A 1 L baffled shake flask containing 300 mL of buffered minimal glycerol yeast extract medium (12.61 g/L glycerol, 10.0 g/L yeast extract, 20.0 g/L soytone, 1.34% YNB, 4×10-5% biotin, 100 mM potassium phosphate, pH 6.0) was inoculated with 0.5 mL of the working cell bank and incubated for 24-28 h at 28℃and 250 rpm until the culture OD600 reached≥5.