Fructose consumption improvement in oenological Saccharomyces cerevisiae yeast strains by CRISPR-assisted genome editing

The yeast Saccharomyces cerevisiae is an important model organism in research, but also widely utilized in industrial applications such as chemical, pharmaceutical and food industry. S. cerevisiae has been mainly utilized in the wine-making industry to produce alcoholic beverages such as beer and wine due to its marked fermentative capabilities. A key feature of yeast strains used in winemaking is their ability to consume the available sugars in the natural grape must, which mainly consist of glucose and fructose. Sugar uptake inside the yeast cell may directly impact on the rate of alcoholic fermentation. Among the hexose transporters genes, HXT3 has been demonstrated to play a key role in determining fructose utilization during fermentation. An allelic variant of the gene, namely HXT3V79, has been reported to enhance fructose utilization and fermentation rate. In this thesis, the function of the HXT3 gene has been further investigated by generating oenological S. cerevisiae mutant strains through CRISPR-assisted genome editing. Notably, genetic modifications introduced either a loss-of-function mutation, or the enhanced allele HXT3V79. Then, the effects of the mutations on the ability of yeast cells to consume sugars were assessed by performing experimental microfermentation assays, in either standard conditions, or simulating a stuck fermentation. Overall, data supported the important role of HTX3 in sugar utilization by yeast cells, as gene deletion decreased fermentation performance, but further indicated that sugar intake may not be the only limiting factor for its utilization by the yeasts. In fact, despite the introduction of the more active HTX3V79 variant, the modified strain was unable to consume more fructose than the parental strain, indicating that other strain-specific metabolic traits may also play a crucial role in determining the yeast’s fermentation phenotype.