Curated Optogenetic Publication Database

Abstract: 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 ADH1 and GPD1 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 ADH1 and GPD1 in a wine yeast strain using light. Initially, we confirmed the light-controlled expression of GPD1 and ADH1 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 GPD1 increased glycerol production under constant illumination without affecting ethanol production. Interestingly, the optogenetic control of ADH1 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.

Abstract: Nitrogen limitations in the grape must is the main cause of stuck fermentations during the winemaking process. In Saccharomyces cerevisiae, a genetic segment known as region A, which harbors 12 protein-coding genes, was acquired horizontally from a phylogenetically distant yeast species. This region is mainly present in the genome of wine yeast strains, carrying genes that have been associated with nitrogen utilization. Despite the putative importance of region A in yeast fermentation, its contribution to the fermentative process is largely unknown. In this work, we used a wine yeast strain to evaluate the contribution of region A to the fermentation process. To do this, we first sequenced the genome of the wine yeast strain known as ‘ALL’ using long-read sequencing and determined that region A is present in a single copy with two possible subtelomeric locations. We then implemented an optogenetic system in this wine yeast strain to precisely regulate the expression of each gene inside this region, generating a collection of 12 strains that allow for light- activated gene expression. To evaluate the role of these genes during fermentation, we assayed this collection using microculture and fermentation experiments in synthetic must with varying amounts of nitrogen concentration. Our results show that changes in gene expression for genes within this region can impact growth parameters and fermentation rate. We additionally found that the expression of various genes in region A is necessary to complete the fermentation process and prevent stuck fermentations under low nitrogen conditions. Altogether, our optogenetics-based approach demonstrates the importance of region A in completing fermentation under nitrogen-limited conditions.

Abstract: In the budding yeast Saccharomyces cerevisiae, the FUN-LOV (FUNgal Light Oxygen and Voltage) optogenetic switch enables high levels of light-activated gene expression in a reversible and tunable fashion. The FUN-LOV components, under identical promoter and terminator sequences, are encoded in two different plasmids, which limits its future applications in wild and industrial yeast strains. In this work, we aim to expand the molecular versatility of the FUN-LOV switch to increase its biotechnological applications. Initially, we generated new variants of this system by replacing the promoter and terminator sequences and by cloning the system in a single plasmid (FUN-LOVSP). In a second step, we included the nourseothricin (Nat) or hygromycin (Hph) antibiotic resistances genes in the new FUN-LOVSP plasmid, generating two new variants (FUN-LOVSP-Nat and FUN-LOVSP-Hph), to allow selection after genome integration. Then, we compared the levels of light-activated expression for each FUN-LOV variants using the luciferase reporter gene in the BY4741 yeast strain. The results indicate that FUN-LOVSP-Nat and FUN-LOVSP-Hph, either episomally or genome integrated, reached higher levels of luciferase expression upon blue-light stimulation compared the original FUN-LOV system. Finally, we demonstrated the functionality of FUN-LOVSP-Hph in the 59A-EC1118 wine yeast strain, showing similar levels of reporter gene induction under blue-light respect to the laboratory strain, and with lower luciferase expression background in darkness condition. Altogether, the new FUN-LOV variants described here are functional in different yeast strains, expanding the biotechnological applications of this optogenetic tool.