Background: Wine fermentation is a harsh ecological niche to which wine yeast are well adapted. The initial high osmotic pressure and acidity of grape juice is followed by nutrient depletion and increasing concentrations of ethanol as the fermentation progresses. Yeast's adaptation to these and many other environmental stresses, enables successful completion of high-sugar fermentations. Earlier transcriptomic and growth studies have tentatively identified genes important for high-sugar fermentation. Whilst useful, such studies did not consider extended growth (>5 days) in a temporally dynamic multi-stressor environment such as that found in many industrial fermentation processes. Here, we identify genes whose deletion has minimal or no effect on growth, but results in failure to achieve timely completion of the fermentation of a chemically defined grape juice with 200 g L-1 total sugar.
Results: Micro- and laboratory-scale experimental fermentations were conducted to identify 72 clones from ~5,100 homozygous diploid single-gene yeast deletants, which exhibited protracted fermentation in a high-sugar medium. Another 21 clones (related by gene function, but initially eliminated from the screen because of possible growth defects) were also included. Clustering and numerical enrichment of genes annotated to specific Gene Ontology (GO) terms highlighted the vacuole's role in ion homeostasis and pH regulation, through vacuole acidification.
Conclusion: We have identified 93 genes whose deletion resulted in the duration of fermentation being at least 20% longer than the wild type. An extreme phenotype, 'stuck' fermentation, was also observed when DOA4, NPT1, PLC1, PTK2, SIN3, SSQ1, TPS1, TPS2 or ZAP1 were deleted. These 93 Fermentation Essential Genes (FEG) are required to complete an extended high-sugar (wine-like) fermentation. Their importance is highlighted in our Fermentation Relevant Yeast Genes (FRYG) database, generated from literature and the fermentation-relevant phenotypic characteristics of null mutants described in the Saccharomyces Genome Database. The 93-gene set is collectively referred to as the 'Fermentome'. The fact that 10 genes highlighted in this study have not previously been linked to fermentation-related stresses, supports our experimental rationale. These findings, together with investigations of the genetic diversity of industrial strains, are crucial for understanding the mechanisms behind yeast's response and adaptation to stresses imposed during high-sugar fermentations.