The solubility of protein complexes and membraneless compartments is maintained by liquid-liquid phase separation (LLPS). Phase transition is induced or dissolved by biological hydrotropes such as ATP and RNA. 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 cellular metabolism can tolerate changes in LLPS, we evolved eight MHCM-resistant strains of S. cerevisiae. We identified thousands of SNPs and indel variants per strain, which was a consistent number between strains that evolved resistance and control strains that remained sensitive. These variants did not show a pattern that would cluster resistant strains together. The many background mutations likely masked any pattern from few large-effect loci or implicated an epistatic effect of many small mutations spread throughout the genome that was undetectable. Among coding variants in the strains that change protein sequence and thereby may alter function, only one gene showed a protein-coding mutation in every resistant strain while showing no variants at all in the control strains. This gene, PDR3, controls transcription for the pleiotropic drug response and is the most significant driver of adaptive MCHM resistance in yeast. While many of the evolved alleles of PDR3 would likely produce functional proteins, a knockout in the parent YJM789 strain was sufficient to produce resistance to MCHM. Normal catabolism of amino acids uses the Pleiotropic Drug Response (PDR) pathway to export breakdown products. The pdr3 resistance is mediated through Med15, a component of the Mediator complex which regulates activation by transcription factors of RNA pol II. Pdr3 can homodimerize or dimerize with Pdr1, another transcription factor and loss of Pdr1 also confers MCHM resistance. Knockouts of other mutated genes in flocculation, glutathione, SAM, and sugar transport mildly affected growth in the ancestral strain. Mutations in PDR3 are first known to increase resistance to this novel hydrotropic chemical.
