In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae . Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn 2 C 6 transcription factors YRR1 and YRM1 ( p < 1 × 10 −100 ). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.

SummaryIn vitroevolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in the model microbe,Saccharomyces cerevisiae. Analysis of 355 curated, laboratory-evolved clones, resistant to 80 different compounds, demonstrates differences in the types of mutations that are identified in selected versus neutral evolution and reveals numerous new, compound-target interactions. Through enrichment analysis we further identify a set of 137 genes strongly associated with or conferring drug resistance as indicated by CRISPR-Cas9 engineering. The set of 25 most frequently mutated genes was enriched for transcription factors and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function, single nucleotide variants found in 170-amino-acid domains in two Zn2C6transcription factors,YRR1andYRM1(p < 1x 10−100). This remarkable enrichment for transcription factors as drug resistance genes may explain why it is challenging to develop effective antifungal killing agents and highlights their important role in evolution.