Abstract:
The subject invention concerns a novel one-stage fermentation process for making the useful steroid intermediate 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid (I). This process is significantly superior to the best prior art process known for making (I).

Description:
DESCRIPTION 
     BACKGROUND OF THE INVENTION 
     U.S. Pat. No. 4,029,549 discloses and claims the use of Mycobacterium fortuitum NRRL B-8119 to make 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid [9-hydroxybisnoracid]. The same microbe is used to make 9-hydroxy-4-androstene-3,17-dione [9-hydroxyandrostenedione] in U.S. Pat. No. 4,035,236; and 9-hydroxy-3-oxo-4-pregnene-20-carboxylic acid methyl ester [9-hydroxybisnoracid methyl ester] in U.S. Pat. No. 4,214,051. 
     European Patent Application 79104372.2 discloses a two-stage fermentation for preparing 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid (I) which is useful as an intermediate in the synthesis of valuable corticoids. This process entails first the conversion of sterols to 3-oxo-4,17(20)-pregnadiene-20-carboxylic acid by fermentation with Mycobacterium strain NRRL B-8054, and then conversion of this compound to (I) by incubation with any one of several microorganisms capable of introducing a hydroxyl group in the 9α position. 
     BRIEF SUMMARY OF THE INVENTION 
     Disclosed and claimed is an efficient one-stage fermentation process for preparing the useful intermediate 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid (I). This process is conducted by use of a novel mutant of M. fortuitum NRRL B-8119. The subject invention process also encompasses the use of novel double mutants obtained from the genera of microorganisms disclosed in U.S. Pat. No. 4,029,549, i.e., Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. The microorganisms of these genera are all well known sterol degraders. The wild type strains of these genera degrade sterols non-selectively to small molecular weight compounds, e.g. CO 2  +H 2  O. Mutants can be made from these wild types by following the procedures disclosed in U.S. Pat. No. 4,029,549, Example 1. This example discloses the preparation of M. fortuitum NRRL B-8119. 
     Mutants of the genera, disclosed above, which can be made by using the procedures of Example 1 of U.S. Pat. No. 4,029,549, can then be subjected to the mutation procedures, disclosed herein, to prepare further mutants. These latter mutants, as exemplified here by M. fortuitum NRRL B-12433, can be used in the one-stage fermentation process, disclosed herein, to prepare compound (I). 
     The subject one-stage fermentation process is a vast improvement over the two-stage process for preparing compound (I) discussed supra. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The Microorganisms 
     Mutants which are characterized by their ability to selectively degrade steroids having 17-alkyl side chains and accumulate 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid (I) as a major product in the fermentation beer can be obtained by mutating microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. 
     Following is an example of the preparation of the novel mutant used in the subject one-stage fermentation process. The mutant prepared in this example is M. fortuitum NRRL B-12433. Similar mutants from other Mycobacterium species and other microbe genera, as recited herein, can be prepared by following the procedures of the following example. 
     Preparation of a mutant which accumulates 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid as a major product of the degradation of sterols 
     Mycobacterium fortuitum NRRL B-8119 is grown at 31° in a medium consisting of (per liter) nutrient broth, 8 g; yeast extract, 1 g; glycerol, 5 g; Tween 80, 0.1% (w/v); and distilled H 2  O. This medium is sterilized by autoclaving at 15 lb/in 2  for 20 min. The cells are grown to a density of about 5×10 8  per ml, and then collected on a sterile 0.2 micron filter. The cells are washed with an equal volume of sterile 0.1 M sodium citrate, pH 5.6 containing 0.1% Tween 80, and then resuspended in 1/2 volume of the same buffer. N-methyl-N&#39;-nitro-N-nitrosoguanidine is added to a concentration of 100 μg/ml and the cell suspension is incubated at 31° C. for 1 hr. The cells are then washed with 2 volumes of sterile, 0.1 M potassium phosphate buffer, pH 7 containing 0.1% Tween 80, and then resuspended in 1 volume of the same buffer. A medium is prepared containing (per liter) nutrient broth, 8 g; NaCl, 5 g; glycerol, 5 g; and distilled H 2  O. Agar is added to 15 g/l and the medium is autoclaved at 15 lb/in 2  for 20 min and then poured into sterile Petri dishes. The mutagenized cells are then plated on this medium and colonies which grow on these plates are subsequently screened in small scale fermentations for their ability to convert sterols to compound (I). Detection of the desired compound is by thin layer chromatography of methylene chloride extracts of the test fermentations, using silica gel and the solvent system methylene chloride-acetone-acetic acid (212-38-1). In this manner, mutant NRRL B-12433 is isolated which accumulates 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid as a major product of the bioconversion of sterols. 
     The key to isolating a mutant like the one described herein is to start with a mutant, such as NRRL B-8119, which is already blocked in steroid ring degradation so that it produces 9α-hydroxyandrostenedione, and introduce into this microorganism a second mutation affecting sterol side chain degradation. 
     Description of the Microorganism 
     The mutant bringing about the biotransformation described herein differs from its parent culture, e.g., Mycobacterium fortuitum NRRL B-8119, only in its action on steroid molecules. In all other respects, such as morphology and drug sensitivities, they are similar if not identical. Both M. fortuitum cultures are acid-fast non-motile, non-sporulating 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 NRRL B-8119 and NRRL B-12433 have been deposited in the permanent collection at the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. M. fortuitum NRRL B-8119 has been available to the public at least since issuance of the above-mentioned U.S. patents disclosing the microbe. M. fortuitum NRRL B-12433 was deposited on May 4, 1981. Subcultures of these microorganisms are available from the NRRL 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. 
     Compound (I) is useful as an intermediate in the synthesis of valuable corticoids. For example, it can be converted to hydrocortisone acetate by following the procedure detailed in published European Patent Application 79104372.2. 
    
    
     Following are examples which illustrate the one-stage fermentation process of the subject invention. These examples are merely illustrative, and, thus, should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. 
     Example 1--Fermentation of Crude Sitosterol 
     The biotransformation medium contains (per liter) Ucon, 8.0 g; Cerelose, 5.0 g; NH 4  Cl, 3.0 g; CaCO 3 , 3.0 g; Na 3  [citrate].2H 2  O, 3.0 g; Tween 80, 2.0 g; soyflour, 1.0 g; KH 2  PO 4 , 0.5 g; urea, 0.5 g and crude sitosterol, 30.0 g in tap water with the pH adjusted to 7.0. Flasks containing 100 ml portions of this medium are innoculated with 10 ml of seed cultures of M. fortuitum NRRL B-12433, grown at 28° in a medium containing (per liter) nutrient broth, 8.0 g; glycerol, 5.0 g; yeast extract, 1.0 g and Tween 80, 1.0 g in distilled water with the pH adjusted to 7.0. The cultures are then incubated at 28° for 336 hr on a rotary shaker. Following incubation, the mixture is extracted and the product isolated as detailed below in Example 3. 
     Example 2 
     Just as in Example 1, but with various steroidal substrates provided singly or in combination and in pure or crude form. Such substrates include sitosterol, cholesterol, stigmasterol and campesterol. 
     Example 3--Isolation of (I) from M. fortuitum NRRL B-12433 Fermentation 
     Fermentation beer (1200 ml) from a sitosterol bioconversion using M. fortuitum NRRL B-12433 is acidified and extracted twice with an equal volume of methylene chloride (MeCl 2 ), giving 29.2 g and 8.9 g crude extract respectively. 
     The first extract is redissolved in MeCl 2  and washed with saturated sodium bicarbonate solution. The aqueous washes are pooled, acidified with dilute HCl (4-N) and back-extracted with MeCl 2 . The extract is dried over magnesium sulfate and filtered. Evaporation of the solvent leaves a yellow solid that is triturated with MeCl 2  to give a pale yellow powder. This is crystallized from a mixture of MeCl 2  and methanol, from which three successive crops are obtained: (a) 3.8 g; (b) 2.1 g; and (c) 2.8 g. These are shown by nmr spectrometry to be mixtures of acids with the Δ 17 (20) -dehydro compound (I) in concentrations of 90%, 35%, and 10%, respectively. 
     Three recrystallizations of the first crop from MeCl 2  /MeOH gives 1.8 g of 9-hydroxy-3-oxo-4,17(20)-pregnadiene-20-carboxylic acid (I), mp 240°-242°, [α] D  40.7° (methanol). The mass spectrum gives a molecular ion at m/e 358 (C 22  H 30  O 4 ). 
     Example 4 
     By substituting a sterol-degrading microorganism from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces, for Mycobacterium fortuitum NRRL B-8119 in the process disclosed for preparing M. fortuitum NRRL B-12433, there are obtained mutant microorganisms which are characterized by their ability to selectively degrade steroids with a C-17 side chain and accumulate compound (I) as a major product. 
     Example 5 
     By substituting the mutants obtained in Example 4 for M. fortuitum NRRL B-12433 in Example 1, there is obtained compound (I).