Curated Optogenetic Publication Database

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.

Abstract: Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Abstract: Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

Abstract: Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.