Monitoring gene expression by TRAC through very high gravity brewing fermentations

Brewers' yeast is subjected to constantly changing environmental conditions during wort fermentation, such as decreasing levels of carbon, nitrogen and oxygen and increasing level of ethanol. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in the genomic expression can indicate the physiological state and condition of yeast in the brewing process. Both conventional and genome-wide transcription analyses have been used to study gene expression during laboratory and industrial scale wort fermentations, but for logistic and economic reasons, data have been collected from only a limited number of samples. Here we have applied to very high gravity brewing fermentations a novel transcriptional analysis method called TRAC (TRanscript analysis with aid of Affinity Capture) for frequent expression analysis of a focused gene set. TRAC enables multiplex mRNA target analysis simultaneously from a large number of samples in a cost- and time-efficient manner. We have selected more than 70 marker genes that code for proteins involved in various pathways relevant to the brewing process, such as maltose, glycerol and lipid metabolism, glucose fermentation, amino acid biosynthesis, aroma formation and flocculation and measured their expression at frequencies up to 2 h. Results showed rapid changes in gene expression during the first hours of fermentations for several genes. For instance genes involved in maltose metabolism (MALx3), glucose fermentation (HXK2, ADH1,3,4), ergosterol (ERG3,13) and glycerol synthesis (GPD1) were strongly up-regulated between 2 to 6 h after pitching. Several genes showed two or more well defined peaks during the first 24 h. Some (not all) glucose-repressible genes (ADH2, HXK1, GLK1) were down-regulated as expected during early fermentation while glucose was present, and up-regulation was observed after 20 h of fermentation. By the time (72 h) yeast growth had stopped and total sugars had dropped by about 50%, most selected genes showed low expression and total mRNA was less than half the levels during growth. However, relatively high transcript levels were found, e.g., for some genes involved in flocculation (FLO8, FLO11) and glucose phosphorylation on a non-fermentable carbon source (GLK1) during the final fermentation stages (120-200 h).