Mixed Culture Bioconversion of 16-Dehydropregnenolone Acetate to Androsta-1,4-diene-3,17-dione: Optimization of Parameters Tushar Banerjee, Aniruddha Shukla, Kirti Shinde, and Shridhar Patil* Applied Microbiology Laboratory, School of Life Sciences, Devi Ahilya University, Takshashila Campus, Khandwa Road, Indore 452 017, India Bioconversion of 16-dehydropregnenolone acetate (16-DPA) to androsta-1,4-diene-3,- 17-dione (ADD), an intermediate for the production of female sex hormones, by mixed culture of Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 is reported. Various physicochemical parameters for the bioconversion of 16- DPA to ADD have been optimized in shake flask cultures. Nutrient broth inoculated with actively growing co-culture proved ideal for bacterial growth and bioconversion. A temperature range of 35-40 °C was most suitable; higher or lower temperatures adversely affected the bioconversion. Dimethylformamide below 2% concentration was the most suitable carrier solvent. Maximum conversion was recorded at 0.5 mg mL -1 16-DPA. A pH of 5.0 yielded a peak conversion of 62 mol % in 120 h incubation period. Addition of 9R-hydroxylase inhibitors failed to prevent further breakdown of ADD to nonsteroidal products. 16-DPA conversion in a 5 L fermenter followed a similar trend. Introduction 16-Dehydropregnenolone acetate (3-acetoxypregna-5,- 16-diene-20-one), commercially known as 16-DPA, is available in large quantities through chemical degrada- tion of steroid alkaloids and sapogenins (1). The produc- tion of male and female sex hormones from 16-DPA through the chemical route requires its conversion to 17- ketosteroids, namely, androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD), respectively (2). However, chemical degradation of 16-DPA to 17-keto- steroids involves its sequential conversion to 16-DPA oxime, DHA acetate, AD, and finally ADD, with an overall yield of 34.5% (1). A preliminary report on the pathway of bioconversion of 16-DPA to ADD by a mixed culture of Pseudomonas diminuta (P. diminuta) MTCC 3361 and Comamonas acidovorans (C. acidovorans) MTCC 3362 has been published earlier (3). The present work reports the optimization of parameters for conver- sion of 16-DPA to ADD by mixed culture of these bacteria. Materials and Methods Microorganisms. Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 were procured from the Microbial Type Culture Collection and Gene Bank, Chandigarh, India. Bioconversion. All bioconversion experiments were carried out in 150 mL Erlenmeyer flasks containing 30 mL culture medium. The medium was supplemented with 10 mg mL -1 16-DPA, adjusted to pH 7.0 with 1 N NaOH/HCl and sterilized at 15 psi pressure for 15 min. The sterile medium was inoculated with a 1 mL actively growing co-culture of Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 raised in the medium of the same composition. After a 12 h growth period on a gyratory incubated shaker (180 rpm) at 30 ( 2 °C, 16-DPA was added. It was suspended in 2 mL of sterile distilled water, sonicated for 1 min using titanium microporbe (3 mm tip diameter, 10 µm amplitude) on a MSE Soniprep-150 sonicator and was aseptically trans- ferred to the culture flask. Alternatively, 16-DPA was dissolved in 0.5 mL of carrier solvent and added to the growth medium. The subsequent bioconversions were carried out adopting the parameters optimized during previous experiments. Medium. The composition (g L -1 ) of various media used during the experiments were as follows: (A) Peptone, 5.0; NH 4 NO 3 , 1.0; MgSO 4 ‚7H 2 O, 0.25. (B) Yeast extract, 5.0; NH 4 NO 3 , 1.0; K 2 HPO 4 , 0.25; MgSO 4 ‚7H 2 O, 0.25. (C) Glycerol, 10.0; soy meal, 10.0; K 2 HPO 4 , 0.5; MgSO 4 ‚7H 2 O, 0.25; FeCl 3 ‚6H 2 O, 0.05. (D) Trisodium citrate, 3.0; 16- DPA, 1.0; urea, 0.5; K 2 HPO 4 , 1.6; KH 2 PO 4 , 0.4; KNO 3 , 1.0; MgSO 4 ‚7H 2 O, 0.1; MnSO 4 ‚7H 2 O, 0.0025; ZnSO 4 ‚ 7H 2 O, 0.0025; FeSO 4 ‚7H 2 O, 0.0025. (E) peptone, 5.0; yeast extract, 2.0; beef extract, 1.0; NaCl, 5.0. Analysis. A 1 mL sample of the culture broth was aseptically drawn at desired intervals and extracted with ethyl acetate (2 + 2 mL). The organic phase separated after centrifugation (5000 rpm, 5 min) was collected and dried over anhydrous sodium sulfate. The residue left after vacuum evaporation was redissolved in 1 mL of ethyl acetate. Aliquots of 0.1 mL each were used for qualitative analysis by thin-layer chromatography (TLC) (4) and quantitative analysis of ADD following the modified Zimmermann reaction (5, 6). Bioconversion in Fermenter. A 3 L aliquot of medium E was dispensed in a 5 L capacity fermenter vessel (BioFlo III, New Brunswick Scientific, New Bruswick, NJ) and sterilized at 15 psi pressure for 45 min. After cooling, the medium was inoculated with 100 mL of co-culture of P. diminuta MTCC 3361 and C. acidovorans MTCC 3362 grown for 12 h. Aeration and agitation were set at 2.5 v v -1 min -1 and 180 rpm, respectively. After a 12 h growth period, 3 g of 16-DPA, dissolved in 30 mL of dimethylformamide, was aseptically * To whom correspondence should be addressed. Phone: ++91 731 477166. E-mail: profspatil@yahoo.co.uk. 662 Biotechnol. Prog. 2003, 19, 662-664 10.1021/bp025699m CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/01/2003