Determination of Transcription Levels

After incubation in shaker flask for 36 and 72 h, 1 mL of fermentation broth was used for the determination of transcription levels. Total RNA was extracted using a Yeast RNA Kit (Omega, Winooski, VT, United States). RNA was diluted with 50 μL of DEPC water and used for reverse transcription with ReverTra RT Master Mix (Toyobo, Osaka, Japan). The cDNA (25 ng) was used as a template to determine transcription levels by qPCR. Each qPCR reaction was performed as described above, with three biological replicates. The transcription level of each target gene was calculated based on the ratio of transcription levels between target mRNA and endogenous control (GAPDH).