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

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.

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

“…In addition, new OS-strains might be generated by controlling MFα2 gene expression to provide different response levels and kinetics of the R-strains. Expanding the repertoire of OS-strains can also be carried out by using FUN-LOV variants to increase the level of production of α-factor by light or improve the LL/DD fold-induction 66 .…”

Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains

“…The luciferase reporter gene was controlled by the P 5XGAL1 synthetic promoter [ 32 ], permitting luciferase expression upon the reconstitution of a two-hybrid system based on PAS-PAS or LOV-LOV interactions in the presence or absence of light [ 32 ]. The reporter gene expression levels were assayed under constant blue light (BL), constant darkness (DD), and a single BL pulse (BLP) of two-hour duration, using a custom LED illumination system recently described by [ 42 ] that provides blue light at 466 nm and applying 20 µmol m 2 s −1 of light intensity. The measurements of optical density at 600 nm (OD 600nm ) and luminescence of the yeast cell cultures over time were simultaneously determined using a Cytation 3 or Synergy H1M microplate readers (BioTek, Winooski, VT, USA), which carry the same monochromator optical configuration.…”

“…In all the experiments, yeast strains were grown overnight in a 96-well plate with 200 µL of SC medium at 30 °C in DD condition. Thereafter, 10 µL of these cultures was used to inoculate a new 96-well plate containing 190 µL of fresh media supplemented with 1 mM of luciferin [ 42 ]. This 96-well plate was incubated inside the plate reader for DD condition, where OD 600nm and the luminescence were acquired at 30 °C every 10 min and during 24 h, running high-resolution kinetic protocols with 30 sec of shaking before data acquisition [ 42 ].…”

“…Thereafter, 10 µL of these cultures was used to inoculate a new 96-well plate containing 190 µL of fresh media supplemented with 1 mM of luciferin [ 42 ]. This 96-well plate was incubated inside the plate reader for DD condition, where OD 600nm and the luminescence were acquired at 30 °C every 10 min and during 24 h, running high-resolution kinetic protocols with 30 sec of shaking before data acquisition [ 42 ]. In the BL and BLP conditions, the 96-well plate was incubated using a discontinuous kinetics protocol, maintaining the 96-well plate outside of the plate reader for illumination and inside of the equipment only for data acquisition [ 42 ].…”

“…This 96-well plate was incubated inside the plate reader for DD condition, where OD 600nm and the luminescence were acquired at 30 °C every 10 min and during 24 h, running high-resolution kinetic protocols with 30 sec of shaking before data acquisition [ 42 ]. In the BL and BLP conditions, the 96-well plate was incubated using a discontinuous kinetics protocol, maintaining the 96-well plate outside of the plate reader for illumination and inside of the equipment only for data acquisition [ 42 ]. The raw data of luciferase expression (luminescence) and OD 600nm for all the assayed experimental conditions are shown in Supplementary Figures S1–S3 and S5 .…”