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.