Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast

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.

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

“…This leads to maximal fold induction along the diagonal as seen in Figure a. This was a crude dialing of protein concentration but suggests that one way to further optimize a split TF system would be by carefully titrating the total and relative dosage of each component, a knob not available in single‐component and homogeneous two‐component optogenetic systems (Benzinger & Khammash, ; Hughes et al, ; Kennedy et al, ; Salinas et al, ; Xu et al, ; Zhao et al, ). It is worth noting that the variability between biological replicates decreased when the ZDBD‐PHR2/VP16AD‐CIB1/pZF(3BS) components were chromosomally integrated using the toolkit (Figure b) rather than maintained on episomal plasmids (Figure b).…”

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

“…In the optogenetic toolkit, the LOV domain has been utilized for optical control of the biofunctional modules through two general strategies [ 31 ]: (i) a single light-activatable protein or peptide that drives a signaling pathway [ 20 , 21 , 57 , 73 ], and (ii) light-induced protein–protein interactions that manipulate the subcellular localization of a protein or a protein activity [ 47 , 49 , 50 , 74 ]. We will put focus on examples of engineering of LOV-based actuators from the perspective of the photocycle, proteins with light-induced allosteric responses, and proteins with light-induced dimerization responses.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…The heterologous protein expression induced by the FUN-LOV switch was 2.5-fold greater than that induced by the commonly used GAL4/galactose chemical switch. The FUN-LOV switch has since been used to control flocculation and other biotechnologically important yeast phenotypes (Salinas et al, 2018). Zhao et al (2018) (Ramsey et al, 2006), by putting the Gal80 repressor under light sensitive control pf VP16-EL222.…”

“…In one study, gene expression in S. cerevisiae was controlled solely based on the application of blue light. They did so by using the FUN‐LOV optogenetic switch, which is based on the photon‐regulated interaction of WC‐1 and VVD, two LOV blue‐light photoreceptors from the fungus Neurospora crassa (Salinas et al, ). In a proof‐of‐principle experiment with a luciferase reporter, the FUN‐LOV switch controlled gene expression with exquisite temporal resolution and a broad dynamic range, inducing differences in expression levels of more than 1,300‐fold.…”