Evolutionary Engineering and Molecular Characterization of an Antimycin A-Resistant Saccharomyces cerevisiae Strain: the Key Role of Pleiotropic Drug Resistance (PDR1).

Antimycin A, an antifungal agent that inhibits mitochondrial respiration, provides a useful model for studying resistance mechanisms. Antifungal resistance is an escalating clinical concern with limited treatment options available. To understand the molecular mechanisms of antimycin A-resistance, a genetically stable, antimycin A-resistant Saccharomyces cerevisiae strain was successfully developed for the first time through an evolutionary engineering strategy, based on long-term systematic application of gradually increasing antimycin A-stress in repetitive batch cultures without prior chemical mutagenesis. Comparative whole genome re-sequencing analysis of the evolved strain ant905-9 revealed two missense mutations in PDR1 and PRP8 genes involved in pleiotropic drug resistance and RNA splicing, respectively. Using CRISPR/Cas9 genome editing tools, the identified mutations were introduced individually and together into the reference strain, and it was confirmed that the Pdr1p.M732R mutation alone confers antimycin A-resistance in S. cerevisiae. Comparative transcriptomic analysis of the reverse engineered Pdr1p.M732R strain showed alterations in PDR (pleiotropic drug response), transmembrane transport, vesicular trafficking and autophagy pathways. Our results highlight the potential key role of PDR1 in antifungal drug resistance. This study provides new insights into mitochondrial drug resistance and the adaptive potential of yeast under respiratory stress.