Loss of PDR3 alters metabolome in response to MCHM, a synthetic hydrotrope

4-methylcyclohexane methanol (MCHM), an alicyclic alcohol, is a synthetic hydrotrope that induces a starvation response by upregulation of biosynthetic pathways despite the availability of nutrients. To investigate how Saccharomyces cerevisiae adapts its metabolism to tolerate MCHM, we evolved MCHM-resistant strains in In-Lab Evolutions (ILEs). We sequenced the parental (YJM789) with long read sequencing and the evolved strain using short sequencing, increasing the quality of the YJM789 genome. We identified thousands of SNPs and indel variants per ILE strain, which was a consistent number between strains that evolved resistance and control strains that remained sensitive. However, one gene, PDR3, was consistently mutated in all resistant strains. Because it controls the pleiotropic drug response, recurrent mutations in PDR3 across resistant strains indicate that it is a key driver of adaptive resistance to MCHM in yeast. While many of the evolved alleles of PDR3 would likely produce functional proteins, a PDR3 knockout in the parent YJM789 strain was sufficient to reproduce resistance to MCHM. We found that the pdr3 resistance is mediated through Med15, a component of the Mediator complex which regulates activation by mediating interactions between transcription factors of RNA Pol II. Pdr3 can homodimerize or dimerize with Pdr1, another transcription factor paralog and loss of Pdr1 also confers MCHM resistance. The pleiotropic drug response (PDR) pathway facilitates the export of amino acid catabolites, and deletion of PDR3 disrupted glutathione metabolism and its intermediates. Thus, mutations in PDR3 represent the first identified mechanism conferring resistance to this novel hydrotropic chemical.