Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation

Abstract

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.

FIG. 1.

Northern blot verification of genome-wide expression analysis. The figure is representative of expression levels of ERG10 and ERG3 measured by using the duplicate total RNA samples isolated from the Lager 1 strain 1 h and 23 h into a pilot-plant fermentation.

FIG. 2.

ERG gene induction in the Lager 1 strain leads to ergosterol accumulation during the initial stages of the pilot-plant fermentation. Quantification of ergosterol in yeast cells was carried out by reversed-phase HPLC.

FIG. 3.

Effect of ERG mutations on the metabolic activity of BY4743 yeast cells. Cells were stored for 2 days, acid washed, and pitched into industrial-grade wort. Metabolic activity was monitored as the amount of gas produced. Shown are results for wild-type strain, BY4743 (⋄), and the Δerg6 (□), Δerg3 (▵), Δerg5 (○), and Δerg4 (X) mutants.

FIG. 4.

Northern blot analysis of ERG10 (▵) and TRR1 (▪) gene expression in the Lager 1 strain during the initial 24 h of a 500,000-liter industrial lager fermentation. The graphical representation of gene expression is relative to that of ACT1.

FIG. 5.

Effect of ERG mutations on the ability of BY4743 yeast cells to respond to oxidative stress. Cells were grown to the stationary phase, diluted to the indicated optical density at 600 nm (OD₆₀₀), and applied as spots to YPD plates containing the indicated oxidants. Plates were photographed after 2 days of growth at 30°C. Shown are results for the wild-type strain, BY4743 (column 1), and the Δerg3 (column 2), Δerg4 (column 3), Δerg5 (column 4), and Δerg6 (column 5) mutants.