Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Andrés Romero,Verónica Delgado,Vicente Rojas,Francisco Salinas,Luis F Larrondo

doi:10.3390/ijms22168538

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.

Highlights

Optogenetics is an approach that began to gain attention when targeted light-activated neurotransmission, utilizing channelrhodopsin-2, was first described [1]
We evaluated the yeast strains carrying the nine different variants of fungal light–oxygen–voltage (FUN-LOV) using a single 120 min blue-light pulse (Supplementary Figure S1), which was administered during the mid-exponential phase (Supplementary Figure S2)
The one carrying the Hap1p DNA-binding domain (DBD), which recognizes the CYC1 promoter (PCYC1) [29], displayed a clear increase in luciferase activity in response to the blue-light pulse compared to the control, and this was rapidly reversed in the absence of the stimulus (Supplementary Figure S1)

Summary

Introduction

Optogenetics is an approach that began to gain attention when targeted light-activated neurotransmission, utilizing channelrhodopsin-2, was first described [1]. The yLightOn optogenetic switch is a single component system based on the light–oxygen–voltage (LOV) domain of the protein VIVID (VVD), a blue-light photoreceptor from the filamentous fungus Neurospora crassa [9] This system can be used to control gene expression and protein stability and has allowed, for instance, fine tuning of the yeast cell cycle upon light stimulation [9]. Another LOV-containing protein in N. crassa is White Collar-1 (WC-1), a blue-light photoreceptor and transcription factor (TF) that interacts with WC-2, forming the White Collar complex (WCC), which is the core component of the Neurospora circadian clock and is responsible for the transcriptional activation of numerous genes in response to light [13,14]. These examples highlight the tremendous potential of optogenetics in the control of different yeast cellular processes

Methods

Results

Conclusion