The oxidation of dehydroepiandrosterone (DHEA), 4-androstene-3,17-dione, and estrone with Xtreptomyces roseochromogenes NRRL B-1233 was studied. The oxidation products were isolated and identified as 16a-hydroxy-DHEA, 16a-hydroxy-4-androstene-3,17-dione and 16a-hydroxyestrone. The yields of these three products were 85%, 41% and ISYO, respectively. This indicates the substrate stereospecificity of 16a-hydroxylase of the organism. An interrelationship between cell growth and the formation of 16a-hydroxylated steroid was observed in any case. For formation of 16a-hydroxy-DHEA, 16a-hydroxylase showed good activity a t DHEA concentration of 3.47 x 10-4 M. I n the case of DHEA, 16a-hydroxy-4-androstene-3,17-dione and 5-androstene-3/3, 16a, 17,!?-triol were obtained after the yield of 168-hydroxy-DHEA reached the maximum yield for about 30 hr. The oxidation pathway of DHEA is discussed.The microbiological transformation of steroids has been well documented (TAMM 1962 1970). We observed the 16ci-hydroxylation of readily available DHEA during the microbial oxidation of some pharmacologically active C,,-steroid substrates. The 16a-hydroxylation of DHEA has not previously been observed.The present paper describes the microbial oxidation of DHEA, 4-androstene-3,17-dione, and estrogen by Streptomyces roseochromogenes (NRRL B-1233).
Materials and methodsCultivation: Xtreptomyces roseochmnogenes NRRL B-1233 was the most appropriate microorganism for our purpose. This stock culture was maintained on BENNET agar medium. Inoculum was prepared for 48 hr-grown cells on 10 ml of the following medium on rotary shaker (220 rpm) a t 27 'C: 3% glucose, 2y0 soy bean meal, 0.22% soy bean oil, and 0.25% CaCO, in distilled water. It is employed for inoculating 100 ml of the same medium placed in a 500 ml Erlenmeyer flask and cultivated a t 27 "C on the shaker under the above conditions. At the logarithmic growth phase, each substrate dissolved in 0.5 ml of N,N-dimethylformamide was added to this culture.Steroid used : DHEA, 16a-hydroxy-DHEA, 4-androstene-3,17-dione, 16a-hydroxy-4-androstene-3,17-dione, estrone, and 16a-hydroxy-estrone were purchased from IKAPHARM Ltd., Ramat-Gan, P. 0. B. 31, Israel.Isolation of oxidation product: After sequence incubation of 30 hr, each reaction mixture was extracted three times with successive equivalent of methylene chloride. The combined methylene chloride phase was washed three times with saturated Na,CO, and with distilled water. The methylene chloride was then dehydrated over anhydrous Na,SO, and evaporated to a dry residue under reduced pressure. The extract dissolved in chloroform was subjected t o thin-layer chromatography (TLC plateSilicage1 60 pre-coated, MERCK) in two migrations of chloroform-methyl
