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cells, approximately 5-mL sample per time interval was rapidly withdrawn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.