Screening the yeast deletant mutant collection for hypersensitivity and hyper‐resistance to sorbate, a weak organic acid food preservative

Abstract

Certain yeasts cause large-scale spoilage of preserved food materials, partly as a result of their ability to grow in the presence of the preservatives allowed in food and beverage preservation. This study used robotic methods to screen the collection of Saccharomyces cerevisiae gene deletion mutants for both increased sensitivity and increased resistance to sorbic acid, one of the most widely-used weak organic acid preservatives. In this way it sought to identify the non-essential, non-redundant activities that influence this resistance, activities that might be the potential targets of new preservation strategies. 237 mutants were identified as incapable of growth at pH 4.5 in presence of 2 mM sorbic acid, while 34 mutants exhibit even higher sorbate resistance than the wild-type parental strain. A number of oxidative stress-sensitive mutants, also mitochondrial mutants, are sorbate-sensitive. This appears to reflect the importance of sustaining a reducing intracellular environment (high reduced glutathione levels and NADH/NAD and NADPH/NADP ratios). Sorbate resistance is also very severely compromised in mutants lacking an acidified vacuole, in vacuolar protein sorting (vps) mutants, in mutants defective in ergosterol biosynthesis (erg mutants) and with several defects in actin and microtubule organization. Sorbate resistance is, however, elevated with the loss of the Yap5 transcription factor; with single losses of two B-type cyclins (Clb3p, Clb5p); and with loss of a plasma membrane calcium channel activated by endoplasmic reticulum stress (Cch1p/Mid1p).

A novel role for the yeast protein kinase Dbf2p in vacuolar H+-ATPase function and sorbic acid stress tolerance

“…These observations are supported by identification of other V-ATPase genes (VMA4, VMA5, VMA7, VMA8, VMA13 and VMA22) as mediators of sorbic acid tolerance in screens of the yeast deletion mutant collection (Mollapour et al, 2004;Schuller et al, 2004), several of which we independently identified in our own, similar screen (data not shown). A functional V-ATPase is therefore required for optimal adaptation to sorbic acid stress, and available evidence supports the hypothesis that V-ATPase function is required to mediate tolerance to a range of growth-inhibitory compounds in yeast.…”

“…Deletion of DBF2 results in other stress-sensitive phenotypes (Liu et al, 1997b), and although DBF2 expression is induced in response to stresses such as salt and heat, we observed no significant change in the level of Dbf2p during growth in the presence of sorbic acid. This conclusion is supported by two independent transcriptional profiling studies of yeast exposed to sorbic acid stress, neither of which detected upregulation of DBF2 (de Nobel et al, 2001;Schuller et al, 2004). Loss of DBF2 also did not affect the increased expression of the ABC-transporter Pdr12p observed during sorbic acid stress (Piper et al, 1998).…”

“…War1p, a novel transcription factor of the Zn 2 Cys 6 family, is the stressactivated regulator of PDR12 (Kren et al, 2003). War1p binds constitutively to the PDR12 promoter irrespective of sorbic acid stress, which is believed to activate War1p through phosphorylation (Kren et al, 2003;Schuller et al, 2004). Notably, the signalling pathway that mediates War1p phosphorylation has not yet been identified.…”

Auxotrophy for uridine increases the sensitivity of Aspergillus niger to weak-acid preservatives

“…In contrast to A. niger, uridine-auxotrophic strains of S. cerevisiae did not show abnormal sensitivity to weak acids, in accordance with a previous report where the authors screened the S. cerevisiae gene-deletion strain collection and did not observe any hypersensitivity to sorbic acid in strains deleted in any gene involved in nucleotide synthesis (Mollapour et al, 2004). It therefore appears that the uridine transporter in A. niger is sensitive to weak-acid inhibition, whereas the S. cerevisiae homologue is not.…”

Stress and Cell Death in Yeast Induced by Acetic Acid

“…The different environmental conditions not only produce a set of common changes, probably accounting for the cross-resistances to different unrelated stress, but also generate specific responses reflecting the particular cell targets for each stress. Genome-wide functional analyses using the yeast disruptome, as well as gene expression profiling, have been exploited to identify key components of stress response induced by different weak carboxylic acids, namely sorbic, citric, benzoic, propionate, lactic and acetic acids (Abbot et al, 2008;Kawahata et al, 2006;Mira et al, 2009Mira et al, , 2010Mollapour et al, 2004;Schuller et al, 2004). The first studies combining the two approaches were performed with sorbic acid.…”