Abstract
ABSTRACTOptogenetic 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.
Highlights
Optogenetic switches permit accurate control of gene expression upon light stimulation
The new optogenetic switch, named FUN-LOV, was developed based on the pairing of White Collar 1 (WC-1) and VVD LOV domains, an interaction known to occur as part of the N. crassa photoadaptation process [17]
Light has been positioned in previous years as a promising tool to control gene expression due to its reduced toxic effects on cells, low costs compared to chemical inducers, and the ability to confer spatiotemporal modulation of biological processes [3]
Summary
Optogenetic switches permit accurate control of gene expression upon light stimulation. Optogenetic switches for heterologous protein expression in yeast would replace the addition of chemical inducers, reducing the cost of industrial-scale bioprocesses, providing a dynamic control of gene expression and the effective temporal control of the on and off states. Another biotechnologically relevant operation in yeast fermentation is flocculation, which allows a fast, accessible, and efficient way to remove remaining yeast cells after fermentation processes [23]. These features position flocculation as an ideal and biotechnologically relevant target for optogenetic control
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