Abstract

Drug resistance evolution is shaped by both selective agent and genetic background in which resistant mutations arise, but most experimental evolution studies rely on a single genetic background. Here we performed parallel experimental evolution across six genetically diverged Saccharomyces cerevisiae strains, propagating 48 replicate populations for ~225 generations under the selection of DNA-damaging chemotherapeutic bleomycin. Over 70% of the evolved populations acquired resistance with the magnitude and repeatability of adaptation varying markedly across genetic backgrounds. Whole-genome sequencing showed that bleomycin elevated single-nucleotide and insertion-deletion mutation rates, and induced background-specific aneuploidies and structural variants, including Ty-element-mediated translocations and tandem duplications. Notably, mutation rates varied up to 10-fold among backgrounds, with the most commonly-used lab strain exhibiting the highest mutagenic susceptibility, underscoring that evolutionary insights derived from single-background models may not universally generalize across a species. Despite this background-dependent genomic heterogeneity, we identified recurrent loss-of-function mutations in PMA1 and PTK2 that arose independently across nearly all genetic backgrounds and conferred strong resistance. Functional and biophysical assays revealed that these mutations impair Pma1p H+-ATPase activity and depolarize the plasma membrane, thereby limiting the cellular uptake of the positively charged drug. Our findings demonstrate that while the mutational routes to drug resistance are heavily shaped by genetic background, a convergent, background-independent mechanism centered on membrane potential regulation can dominate phenotypic adaptation. This work underscores the necessity of integrating genetic diversity into experimental evolution paradigm and nominates plasma membrane potential as an important lever for modulating chemotherapeutic efficacy.