Divergent metallothionein strategies underlie copper tolerance in Saccharomyces species relevant to winemaking

Copper is an essential redox-active metal that serves as a cofactor in fundamental biological processes, including mitochondrial respiration and iron homeostasis. However, excess copper promotes the formation of reactive oxygen species (ROS), leading to cellular damage and death. Owing to its antimicrobial properties, copper is widely used in winemaking. Saccharomyces cerevisiae, the primary fermentative yeast, responds to copper excess by transcriptionally inducing the expression of metallothioneins CUP1 and CRS5, along with antioxidant defenses. In this study, we examined copper tolerance across the Saccharomyces genus. Consistent with previous work, CUP1 copy number variation (CNV) and expression were the main determinants of copper tolerance in wine S. cerevisiae strains. In contrast, wild Saccharomyces species, harboring only a single CUP1 copy, showed no correlation between copper tolerance and CUP1 expression levels. Notably, species within the Saccharomyces clade closely related to S. cerevisiae predominantly expressed CUP1, whereas more distantly related species preferentially expressed CRS5. These findings highlight a substantial role for Crs5 metallothionein in copper detoxification when CUP1 amplification is absent and suggest that additional Cup1-independent mechanisms contribute to copper tolerance in non-domesticated Saccharomyces species. KEY POINTS: Copper-sensitive wine yeasts exhibit increased reliance on Crs5 metallothionein.Saccharomyces species closely related to S. cerevisiae preferentially express CUP1.Saccharomyces species distantly related to S. cerevisiae predominantly express CRS5.