2.3 Fermentation, analyses of cell growth and product accumulation
For seed culture, strains were grown in SC-His-Leu-Ura at 30 °C and 220 rpm to an OD600 of 2-3. For shake-flask fermentation, the seeds were inoculated into 250 mL flasks containing 50 mL of YPD medium with appropriate concentrations of G418 and Hyg at an initial OD600 of 0.05 and then grew at 30 °C and 220 rpm. The fermentation data represented the means ± S.D. of three independent samples.
For detection of compound A , 15 mL of yeast culture was mixed with an equivalent volume of ethyl acetate and vortexed for 30 min. After centrifugation (12,000 g, 4 °C, 10 min), the supernatant was collected and evaporated (-0.1Mpa, 40 °C). Then, the dried product was re-dissolved in methanol for HPLC (Agilent, Waldbronn, Germany) analysis. Samples were assayed on an Agilent SB-C18 column (5 μm, 4.6 mm × 250 mm). Mobile phase A was 100% water, and mobile phase B contained methanol/acetic acid (100:0.1 v/v). A linear gradient for 80% to 100% B in 30 min at 1 ml/min was adopted.