Evolutionary Engineering and Molecular Characterization of a Sulfur Dioxide-Stress-Resistant Saccharomyces cerevisiae Strain

Sulfiting agents are common preservatives in the food and beverage industry to inhibit spoilage microorganisms. Sulfite produced by the dissolution of sulfur dioxide (SO₂) in water is used as a microbial inhibitor and antioxidant during winemaking. Thus, sulfite resistance is a desirable trait for wine yeasts. However, consumer health concerns regarding SO₂ exposure require a better understanding of the molecular basis of sulfite resistance/response. In this study, we have developed a highly SO₂-stress-resistant Saccharomyces cerevisiae strain (F3) using evolutionary engineering by repeated batch selection at gradually increased potassium metabisulfite (K₂S₂O₅) levels. F3 was resistant to 1.1 mM K₂S₂O₅ stress, which was strongly inhibitory to the reference strain, and cross-resistant to oxidative, heat, and freeze–thaw stresses. F3 also had enhanced cell wall integrity and altered carbon metabolism, indicating its potential for industrial applications, including winemaking. Comparative whole genome sequencing revealed point mutations in SSU1 and FZF1 that are related to SO₂ transport; ATG14, related to autophagy; and other genes involved in vacuolar protein sorting. Comparative transcriptomic analysis showed significant upregulation of SSU1 and differential expression of genes related to transport and carbohydrate metabolism. These findings may shed light on the molecular mechanisms contributing to SO₂ resistance and industrial robustness in S. cerevisiae.