16:30-17:00 / Keynote Lecture — LUIS LARRONDO

Luis F. Larrondo

OPTOGENETIC CONTROL OF GENE EXPRESSION: PUTTING SOME LOV AND RED-LIGHT ACTION INTO YEAST BIOTECHNOLOGY
Light is a strong environmental cue. Learning how to harness it, as a means to control gene expression, opens the door to new strategies to reprogram cell function. Thus, we have adopted different optogenetics strategies to utilize light as an orthogonal signal to control transcription in yeast, as Saccharomyces cerevisiae is an organism naturally incapable of seeing light. Therefore, we have implemented in yeast an optogenetic system based on the molecular interaction of two blue-light photoreceptors from the filamentous fungus Neurospora crassa, WC-1 and VVD. This synthetic optogenetic switch, which we called FUN-LOV, can directly drive transcription of a gene of interest, providing low background levels in the dark, and over 1300-fold of induction upon blue-light, as measured with a luciferase reporter. We tested the efficiency of FUN-LOV for heterologous protein expression, obtaining higher levels of a tagged protein, when compared to a classical galactose induction system. Additionally, we have utilized FUN-LOV to control flocculation with the click of a light. Depending of the target gene controlled by FUN-LOV, Flocculation in Light (FIL) or Flocculation in Darkness (FID) were achieved. In addition, we have also implemented a red-light toggle switch, which activates transcription upon red-light, and cease such activity when far-red light is shined. This two-positions switch also shows low background in the dark, and fast ON /OFF dynamics. We are presently trying to combine both systems, in order build complex logic gates, combining red and blue light switches, in order to efficiently manipulate metabolic pathways. Current efforts, are also focused on the implementation of these optogenetic switches under bioreactor conditions, in a way that they can be easily utilized under industrial settings. Funding: MI-iBIO, FONDECYT 1171151, HHMI International Research Scholar Research Program.