Grant: Discovery Grants Program - Individual

Icewine is a sweet dessert wine of critical importance to the Canadian wine industry and for international trade. Concentrated solutes (sugars and salts) in Icewine juice place wine yeast under hyperosmotic stress, leading to reduced cell growth, altered metabolism, 10-fold higher acetic acid, 2-fold higher glycerol, fermentation difficulties and sometimes organoleptic faults in wine. New wine styles for Canada are now made from dried grapes (appassimento wines), resulting in concentrated juices with similar issues. Fermentation problems are becoming increasingly relevant in common table wine production due to increased sugar in fruit due to global warming. Saccharomyces cerevisiae wine yeast display properties and DNA sequences distinct from well-studied S. cerevisiae laboratory yeast, and these differences impact yeast performance during fermentation and wine organoleptic properties. It is critical to understand these differences in selecting wine yeast best suited for high Brix fermentations. Intracellular accumulation of glycerol is essential for cell survival when yeast are subjected to hyperosmotic stress. Glycerol accumulation is accomplished through uptake of glycerol from the growth media via Stl1p, closing the glycerol export channel Fps1p, and increasing metabolic production from dihydroxyacetone phosphate. When cells produce glycerol internally, the NAD+ produced must be reduced to NADH by metabolite formation to maintain cellular redox balance. Acetic acid formation by yeast may be this mechanism but it is not clear which cofactor system its production is linked to, NAD+ or NADP+ via aldehyde dehydrogenases. While gene expression analysis is a useful tool for identifying potential gene targets associated with a given phenotype, further investigation is required to confirm protein function and phenotypic impact, which is lacking in the study of wine yeast under hyperosmotic stress. Constructing engineered wine yeast strains would be optimal for studying hyperosmotic stress during fermentation given the differing responses of laboratory and wine yeast strains and difficulties in separating isozymes from cellular compartments along with similar cofactor requirements. The central goal of my research program is to gain a comprehensive understanding of factors influencing wine yeast stress responses during fermentation of concentrated grape juices and how these responses relate to cellular redox balance, glycerol and organic acid homeostasis and wine quality. We will further advance this goal by confirming there is no glucose repression of membrane transporter STL1 in wine yeast under sugar-induced osmotic stress; determine if increasing [NAD+] while wine yeast are under hyperosmotic stress leads to increased acetic acid production by adding exogenous nicotinamide riboside; and quantifying roles of Ald 2-6p, alone and in combination, during wine fermentations by constructing wine yeast deletion mutants of ALD genes using CRISPR-Cas9.