Thiol release improvement in Saccharomyces cerevisiae oenological strains by CRISPR/Cas9-based IRC7 molecular cisgenesis

BACKGROUND: Polyfunctional thiols are essential contributors to the aromatic profile of varietal white wines like Sauvignon Blanc. These compounds are released during fermentation by Saccharomyces cerevisiae cells from grape-derived precursors, with the carbon-sulfur β-lyase encoded by the IRC7 gene playing a crucial role. However, most oenological yeast strains lack the fully functional IRC7 allele, limiting their thiol-releasing ability. RESULTS: variant into four oenological S. cerevisiae strains, commonly used to produce different white and red wines. Interestingly, all cisgenic strains showed enhanced thiols release, confirming the direct role of IRC7 in their biosynthesis. Fermentation performance, including the production of ethanol and multiple metabolites, remained however unchanged. GC-MS analyses then confirmed that strain-specific profiles of aromatic molecules were also preserved, indicating that genome editing did not affect other relevant oenological traits. Importantly, the cisgenic strains released thiols even when fermenting musts with low precursors content, without the need for additional supplements. CONCLUSIONS: This work demonstrates that targeted IRC7 gene editing via CRISPR/Cas9 is a precise and efficient strategy to enhance thiol production in oenological yeasts, without compromising fermentative behavior or aromatic identity. Importantly, although the strains are formally GMOs, the modification mimics natural allelic variation. Our findings provide a foundation for developing next-generation wine yeasts optimized for high-aroma varietal wines and support the broader application of genome editing in fermentation biotechnology.