Optogenetic Control\nof Phosphate-Responsive Genes\nUsing Single-Component Fusion Proteins in Saccharomyces\ncerevisiae

Blue light illumination can be detected\nby light-oxygen-voltage\n(LOV) photosensing proteins and translated into a range of biochemical\nresponses, facilitating the generation of novel optogenetic tools\nto control cellular function. Here, we develop new variants of our\npreviously described VP-EL222 light-dependent transcription factor\nand apply them to study the phosphate-responsive signaling (<i>PHO</i>) pathway in the budding yeast Saccharomyces\ncerevisiae, exemplifying the utilities of these new\ntools. Focusing first on the VP-EL222 protein itself, we quantified\nthe tunability of gene expression as a function of light intensity\nand duration and demonstrated that this system can tolerate the addition\nof substantially larger effector domains without impacting function.\nWe further demonstrated the utility of several EL222-driven transcriptional\ncontrollers in both plasmid and genomic settings, using the <i>PHO5</i> and <i>PHO84</i> promoters in their native\nchromosomal contexts as examples. These studies highlight the utility\nof light-controlled gene activation using EL222 tethered to either\nartificial transcription domains or yeast activator proteins (Pho4).\nSimilarly, we demonstrate the ability to optogenetically repress gene\nexpression with EL222 fused to the yeast Ume6 protein. We finally\ninvestigated the effects of moving EL222 recruitment sites to different\nlocations within the <i>PHO5</i> and <i>PHO84</i> promoters, as well as determining how this artificial light-controlled\nregulation could be integrated with the native controls dependent\non inorganic phosphate (P<sub>i</sub>) availability. Taken together,\nour work expands the applicability of these versatile optogenetic\ntools in the types of functionalities that they can deliver and the\nbiological questions that can be probed.