PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress

Abstract

In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity.

Microbial lag phase can be indicative of, or independent from, cellular stress

“…1a,b, 3a, 4a,b, 5a-c, 6a-c, 7a,b, 8a,b, S1b, S3a,b, S7a,b, S8d,g and S9c,d are consistent with the findings of some other studies. For instance, Ding et al 79 , characterized lag-phase adaptation in mutants of S. cerevisiae which over-expressed genes implicated in acid-stress tolerance. Over-expression of genes which code for a protein thought to increase vacuolar proton-pumping ATPase activity, PEP3, also shortened the lag phase.…”

Adaptive Response and Tolerance to Acetic Acid in Saccharomyces cerevisiae and Zygosaccharomyces bailii: A Physiological Genomics Perspective

“…The genetic engineering of laboratory S. cerevisiae strains through the overexpression of single genes has yielded strains with increased acetic acid tolerance. Specifically, the overexpression of WHI2 , encoding a protein required for full activation of the general stress response ( Chen et al, 2016b ), of the genes PEP3 , encoding a vacuolar membrane protein involved in vesicular tethering/docking/fusion, STM1 , encoding a protein required for optimal translation under nutrient stress, PEP5 , encoding the E3 ubiquitin protein-ligase involved in the catabolism of histones ( Ding et al, 2015a ), or the gene ACS2 , encoding acetyl-coA synthetase isoform ( Ding et al, 2015b ), decreased the duration of lag phase of S. cerevisiae cells cultured with acetic acid. Improvement of S. cerevisiae growth and alcoholic fermentation performance in the presence of acetic acid also resulted from the overexpression of SET5 and PPR1 , coding for a methyltransferase for the methylation of histone H4 at Lys5, -8, and -12 and a transcription factor involved in the regulation of pyrimidine pathway, respectively ( Zhang et al, 2015 ).…”

A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

“…For example, generation of haploid segregants from crosses of highly tolerant and less tolerant strains, followed by quantitative-trait-locus (QTL) analysis, enabled the identification of multiple alleles that affect acetic acid tolerance in this yeast [ 30 ]. Increased acetic acid tolerance has also been reported for strains carrying targeted genetic modifications, such as overexpression of TAL1, encoding a transaldolase [ 31 ], overexpression of PEP3 , encoding a protein subunit involved in vacuolar biogenesis [ 32 ], overexpression of the transcription factor HAA1 [ 33 ], introduction of an artificial zinc-finger based transcription factor [ 34 ] and introduction of an ascorbic-acid production pathway [ 35 ]. However, the levels of tolerance reached by these approaches are not sufficient for the high concentrations of acetic acid present in lignocellulosic hydrolysates at industrially relevant pH values.…”