Microbial transformation of steroids
BACKGROUND OF THE INVENTION 
     The transformation of steroids by microorganisms has been widely studied and documented. Apparently, the earliest such work was by Mamoli and Vercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed the reduction of 17-ketosteroids to 17β-hydroxysteroids by fermenting yeast. More recently, Peterson and Murray disclosed the 11α-hydroxylation of progesterone with the fungus Rhizopus nigricans; see, U.S. Pat. No. 2,602,769 (1952). Also recently, Kraychy et al. in U.S. Pat. No. 3,684,657 (1972) discloses the selective microbiological degradation of steroidal 17-alkyls by fermenting a steroid containing at least 8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 to prepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and 20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck et al. in U.S. Pat. No. 3,759,791 (1973) disclose the selective microbiological preparation of androst-4-ene3,17-dione by fermenting a steroid of the cholestane or stigmastane series containing at least 8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805. 
     Also pertinent in the background of the subject invention is the article by Schubert, K., Bohme, K-H., and Horhold, C. entitled &#34;Formation of low molecular degradation products from progesterone by microorganisms&#34;. Steroids 4, 581-586 (1964). These authors described the formation of tricyclic intermediates during the degradation of progesterone by Mycobacterium smegmatis. 
     The culture of the subject invention process, Mycobacterium fortuitum NRRL B-8129 is disclosed in pending Applications Ser. Nos. 632,635, filed on Nov. 17, 1975, now U.S. Pat. No. 4,039,381, and 745,113 filed on Nov. 26, 1976. The process conditions disclosed in Ser. Nos. 632,635 and 745,113 are not conducive to the optimal production of compound I of the subject application. 
     BRIEF SUMMARY OF THE INVENTION 
     A novel microbial transformation process using mutants which are characterized by their ability to selectively degrade steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, and accumulate predominantly compound I in the fermentation beer. 
     These mutants can be obtained from sterol-degrading microorganisms of the following genera by using the mutation procedures disclosed herein or other mutation procedures Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. A preferred genus is Mycobacterium. Exemplary species of this genus are M. phlei, M. smegmatis, M. rhodochrous, M. mucosum, M. fortuitum, and M. butyricum. Specifically exemplified herein is a mutant microorganism, Mycobacterium fortuitum, NRRL B-8129, which is used to selectively degrade steroids with or without 17-alkyl chains containing from 2 to 10 carbon atoms, inclusive, to compound I. Examples of suitable steroids are sitosterols, cholesterol, stigmasterol, campesterol, and like steroids with 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, and androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, testosterone, and bis-nor acid (3-ketobisnorchol-4-en-22-oic acid). These steroid substrates can be in either the pure or crude form. 
     Also produced in the fermentation beer are lesser amounts of 3aα-H-4α- 3&#39;-propanal!-5α-hydroxy-7aβ-methylhexahydro-1-indanone hemiacetal ##STR2## hereinafter referred to as Compound II; 
     3aα-H-4α- 3&#39;-propionic acid!-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone ##STR3## hereinafter referred to as Compound III; 
     3aα-H-4α- 3&#39;-propanol!-5α-hydroxy-7aβ-methylhexahydro-1-indanone ##STR4## hereinafter referred to as Compound IV; and 
     3aα-H-4α- 3&#39;-propionic acid!-7aβ-methylhexahydro-1,5-indanedione ##STR5## hereinafter referred to as Compound V. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The Microorganisms 
     Mutants which are characterized by their ability to selectively degrade steroids with or without 17-alkyl side chains containing from 2 to 10 carbon atoms, inclusive, and accumulate predominantly compound I in the fermentation beer, under controlled fermentation conditions, as disclosed herein, can be obtained by mutating sterol-degrading microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. Mycobacterium fortuitum, ATCC 6842, has been mutated, as disclosed herein, to give a laboratory mutant microorganism. The 1974 ATCC Catalogue discloses the following alongside the listing of ATCC 6842: &#34;J.C. Cruz 2. Cold abscess. Acta Med. Rio de Janeiro 1:1 (1936). Medium 90 37C&#34;. M. fortuitum, ATCC 6842, degrades sterols non-selectively to small molecular weight compounds, e.g., CO 2  +H 2  O. Tnus, this microorganism is not suitable as a selective steroid degrader. 
     Mutation of M. fortuitum, ATCC 6842, using nitrosoguanidine has resulted in the production of a mutant which selectively degrades steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, to produce predominantly compound I in the fermentation beer. This mutant microorganism of M. fortuitum has been given the accession number NRRL B-8129, by the Northern Regional Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A., where it has been deposited in the permanent collection. A subculture of this microorganism is freely available from this depository by request made thereto. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action. 
     The morphology and drug sensitivities of M. fortuitum, NRRL B-8129, are indistinguishable from that of the parent M. fortuitum, ATCC 6842. Both M. fortuitum cultures are acid-fast non-motile, non-spore-forming bacilli belonging to the family Mycobacteriaceae of the order Actinomycetales. According to Runyon&#39;s classification (Runyon, E. H. 1959 Med. Clin. North America 43: 273) it is a nonchromogenic group IV mycobacterium, i.e., it grows rapidly at low temperature to produce nonpigmented colonies on relatively simple media. 
     M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8129, are clearly distinguishable in their action on steroid molecules. As disclosed above, M. fortuitum ATCC 6842 is a degrader of steroids, whereas M. fortuitum NRRL B-8129 is a selective degrader. This property of M. fortuitum NRRL B-8129 makes it highly useful, as disclosed herein. 
     The mutation of M. fortuitum ATCC 6842 to give M. fortuitum NRRL B-8129 was accomplished by the use of nitrosoguanidine. The details of the procedure are described infra. Though mutation procedures are generally known in the art, there is no known art which teaches or even suggests the type of mutants, if any, which might be obtained by use of the subject mutation procedure. Also, though the mutation and transformation procedures, disclosed herein, are detailed for a Mycobacterium, it should be understood that similar or equivalent procedures can be used with microorganisms of the other genera, as disclosed herein. 
     The Transformation Process 
     The selective transformation of the subject invention can be effected in a growing culture of M. fortuitum NRRL B-8129 by either adding the selected steroid substrate to the culture during the incubation period, or incorporating it in the nutrient medium prior to inoculation. The steroid can be added singly or in combination with another steroid. The preferred, but not limiting, range of concentration of the steroid in the culture is about 0.1 to about 100 grams per liter. The culture is grown 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, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, urea, distillers&#39; solids, animal peptone liquors, meat and bone scraps, ammonium salts and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium. Prior to sterilization of the medium the pH is adjusted to a pH of about 6.5. The pH adjustment can be made with a strong base, for example, NaOH, KOH, and the like. 
     A critical feature in the invention process is to maintain the pH at about 5.5 or lower during the fermentation in order to produce predominantly compound I. Preferably the pH is maintained at about 3.0 to about 6.0. This pH maintenance can be done by procedures well known in the art, for example, by use of CaCO 3  or phosphate buffer in the medium, or by pH control with a base, for example, sodium hydroxide, ammonium hydroxide, and the like, or with an acid, for example, HCl, H 2  SO 4 , and the like. Further, the pH can be maintained at the desired level by the use of excess amounts of nutritional ingredients the metabolism of which results in a lowered pH. Examples of such compounds include soluble ammonium salts, carbohydrates such as glucose and other mono- and disaccharides, starch and other polysaccharides, and oils and fats such as lard oil and soybean oil. 
     The transformation process can range from about 72 hours to 15 days. The incubation temperature during the transformation process can range from about 25° C. to about 37° C., with 31° C. being preferred. The contents of the transformation vessel are aerated with sterilized air 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 (E. Merck, Darmstadt) and a solvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane, 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, trichloroethylene, ether, amyl acetate, benzene 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-miscible or 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 vacuum distilled to dryness. The resulting residue containing the desired transformed steroid then can be dissolved in 10% chloroform in Skellysolve B and chromatographed on silica gel, using Skellysolve B (isomeric hexanes) and mixtures thereof with increasing amounts of ethyl acetate as developing solvent. This procedure elutes the desired compound. Upon recrystallization from ethyl acetate, compound 1, a 1:1 mixture of two epimers, melts at 100°-112° C. A crystalline product can be obtained by use of a solvent, for example, ethyl acetate. The desired transformed steroid can also be obtained from the remaining supernatant upon evaporation of the solvent in the supernatant. 
     Compound I is useful as an intermediate in the chemical synthesis of useful steroids. For example, it can be converted to starting material for the process disclosed in U.S. Pat. No. 3,880,884 which discloses a process for the total synthesis of useful 19-nor steroids. This conversion to starting material can be done by procedures known in the art. See J.A.C.S. 85: 2135-2137. 
     The following examples are illustrative of the process of the subject invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. 
    
    
     EXAMPLE 1 
     Preparation of Mutant M. fortuitum NRRL B-8129 From M. fortuitum ATCC 6842 
     (a) Nitrosoguanidine Mutagenesis 
     Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the following sterile seed medium: 
     
         ______________________________________                                    
Nutrient Broth (Difco)                                                    
                     8       g/liter                                      
Yeast Extract        1       g/liter                                      
Sodium Propionate    0.5     g/liter                                      
Distilled Water, q.s.                                                     
                     1       liter                                        
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     The pH is adjusted to 7.0 with 1N NaOH prior to sterilization at 121° C. for 20 minutes. 
     The cells are grown to a density of about 5 × 10 8  per ml, pelleted by centrifugation, and then washed with an equal volume of sterile 0.1 M sodium citrate, pH 5.6. Washed cells are resuspended in the same volume of citrate buffer, a sample removed for titering (cell count), and nitrosoguanidine added to a final concentration of 50 μg/ml. The cell suspension is incubated at 37° C. in a water bath for 30 minutes, after which a sample is again removed for titering and the remainder centrifuged down and washed with an equal volume of sterile 0.1 M potassium phosphate, pH 7.0. Finally, the cells are resuspended in a sterile minimal salts medium, minus a carbon source, consisting of the following: 
     
         ______________________________________                                    
NH.sub.4 NO.sub.3    1.0      g/liter                                     
K.sub.2 HPO.sub.4    0.25     g/liter                                     
MgSO.sub.4  . 7H.sub.2 O                                                  
                     0.25     g/liter                                     
NaCl                 0.005    g/liter                                     
FeSO.sub.4  . 7H.sub.2 O                                                  
                     0.001    g/liter                                     
Distilled Water, q.s.                                                     
                     1        liter                                       
______________________________________                                    
 
    
     The pH is adjusted to 7.0 with 1N HCl prior to sterilization at 121° C. for 20 minutes. The cells are then plated out to select for mutants. 
     (b) Selection And Isolation Of Mutant M. fortuitum NRRL B-8129 
     Mutagenized cells, as described above, are diluted and spread onto plates containing a medium consisting of the following (modified from Fraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295): 
     
         ______________________________________                                    
Glycerol             10.0     g/liter                                     
Na.sub.2 HPO.sub.4   8.4      g/liter                                     
KH.sub.2 PO.sub.4    4.5      g/liter                                     
NH.sub.4 Cl          2.0      g/liter                                     
MgSO.sub.4 . 7H.sub.2 O                                                   
                     0.3      g/liter                                     
FeCl.sub.3  . 6H.sub.2 O                                                  
                     0.05     g/liter                                     
Distilled Water, q.s.                                                     
                     1        liter                                       
______________________________________                                    
 
    
     Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for 30 minutes and then poured into sterile Petri plates. 
     Growth on this medium eliminates most nutritional auxotrophs produced by the mutagenesis procedure, e.g. cultures that require vitamins, growth factors, etc. in order to grow on chemically defined medium are eliminated. After incubation at 28° C. for about 7 days, the resulting colonies are replicated to test plates suitable for selecting mutants and then back onto control plates containing the glycerol-based medium. The test plates are prepared as described by Peterson, G. E., H. L. Lewis and J. R. Davis. 1962. &#34;Preparation of uniform dispersions of cholesterol and other water-insoluble carbon sources in agar media.&#34; J. Lipid Research 3: 275-276. The minimal salts medium in these plates is as described above in section (a) of Example 1. Agar (15 g/liter), and an appropriate carbon source (1.0 g/liter), such as sitosterol or androstenedione (AD), are added and the resulting suspension autoclaved for 30 minutes at 121° C. The sterile, hot mixture is then poured into a sterile blender vessel, blended for several minutes, and then poured into sterile Petri plates. Foaming tends to be a problem in this procedure but can be reduced by blending when the mixture is hot and by flaming the surface of the molten agar plates. In this manner uniform dispersions of water-insoluble carbon sources are obtained which facilitates the preparation of very homogenous but opaque agar plates. 
     Colonies which grew on the control plates, but not on test plates containing AD as the sole carbon source, are purified by streaking onto nutrient agar plates. After growth at 28° C., individual clones are picked from the nutrient agar plates with sterile toothpicks and retested by inoculating gridded plates containing AD as the carbon source. Purified isolates which still exhibit a phenotype different from the parental culture are then evaluated in shake flasks. 
     (c) Shake Flask Evaluation 
     Shake flasks (500 ml) contain 100 ml of biotransformation medium consisting of the following ingredients: 
     
         ______________________________________                                    
Glycerol             10.0     g/liter                                     
Na.sub.2 HPO.sub.4   8.4      g/liter                                     
KH.sub.2 PO.sub.4    4.5      g/liter                                     
NH.sub.4 Cl          2.0      g/liter                                     
MgSO.sub.4 . 7H.sub.2 O                                                   
                     0.3      g/liter                                     
FeCl.sub.3  . 6H.sub.2 O                                                  
                     0.05     g/liter                                     
Distilled Water, q.s.                                                     
                     1        liter                                       
______________________________________                                    
 
    
     Soyflour (1 g/liter) is blended into the medium and then sitosterol (10 g/liter) is also blended into the medium. After the flasks are autoclaved for 20 minutes at 121° C., they are cooled to 28° C. and then inoculated with 10 ml of seed growth prepared as follows: 
     The purified isolates from part (b) are grown on agar slants at 28° C. A loop of cells taken from a slant is used to inoculate a 500-ml flask containing 100 ml of sterile seed medium consisting of the following ingredients: 
     
         ______________________________________                                    
Nutrient Broth (Difco)                                                    
                     8      g/liter                                       
Yeast Extract        1      g/liter                                       
Glycerol             5      g/liter                                       
Distilled Water, q.s.                                                     
                     1      liter                                         
______________________________________                                    
 
    
     The pH is adjusted to 7.0 with 1N NaOH prior to autoclaving the flasks at 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for 72 hours. 
     As disclosed above, 10 ml of seed growth is then used to inoculate each 500-ml flask containing 100 ml of sterile transformation medium. The flasks are then incubated at 28° C. to 30° C. on a rotary shaker and sampled at various intervals. Ten ml samples are removed and extracted by shaking with 3 volumes of methylene chloride. Portions of the extracts are analyzed by thin layer chromatography using silica gel and the solvent system described above, i.e., 2:3 (by volume) ethyl acetate-cyclohexane, and by gas-liquid chromatography. Evidence of the presence of compounds I and V confirms the selective degradation of sitosterol by the novel mutant produced from the parent M. fortuitum ATCC 6842. 
     EXAMPLE 2 
     Transformation of Sitosterol To Compound I 
     The medium used is the same as in Example 1 (c), except that it is adjusted to a pH of about 6.5 with 4N NaOH. This medium is sterilized by heating 30 minutes at 121° C., whereupon it is cooled to 28° C. and then inoculated with 10 parts of a seed culture of the mutant M. fortuitum NRRL B-8129, prepared as described in Example 1 (c). The inoculated mixture is incubated at 28° C. for 168 hours with agitation to promote submerged growth. As required, the pH is maintained at about 5.0 by the addition of NaOH, or an acid, for example, HCl. Progress of the biotransformation is followed by chromatography on thin layer silica gel plates in a solvent system consisting of 3:2 (by volume) cyclohexane-ethyl acetate. 
     Upon completion of the biotransformation the desired product is isolated by the following procedure. The reaction mixture is extracted with methylene chloride. This extract is filtered through diatomaceous earth and the filtrate is distilled to dryness. The residue from the filtrate is taken up in 10% chloroform in Skellysolve B and chromatographed on silica gel, using Skellysolve B and mixtures thereof with increasing amounts of ethyl acetate as developing solvent. This procedure elutes the desired compound I which has an R f  value of 0.37 on thin layer chromatography (cyclohexane-ethyl acetate; 3:2). Compound I melts at 100°-112° C. 
     EXAMPLE 3 
     By substituting cholesterol for sitosterol in Example 2 there is obtained compound I. 
     EXAMPLE 4 
     By substituting stigmasterol in Example 2 for sitosterol there is obtained compound I. 
     EXAMPLE 5 
     By substituting campesterol for sitosterol in Example 2 there is obtained compound I. 
     EXAMPLE 6 
     By adding a combination of any of the sterols in Examples 2-5, in addition to sitosterol, or in place of sitosterol, in Example 2 there is obtained compound I. 
     EXAMPLE 7 
     By substituting a sterol-degrading microorganism from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces, in Example 1 for Mycobacterium fortuitum ATCC 6842 there are obtained mutant microorganisms which are characterized by their ability to selectively degrade steroids with or without a 17-alkyl side chain of from 2 to 10 carbon atoms, inclusive, and accumulate predominantly compound I in the fermentation beer. 
     EXAMPLE 8 
     By substituting the mutants obtained in Example 7 for M. fortuitum NRRL B-8129 in Examples 2-6, there is obtained compound I. 
     EXAMPLE 9 
     By substituting a sterol-degrading microorganism selected from the group consisting of Mycobacterium phlei, M. smegmatis, M. rhodochrous, M. mucosum, and M. butyricum for M. fortuitum ATCC 6842 in Example 1 there are obtained mutant microorganisms which are characterized by their ability to selectively degrade steroids with or without a 17-alkyl side chain of from 2 to 10 carbon atoms, inclusive, and accumulate predominantly compound I in the fermentation beer. 
     EXAMPLE 10 
     By substituting the mutants obtained in Example 9 for M. fortuitum NRRL B-8129 in Examples 2-6, there is obtained compound I. 
     EXAMPLE 11 
     By substituting a compound selected from the group consisting of androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, testosterone, and bis-nor acid for sitosterol in Example 2 there is obtained compound I. 
     EXAMPLE 12 
     By substituting a combination of two or more compounds selected from the group consisting of sitosterol, cholesterol, stigmasterol, androst-4-ene-3-17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, testosterone, and bis-nor acid for sitosterol in Example 2 there is obtained compound I. 
     EXAMPLE 13 
     By substituting the mutants obtained in Example 7 for M. fortuitum NRRL B-8129 in Examples 11 and 12, there is obtained compound I. 
     EXAMPLE 14 
     By substituting the mutants obtained in Example 9 for M. fortuitum NRRL B-8129 in Examples 11 and 12, there is obtained compound I.