Sulphur dioxide (SO₂) is mostly used as an antioxidant additive in winemaking, but excessive levels may be harmful to both wine quality and consumers health. During fermentation, yeast Saccharomyces cerevisiae contributes significantly to final SO₂ levels, and low-producing strains become especially interesting for the wine industry. Recent evidence implicating the impairment of sulphate transport in the SO₂ decrease prompted us to further investigate the sulphate/sulphite metabolic connection in multiple winery yeast strains. Here, we inactivated by CRISPR/Cas9 the high-affinity sulphate permeases (Sul1p and Sul2p) in four strains normally used in winemaking, selected by their different abilities to produce SO₂. Mutant strains were then used to perform fermentation assays in different types of natural must, and the final levels of SO₂ and other secondary metabolites, crucial for wine organoleptic properties, were further determined for all fermentation products. Overall, data demonstrated the double ΔSUL1/ΔSUL2 inactivation in winery strains significantly decreases the levels of SO₂ produced by mutant cells, without however altering both yeast fermentative properties and the ability to release relevant metabolites. Since similar effects were observed in diverse must types for strains with different features, the data strongly support that sulphate assimilation is the determining factor in SO₂ production during oenological fermentations.

While a recent characterization of non-Saccharomyces thermally inactivated yeasts (TIYs) in a wine-like solution highlighted the release of oenologically relevant compounds and different oxygen consumption rates and antioxidant activity, here the impact of TIYs derived from Saccharomycodes ludwigii (SL), Metschnikowia pulcherrima (MP), Torulaspora delbrueckii (TD), and Saccharomyces cerevisiae (SC), as the reference strain, was evaluated in white wine. Wine treatment with TIYs resulted in an increase in polysaccharide concentration compared to the untreated wine, with SL-TIY exhibiting the highest release. Additionally, all TIYs, particularly SL-TIY, improved protein stability by reducing heat-induced haze formation. The addition of TIYs also demonstrated an effect on color parameters through phenolic compound adsorption, preventing potential browning phenomena. All TIYs significantly impacted the wine’s volatile profile. Overall, it was shown that an improvement in wine quality and stability may be obtained by using TIYs in the winemaking process.

Efficient yeast breeding remains a major bottleneck in industrial biotechnology, particularly in food and beverage applications, where strain improvement must balance phenotypic innovation with regulatory constraints. Traditional hybridization procedures for natural Saccharomyces strains are experimentally limited by their diploid and homothallic nature, often resulting in labor-intensive processes of unpredictable duration. Here, we developed and validated a CRISPR/Cas9-assisted breeding framework that enables rapid, systematic, and informative hybridization by transiently converting homothallic strains into stable heterothallic mating partners through targeted HO gene inactivation. Using this strategy, we generated and characterized multiple intraspecific and interspecific hybrids challenging some industrially relevant traits, as sulfur dioxide release, acetic acid production, and the ability to restart stuck fermentations. In all cases, we generated the hybrids and assessed their performance within three months. Hybrid phenotypes followed inheritance patterns including parental-like, intermediate, and combinatorial traits, enabling rapid evaluation of cross utility. Moreover, cisgenic hybrids were generated by the restoration of the native HO locus, maintaining identical fermentative performance. Application to Saccharomyces cerevisiae × Saccharomyces uvarum interspecific crosses further demonstrated the versatility of the approach while revealing intrinsic biological constraints on trait combination. Overall, CRISPR/Cas9-assisted heterothallic conversion emerges as a versatile platform for accelerated yeast breeding, providing decision-enabling insights that support informed strain development across fermentation-based industries.