Sampling and extraction for extracellular metabolites and total RNA
For the sampling of total RNA and extracellular metabolites in yeast cells, approximately 5-mL sample per time interval was rapidly withdrawn from the fermenter. Cell pellets were immediately harvested by centrifugation at ambient temperature and 10,000 rpm for 2 min, washed with 0.9% (w/v) NaCl, centrifuged at ambient temperature and 10,000 rpm for 2 min, and then frozen at –80°C for further analysis. The supernatant was frozen at –20°C for determination of extracellular metabolites.
Total RNA was extracted by the RNeasy Mini Kit (Qiagen, Germany). RNA quality was confirmed by A260/A280 ratio (>2.0) and agarose gel electrophoresis (28S:18S ratio=1.5-2.0). RNA samples were dissolved in Milli-Q water and frozen at –80°C for transcriptome analysis.