Genome-wide identification of genes involved in the positive and negative regulation of acetic acid-induced cell death in saccharomyces cerevisiae

Abstract

Acetic acid is a weak organic acid that is a normal by-product of the alcoholic fermentation carried out by Saccharomyces cerevisiae. It is a common toxic agent present in different biotechnological processes, such as in wine or bioethanol production, and is frequently associated with impairment of fermentation 1,2. On the other hand, its toxic effects have prompted its use as a food preservative, but the resistance of some spoilage yeast to this acid limits its action, causing great economic losses3. The more recent finding that acetic acid triggers a programmed cell death (PCD) process provides a new basis for future breeding strategies of industrial strains with improved cell survival, and may also give clues for more efficient food preservation strategies. Therefore, we aimed to identify genes involved in the execution and regulation of acetic acid-induced PCD through a genome-wide analysis. To this end, we optimized and carried out a functional analysis of the Euroscarf knock-out mutant collection. For the phenotypic screening, deletion strains representing haploid (viable) single deletions of approximately 4800 yeast ORFs were grown in YPDA media for 48 hours. Cells were then replicated into 96-well plates with YPD medium and grown for an additional 24 hours at 30 °C, after which they were inoculated in YPD medium (pH 3.0) with 400 mM acetic acid. After 100, 300 and 330 minutes, cells were replicated into 96-well plates containing YPD medium without acetic acid, and optical density (640 nm) was read to assess growth after two days of incubation at 30 °C. This screening uncovered a set of genes involved in resistant and sensitive phenotypes that were clustered according to biological function and known physical and genetic interactions. The results obtained contribute to further characterize acetic acid-induced PCD, and provide information on new putative targets for its control.

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