A New Set of Optogenetic Switches in Yeast Based on the BcWCL1 Photoreceptor.

Optogenetic switches are molecular systems enabling light-controlled gene expression. These systems are based on the reconstitution of chimeric Transcription Factors (TFs) including a DNA-binding Domain (DBD), a photoreceptor domain, and an Activation Domain (AD). Thus, depending on the light-mediated homodimerization or heteromerization of the chimeric TF, the optogenetic switches can be classified as single-component or two-component systems, respectively. In the budding yeast Saccharomyces cerevisiae, optogenetic switches have shown multiple applications in metabolic engineering and biotechnology. Here, we expand the repertory of optogenetic switches available in yeast, developing a collection of plasmids and yeast strains carrying single-component or two-component optogenetic switches based on the BcWCL1 protein, a blue-light photoreceptor from Botrytis cinerea that contains a LOV (Light Oxygen Voltage) domain. By dissecting the N-terminal, C-terminal, and both protein regions simultaneously, we developed 18 plasmids encoding the BcWCL1 versions fused to the Gal4-DBD and Gal4-AD. Assembled plasmids were combined and transformed into yeast to generate single-component and two-component optogenetic switches, whose blue-light response was measured as transcriptional activity of the luciferase reporter. In general, we observed that the two-component configuration increased the luciferase expression in response to blue-light compared to single-component switches. Importantly, BcWCL1 versions with deletions in the C-terminal region showed the highest luciferase expression upon blue-light stimulation with the lowest background in the dark condition, suggesting that the C-terminal region modulates the blue-light response of this protein. Altogether, we generated new single-component and two-component optogenetic switches, each of them achieving different levels of light-activated luciferase expression, enabling their potential applications in yeast biotechnology.