Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Here we report the mode of action of theonellamides, bicyclic peptides derived from marine sponges. We generated a chemical-genomic profile of theonellamide F using a collection of fission yeast strains in which each open reading frame (ORF) is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology (GO) terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-beta-D-glucan synthesis. Indeed, theonellamide F induced overproduction of 1,3-beta-D-glucan in a Rho1-dependent manner. Subcellular localization and in vitro binding assays using a fluorescent theonellamide derivative revealed that theonellamides specifically bind to 3beta-hydroxysterols, including ergosterol, and cause membrane damage. The biological activity of theonellamides was alleviated in mutants defective in ergosterol biosynthesis. Theonellamides thus represent a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis.
The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single‐gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high‐throughput genetics. We characterized YETI using growth phenotyping and BAR‐seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter‐perturbing screens, including CRISPRi. We engineered a second expression system, Z
3
EB42, that gives lower expression than Z
3
EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.
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