Strain GDN1 was aerobically grown in a phosphate-buffered shake-flask (300 ml in 1 L-Erlenmeyer flask) containing 1 mM testosterone. 17α-Ethinylestradiol (50 μM; indigestible by strain GDN1) was added as an internal control. Nitrate was omitted from the medium. In 1 L of distilled water, the medium contained the following: 0.29 g testosterone, 2.0 g NH4Cl, 0.5 g MgSO47 H2O, and 0.1 g CaCl22 H2O. After autoclaving, sterile 50 ml KH2PO4-K2HPO4 buffer solution (1 M, pH 6.0), vitamins (1 mL/L)54 (link), EDTA-chelated mixture of trace elements (1 mL/L)55 (link), and selenite and tungstate solution (1 mL/L)56 (link) were added. The aerobic culture was incubated at 37°C in an orbital shaker (180 rpm). The cultural samples (5 mL) were withdrawn every 6 h (0 ~ 30 h). The testosterone-derived intermediates extracted from the cultural samples were identified and quantified using UPLC – HRMS The androgenic activity of the cultural samples was determined as described later.
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