FUN-LOV: Fungal LOV 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 1300-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 co-repressor TUP1 provided Flocculation in Darkness (FID). Overall, the results revealed 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.ImportanceOptogenetic 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. Herein, 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.

Coupling cell communication and optogenetics: Implementation of a light-inducible intercellular system in yeast

“…Only certain co-cultures at 1:1 ratio and exposed to BL showed activation of the normalized response during the first 10 h, which did not occur for the same cell mixtures grown in DD (Figure 4). Specifically, co-cultures involving R2 reached peaks between 3.5 and 4 h. On the other hand, the kinetics of cell mixtures involving R3 were slower, exhibiting the greatest values between 9 and 9.5 h. Interestingly, these maximum levels were similar to those ones obtained in BL for direct optogenetic control of a chromosomal luciferase using the FUN-LOV switch 50 . Although blue-light is the stimulus that triggers the split genetic circuit that leads to luciferase induction in the R-strains, the response of the system was surprisingly higher when examining co-cultures going through an illumination transition such as DL4h:20h, compared to ones grown in BL (Figure 4).…”

“…FUN-LOV, an optogenetic switch we recently described by exploiting the blue-light dependent interaction between WC-1 and VVD photoreceptors from the filamentous fungus Neurospora crassa, has enabled a fine temporal resolution of transcriptional control yielding a broad range of expression in yeast 50 . In this work, we used this switch to implement a light-inducible intercellular system based on two yeast strains (Figure 1).…”

“…First, the cells were evaluated at OS-R 1:1 ratio under three different illumination conditions: 24 h of DD, 24 h of blue-light (BL) and a transition of 4 h of DD followed by 20 h of BL (DL4h:20h) in a temperature-controlled dark room. Blue-light (BL) experiments were carried out using an LED lamp (model i5038) at ~ 20 µmol m -2 s -1 of light intensity 50 . Briefly, OS-and R-strains were grown separately overnight in a 96-well plate with 200 µL of SC medium at 30ºC in DD.…”

“…Thereafter, 5 µL of the OS-and R-cell cultures were co-inoculated in nine combinations in a new 96-well plate containing 190 µL of fresh SC media supplemented with luciferin (1 mM). The measurements involving BL used a discontinuous protocol, that allows keeping the plate outside of the microplate reader to expose the co-cultures to the LED lamp between each measurement 50 . Then, different dark/blue-light transitions (DL1h:23h, DL2h:22h, DL6h:18h, DL8h:16h, DL10h:14h, DL12h:12h) were assessed.…”

Expanding the molecular versatility of an optogenetic switch in yeast

“…Optogenetics is based on photosensitive proteins known as photoreceptors, which are capable of light sensing at different wavelengths (Losi et al, 2018;Tang et al, 2021). Currently, optogenetic tools include a large set of different photoreceptors, which have been implemented in a variety of biological platforms (Kolar et al, 2018), including microorganisms such as the budding yeast Saccharomyces cerevisiae (Salinas et al, 2017;Figueroa et al, 2021).…”