Genome-wide identification of Saccharomyces cerevisiae genes required for maximal tolerance to ethanol

Abstract

The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H(+)-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme beta-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [(3)H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

FIG. 1.

Categorization, based on the biological process taxonomy of GO, of the genes required for ethanol stress resistance. Genes were clustered using GOToolBox, and only classes found to be statistically overrepresented in our data set are displayed (P value below 0.01). Striped bars, gene frequency within each class; black bars, frequency registered for the whole genome.

FIG. 2.

Main interaction network maps of the determinants of ethanol tolerance. The map was created using the OSPREY software and shows protein and genetic interactions within three groups of genes associated with vacuolar protein sorting, V-ATPase complex assembly, and peroxisome protein import machinery. The existence of an interaction between two genes or proteins is represented by a connection between two nodes.

FIG. 3.

pH_v, assessed as fluorescence intensity values as described in Materials and Methods, upon 1 h of BY4741 cell cultivation in growth medium in the absence (control) or presence of inhibitory concentrations of ethanol (6% or 8% [vol/vol]). Values are the averages of at least three independent experiments; standard variations are displayed as error bars.

FIG. 4.

Comparison of the susceptibilities to lyticase of cells of S. cerevisiae parental strain BY4741 grown in the absence of ethanol and then incubated in a growth medium that was either left unsupplemented (⧫) or supplemented with 6% ethanol (▪). Cells were harvested after 3 h of incubation. The different cell populations were washed with water and resuspended in 0.1 M sodium phosphate buffer at pH 7.5. After the addition of 20 μg of lyticase (Sigma) per ml, the decrease in the OD₆₀₀ of the cell suspension was measured periodically.

FIG. 5.

Comparison of the susceptibilities to ethanol-induced stress of S. cerevisiae parental strain W303-1A, harboring the FPS1 expression plasmid (□) or the corresponding empty vector (⋄), to those of the derived deletion mutant Δfps1, harboring the FPS1 expression plasmid (○) or the corresponding empty vector (▵). Cells were grown in the absence of ethanol and then incubated in MM4 liquid medium supplemented with 6% ethanol. Growth curves are representative of at least three independent experiments.

FIG. 6.

Comparison of levels of [³H]ethanol accumulation in nonadapted cells of S. cerevisiae parental strain W303-1A, harboring the FPS1 expression plasmid (○) or the corresponding empty vector (□), and the derived Δfps1 deletion mutant, harboring the FPS1 expression plasmid (▪) or the corresponding empty vector (•), during cultivation in MM4 liquid medium suddenly supplemented with 6% ethanol under conditions identical to those used for Fig. 5. The accumulation values are representative of at least three independent experiments. [³H-Et]_intra and [³H-Et]_extra, intracellular and extracellular [³H]ethanol concentrations, respectively.

FIG. 7.

Comparison of extracellular concentrations of ethanol and glucose, accumulated during cultivation in fermentation medium of BY4741 yeast cells harboring the pYEP-FPS1 plasmid (▪) or the corresponding cloning vector (⋄). Growth was followed by measuring culture OD₆₀₀.