst-4-ene-3,17-dione with good yield and c-hydroxymethyl-pregna-4-en-3-one with lower yield by fermentation of a steroid Tde containing at least 8 carbon atoms in the 17-alkyl side-chain

Mycobacterium sp NRRL B-3805.

  Microorganisms and their mutants involved in the selective degradation of steroids are largely

  known to those skilled in the art as indicated above.

  U.S. Patent No. 3,759,791 discloses a process for producing precursor androst-4-ene-3,17-dione

  of testosterone, using Mycobacteriumum sp NRRL B-3805.

  Japanese Patent No. 5,5037-121 discloses a process for the production of 1,2-dehydrotestosterone using an oxidative inhibitor and

  a microorganism of the species Mycobacterium or Brevibacterium.

  The present invention particularly relates to a method for the production of testosterone which comprises the following steps:

  a) a microorganism producing testosterone

  terone in an aqueous nutrient medium under anaerobic conditions in

  the presence of AD or a steroid having from 2 to 10 carbon atoms included in the 17-alkyl side chain to produce testosterone in the fermentation broth; and

  b) isolating said testosterone from the fermentation broth.

  The invention also relates to a process for producing

  1,2-dehydrotestosterone which comprises the following steps:

  a) a microorganism producing 1,2-dehydro-

  testosterone in an aqueous nutrient medium under aerobic conditions in the presence of ADD to produce 1,2-dehydrotestosterone in the fermentation broth; and b) said 1,2-dehydrotestosterone is isolated from the broth

fermentation.

  Finally, the invention relates to a biologically cultured

  of the microorganism Mycobacterium sp having the characteristics

  identification of NRRL B-12472, said culture being capable of producing testosterone from androstenedione or a sterotide having from 2 to 10 carbon atoms included in the alkyl side chain and producing the 1, 2-dehydrotestosterone

  from androstadienedione, in recoverable quantities by fermentation.

  in an aqueous nutrient medium containing

  milable carbon, nitrogen and inorganic substances.

  The interest of this method stems in particular from the fact that the steroids of the products are pharmacologically interesting products. See Goodman and Gilman 4th Edition, pages 1566-1580 and Japanese Patent J 55037-121, abbreviated Derwent under No. 66 629 C / 38 Furthermore, the method of the invention gives

  higher yields than the previous microbiological processes

  Finally, this process can be used without

  needs an inhibitor such as a chelating agent.

  The new microorganism Mycobacterium sp NRRL B-12472 was obtained by mutagenesis of the known microorganism Mycobacterium sp NRRL B-3805. The microorganism thus uniquely adapting to the selective microbial degradation according to the invention can be obtained from ARS Culture Collection Research, Fermentations Laboratory, Northern Regional Research Laboratory,

  18 N University Ave, Peoria, IL 61604.

  Other mutants of Mycobacterium and the following genera of microorganisms may be used according to the invention, provided that they are capable of selectively degrading steroids having a C 2 -C 10 17 -alkyl side-chain. including, and accumulating appreciable amounts of testosterone or 1,2-dehydrotestosterone in the fermentation broth. The kinds of microorganisms that fall within the scope of the invention are Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia and Streptomyces The preferred genus is Mycobacterium. Characteristic species of this genus are M. phlei, M. smegmatis, M. rhodochrous, M. mucosum, M. fortuitum, M. butyricum and M. vaccae.

  procedures are described below for the mutation of micro-

  Bodies of the above genera to provide the desired mutants Examples of suitable steroid substrates are sitosterol, cholesterol, stigmasterol, campesterol, and other steroids having C 1 -C 12 -alkyl-side chains contained therein. substrates

  can be either in pure form or in raw form.

  In general, the conditions for the production of testos-

  terone or 1,2-dehydrotestosterone by selective microbial degradation in the process according to the invention are the same as for

  the organism's culture of normal growth, apart from the incor-

  poration of the steroid to be converted, AD or ADD.

  The method according to the invention can be implemented in

  a culture of Mycobacterium sp NRRL B-12472 to another micro-

  organism as described above, either by adding the selected substrate to the culture during the incubation period or by incorporating said substrate into the nutrient medium prior to inoculation. The steroid, AD or ADD, may be added either alone or or in combination with a different AD, ADD and steroid Tde The preferred but non-limiting concentration range of the steroid in the culture is from about 0.1 to 100 g / l. The culture is grown in a nutrient medium containing a steroid. carbon source, for example an assimilable carbohydrate, and a nitrogen source, for example an assimilable nitrogen compound, or a proteinaceous material. The preferred carbon sources include glucose, brown sucrose, sucrose, glycerol, starch, fructose syrup, corn starch, lactose, dextrin, molasses, etc. The preferred nitrogen sources include the quench liquor, yeast, autolysis beer yeast with solids of milk, soy flour, urea, cottonseed meal, corn flour, milk solids, pancreatic digest of casein, fish meal, distillery liquids, animal peptone liquors , meat and bone waste, ammonium salts, nitrates It is advantageous to use combinations of these sources of carbon and nitrogen. It is not necessary to add trace elements to the fermentation medium. eg zinc, manganese, cobalt, iron, etc., since tap water and other ingredients containing these metals are used

as components of the medium.

  The fermentation process can last from about 3 hours to

  4 days The incubation temperature during the fermentation process

  The temperature can be varied from about 25.degree. C. to about 37.degree. C., preferably at 30.degree. C.. The contents of the fermentation vessel are aerated and stirred to facilitate the growth of the microorganism.

  and thus increase the efficiency of the fermentation process.

  When the fermentation is complete, as seen by thin layer chromatography using silica gel plates and a suitable solvent system, the desired transformed steroid is recovered by well known

  Those skilled in the art. For example, the fermentation mixture can be extracted.

  containing the fermentation liquor and the cells with an organic solvent of steroids that is immiscible with water Solvents

  are methylene chloride, chloroform, tetra-

  carbon chloride, ethylene chloride, trichlorethylene,

  ether, amyl acetate, toluene and the like, preferably

methylene chloride.

  We can also separate the fermentation liquor first

  and cells by conventional techniques, for example filtering

  The cells can be extracted with solvents which are either miscible or immiscible with water. The cell-free fermentation liquor can be extracted by means of suitable solvents.

solvents immiscible with water.

  It is possible to filter extracts on diatomaceous earth

  and take the filtrate to dryness under vacuum or by other means.

  The resulting residue containing the desired transformed steroid can then be dissolved in 10% chloroform in methanol and this solution then concentrated with a stream of nitrogen in the steam bath until the crystals appear. The solution can then be cooled. at room temperature and filter to separate the precipitated steroid T It is also possible to obtain the desired transformed steroid Yde from the remaining supernatant liquid after evaporation

solvent of the supernatant liquid.

  The yields are calculated by the formula F

Y O (S-R)

  in which Y represents the yield in%, P the weight of product recrystallized, Q the molecular weight of the product divided by the molecular weight of the substrate, S the initial weight of the substrate and R the weight of recovered substrate. The conversions are calculated by the formula c _ lop QS

  in which C represents the conversion in% and P, Q and S have the.

  same meanings as above.

  The following examples illustrate the invention without, however, limiting its scope. In these examples, the relative amounts of substances are by weight per unit volume, except

otherwise stated.

EXETMPLE 1

  Preparation of the mutant Microbacterium sp NRRL B-12472 from M sp NRRL B-3805

  (a) muta & teninearanitroso & uanidine -

  PH 7.7 is adjusted by potassium hydroxide to

  medium consisting of: 1 g ammonium chloride, 0.5 g phos-

  dipotassium lighthouse, 0.5 g of magnesium sulphate heptahydrate, 0.001 g of ferrous sulphate heptahydrate, 20 g of glycerol, 0.01% of "Tween 80" (polyoxyethylene-20-sorbitan monooleate) and 1 liter of water of The resulting medium (MGYB + "Tween 80") was added in 50 ml portions to 250 ml shake flasks and sterilized by heating for 20 minutes at 121 ° C., this medium was inoculated with 2.5 ml of brine. a liquid culture of Mvcobacterium sp NRRL B-3805 grown on the same medium is incubated culture at + 1 C on a rotary shaker having a circular stroke of

  5.04 cm at 200 rpm for 2 days.

  The crop is pelletisle by centrifugation and on-puts

  in suspension the cells in an equal volume of hard buffer

  0.05 M maleic acid (p H 8.0) containing 0.01% "Tween 80".

  15 ml of tris-maleic acid buffer containing 1 mg / ml of nitrosoguanidine (NTG) is added to 15 ml of the cell suspension to give a final concentration of 500 mg / ml of NTG. The cell suspension is incubated in a shaker in a water-bath for 15 minutes at 300 ° C., after which the suspension is centrifuged once more. The cells are washed three times with a solution of 0.85% NaCl and 0.01% Tween 80 and the mixture is re-opened. suspension

in the medium MGYB + Tween 80.

  (b) Isolation of the mutant MS NRRL Bi-12472 After incubation on the shaker for 1 day at 30 1 C to allow the segregation of the putative mutants of the clumps of cells, the serial culture is diluted with the tris-maleic acid buffer with Tween 80 Aliquots are placed at appropriate dilutions on the previously prepared plate surface containing 20 to 25 ml of CGYA medium of the following composition: 20 g of Trypticase soy broth (Baltimore Biological Laboratories), 5 g of yeast extract, 20 g of glycerol, 15 g of agar and 1 liter of tap water and which are sterilized for 20 min at 121 ° C. The

  plates at 30 + 1 C for 7 days.

(c) Shaker tube evaluation

_______________________________

  The resulting colonies that occur on the agar plates are randomly removed and transferred to test tubes containing 4 ml of CGYB medium (CGYA medium in which the agar has been removed). The cultures are incubated statistically for 7 days. days at 30 / + 1 C. 1/2 ml portions of the inoculum cultures are then transferred to test tubes (25 x 125 mm) containing 10 ml of MGYB medium which has been

  added about 150 mg of sitosterols, N F before sterilization.

  After 7 days of incubation on the shaker at 30 + 1 C, the cultures are extracted in shaker tubes with 10 ml of methylene chloride. The chloromethylenic extracts are analyzed by thin layer chromatography using silica gel and

  a toluene-ethanol solvent system 90:10 (by volume).

  The presence of appreciable amounts of testosterone confirms the selective degradation of sitostero Yde by the new mutant produced

  from the parent strain of M sp NRRL B-3805.

EXAMPLE 2

  Transformation of AD into testosterone 50 ml portions of MGB medium are added to vials

  250 ml shaker containing 500 mg AD The MGB medium is preferably

  Same as the MGYB medium except that the glycerol is removed and 2 ml of sterile 50% dextrose solution is added to each vial after autoclaving. Five vials of 14 GB M medium are inoculated with 2.5 ml of liquid culture of M sp NRRL B-12472 and

  Incubate at 30 + 1C for 7 days on the shaker.

  The combined culture broth (250 ml) is extracted three times with methylene chloride. The combined extracts are dried over anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo. The residue is redissolved in the reaction mixture. ethyl-toluene (1: 5 by volume) and chromatographed on a column containing 80 g of Woelm silica gel. The column is eluted with ethyl acetate-toluene (1: 5 by volume) and the fractions are monitored. by thin layer chromatography The fractions containing testosterone are evaporated under vacuum to obtain the product

  which is analyzed by gas chromatography (determined by

  quantitative determination by gas chromatography: 3% OV-17 / OV-210 on "Chromosorb W / HP", O, 149-0.177 mm; at 240 C; using a

  flame ionization detector for a purity of more than 80%.

  The conversion efficiency is 28% for 1 AD Spectra

  physicochemical, IR and NMR, are consistent for testosterone -

EXAMPLE 3

  Transformation of Sistosterol into Testosterone The method according to Example 2 is used, except that V'AD is replaced by 750 mg of sitosterol N F. The whole of the combined culture broth is also treated as in Example 2.

conversion efficiency 19%.

EXAMPLE 4

  Transformation of & DD into testosterone.

  A method according to Example 2 is used except that the AD is replaced by V'ADD. The combined culture broth of 5 shake flasks is extracted three times with an equal volume of methylene chloride. organic extracts combined on anhydrous sodium sulphate, filtered, then evaporated to dryness under vacuum Testosterone is determined in the residue by

  gas chromatography, conversion efficiency 69%.

EXAMPLE 5

  A process according to Example 2 is used except that 1.050 g of the dried chloromethylenic extract is chromatographed (39% assayed by gas chromatography) and the Woelm silica column is successively eluted using proportions and volumes. 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, determined by thin layer chromatography, are pooled and evaporated to obtain 0.3487 g of solids. Recrystallization from aqueous acetone gives 0.307 g of testosterone; F 153.5-154.5 ° C, Amax 241 mm (E = 16000) λ1O = + 120.7 (c = 1.007 in CHCl3) IR spectrum (K Br pellet) and the NMR spectrum (CDC 13 ) are identical to those of a

  testosterone sample known.

EXAMPLE 6

  A process according to Example 2 is used except that AD is replaced by 500 mg of NF sitosterols. One half of the residue of the dried extract is treated with 0.445 g (25% testosterone and% AD). determined by gas chromatography) as in Example 4. Testosterone-containing fractions, determined by thin layer chromatography, were combined and evaporated to give 0.135 g of dry residue. Recrystallized from aqueous acetate to give

  0.074 g of testosterone, F 153-153.5 ° C, Xmax 241 mm (t = 16,600).

  The IR spectrum (K Br pellet) and the NMR spectrum are identical

  to those of a known sample of testosterone.

EXAMPLE 7

  A method according to Example 2 is used except that the AD is replaced by 500 mg of ADD. Thin layer chromatography of the dried methylene chloride extract, 0.957 g, indicates that the main constituent is 1 2-dehydrotestosterone, with lower amounts of ADD and testosterone. The extract is chromatographed in the same manner as in Example 4.

  evaporates the fractions containing 1,2-dehydrotestosterone, deter-

  The residue was recrystallized from 0.719 g in ethyl acetate to give 0.510 g of 1,2-dehydrotestosterone, F 170-172 ° C, and 21.7 ° C (1.012 in CHRC 13). kmax 244.5 mm (t = 14 640) The IR spectrum (K Br pellet) and the NMR spectrum (CD C 13) are identical to those of a known sample of 1,2-dehydrotestosterone No impurities are detected in products isolated by gas chromatographic analysis It is understood that the invention is not limited to the preferred embodiments described above by way of illustration and that one skilled in the art can make use of it. various modifications and changes without departing from the scope and spirit of the invention

Claims (8)

CLAIMS _____________ _____________   A process for the production of testosterone, characterized in that it comprises the following steps: a) a testosterone-producing microorganism is cultured in an aqueous nutrient medium under aerobic conditions in the presence of 4-androstene-3-17-dione or a Yde steroid having a C 2 -C 18 17 -alkyl side chain included to produce testosterone in the fermentation broth; and   b) isolating said testosterone from the fermentation broth.   2 Process according larevendication 1, characterized in that the microorganism is cultured in the presence of 4-androstene 3,17-dione 3 Process according to claim 1, characterized in that the microorganism is cultured in the presence of a Yde steroid having a   17 C-C 1 -C alkyl side-chain included.   4 Process according to claim 3, characterized in that said steroid Mde is chosen from sitosterol, cholesterol, stigmasterol and campesterol.   Process according to Claim 1, characterized in that the said testosterone-producing microorganism is a mutant chosen from the Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacter, Serratia and Streptomyces species, capable of selectively degrading 4-androstene-3. , 17-dione or a steroid Tde having a 17-alkyl side chain including C 2 -C 10 and accumulating testosterone in the fermentation broth.   Process according to claim 5, characterized in that   said mutant is a mutant of Mycobacterium.   Method according to claim 6, characterized in that   said mutant is Mycobacterium sp NRRL B-12472.   Process for the production of 1,2-dehydrotestosterone, characterized in that it comprises the following steps:   a) a microorganism producing 1,2-dehydrotestos is cultivated   terone in an aqueous nutrient medium under aerobic conditions in the presence of 1,4-androstadiene-3,17-dione to produce 1,2-dehydrotestosterone in the fermentation broth and   b) said 1,2-dehydrotestosterone is isolated from the fermentation broth   tion. 11. Process according to claim 8, characterized in that said 1,2-dehydrotestosterone-producing microorganism is chosen from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Protaminobacterium Serratia and Streptomyces and capable of selectively degrading. the   1,4-androstadiene-3,17-dione and accumulate 1,2-dehydrotestos-   terona in the fermentation broth.   Process according to claim 9, characterized in that said microorganism producing 1,2-dehydrotestosterone is a Mycobacterium mutant capable of selectively degrading the   1,4-androstadiene-3,17-dione and accumulate 1,2-dehydrotestos-   terona in the fermentation broth.   Process according to claim 10, characterized in that   said mutant is Mycobacterium sp NRRL B-12472.   12 Biologically pure culture of microorganism Mycobacterium sp having the identification characteristics of N RL B-12472, characterized in that it is capable of producing testosterone from 4-androstene-3,17-dione or a stero Tde having a chalne   side 17-C 2 -C 10 alkyl and to produce 1,2-dehydro-   testosterone from 1,4-androstadiene-3,17-dione, in amounts recoverable by fermentation in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen and inorganic substances.