Optogenetic Modificationof Glycerol Production inWine Yeast

The wine strains of Saccharomyces cerevisiae transform glucose into ethanol and other byproducts such as glycerol and acetate. The balance of these metabolites is important during the fermentation process, which impacts the organoleptic properties of wines. Ethanol and glycerol productions are mainly controlled by the <i>ADH1</i> and <i>GPD1</i> genes, which encode for the alcohol dehydrogenase and glycerol-3-phosphate-dehydrogenase enzymes, respectively. Genetic modification of these genes can thus be used to alter the levels of the corresponding metabolites and to reroute fermentation. In this work, we used an optogenetic system named FUN-LOV (FUNgal-Light Oxygen Voltage) to regulate the expression of <i>ADH1</i> and <i>GPD1</i> in a wine yeast strain using light. Initially, we confirmed the light-controlled expression of <i>GPD1</i> and <i>ADH1</i> in the engineered strains via RT-qPCR and a translational reporter, respectively. To characterize the generated yeast strains, we performed growth curve assays and laboratory-scale fermentations, observing phenotypic differences between illumination conditions that confirm the optogenetic control of the target genes. We also monitored glucose consumption and ethanol and glycerol productions during a fermentation time course, observing that the optogenetic control of <i>GPD1</i> increased glycerol production under constant illumination without affecting ethanol production. Interestingly, the optogenetic control of <i>ADH1</i> showed an inverted phenotype, where glycerol production increased under constant darkness conditions. Altogether, our results highlight the feasibility of using optogenetic tools to control yeast fermentation in a wine yeast strain, which allows changing the balance of metabolic products of interest in a light-dependent manner.