Wine Yeast Engineering for Improved Polysaccharide Depolymerization and Reduced Urea Accumulation

Engineering wine yeasts to combine efficient extracellular polysaccharide degradation with reduced urea accumulation may support improvements in winemaking. This study aimed to replace the arginase-encoding CAR1 locus in the genome of the triploid industrial wine yeast Saccharomyces cerevisiae I-328 with an expression cassette for the endopolygalacturonase gene PGU1, placed under the control of the PGK1 gene promoter and terminator. CRISPR/Cas9-mediated genome editing was performed using an acetamidase-selectable plasmid followed by phenotypic screening and molecular genetic verification. Whole genome sequencing confirmed the replacement of all three CAR1 alleles by the PGU1 expression cassettes. The modified strain I-328-Y21 showed enhanced degradation of polygalacturonic acid relative to the parental strain. Consistently, RT-qPCR analysis showed that the transcription level of the PGU1 gene in strain I-328-Y21 was about 300 times higher than in I-328. Disruption of arginase—the enzyme that generates urea from arginine—reduced urea content in the growth medium by about ninefold. As ethyl carbamate is formed from ethanol and yeast-excreted urea, this strain could help lower ethyl carbamate in wine. Fermentation performance and other technological characteristics remained unaffected by the genome editing. The new strain, in which the arginase gene is replaced by an endopolygalacturonase expression cassette, is a promising candidate for winemaking.