Role of a conserved family of serine hydrolases in ethanol toxicity

Abstract

A recent CRISPR screen for genes involved in controlling acetaldehyde dependent ethanol toxicity, identified OVCA2, a conserved serine hydrolase, as the highest protectant against acetaldehyde toxicity. OVCA2 is a member of a larger, diverse group of proteins responsible for metabolic activities known as the family of serine hydrolases (FSH). In S. cerevisiae,three FSH homologues (FSH1, FSH2, and FSH3) are present, but their overlapping functions and their connection to acetaldehyde toxicity are unknown. In this study, we worked to understand the role of FSH proteins in controlling acetaldehyde toxicity in two divergent ways. First, we compared the substrate specificity of the three FSH homologues from S. cerevisiae against a library of ester substrates at varying pH environments. Second, we constructed combinatorial deletions of FSH genes in S. cerevisiae and screened deletion strains for changes in growth under high ethanol, acetaldehyde, and varying other toxic aldehydes. For enzyme activity, both FSH2 and FSH3 had a preference for alkaline conditions and thus the comparative substrate specificity screen was completed at pH 7.5. In this screen, FSH2 and FSH3 showed substrate preferences in line with FSH1, as they both preferred short chain esters versus longer chain esters (>8C). To further evaluate the metabolic activity of FSH enzymes in acetaldehyde toxicity, single, double, and triple deletion strains of the three FSH genes were made in S. cerevisiae. Proper gene deletions for each strain were confirmed by colony PCR. Each of the deletion strains were then grown on agar plates with varying concentrations of alcohol and aldehyde toxins. Each of the single deletion mutants showed similar growth rates to each other and to the wild‐type strain by spot dilution but exhibited slightly different colony morphology from the wild‐type strain. Further testing on the ability to withstand toxins produced by the alcohol metabolism pathway will be conducted to determine whether these enzymes and OVCA2 can metabolize the toxic byproducts of ethanol production.