Aims: The present work aimed at identifying the metabolic response to acid stress and the mechanisms that lead to cell tolerance and adaptation. Methods and Results: Two strategies were used: screening deletion mutants for cell growth at neutral and acid pH compared to wild type and measurement by qPCR of the expression of yeast genes involved in different pathways. Conclusions:The results complement our previous findings and showed that the Cell Wall Integrity pathway is the main mechanism for cell tolerance to acid pH, and this damage triggers the protein kinase C (PKC) pathway mainly via the Wsc1p membrane sensor. In addition, cell wall injury might mimic the effects of high osmotic shock and activates the High Osmolarity Glycerol pathway, which amplifies the signal in the upper part of PKC pathway and leads to the activation of Ca 2+ channels by SLT2 overexpression and this Ca 2+ influx further activates calcineurin. Together, these mechanisms induce the expression of genes involved in cell cycle regulation and cell wall regeneration. Significance and Impact of the Study: These interactions are responsible for long-term adaptation of yeast cells to the acidic environment, and the results could drive future work on the genetic modification of yeast strains for high tolerance to the stresses of the bioethanol fermentation process.

In bioethanol production plants, yeast cells are generally recycled between fermentation batches by using a treatment with sulphuric acid at a pH ranging from 2.0 to 2.5. We have previously shown that Saccharomyces cerevisiae cells exposed to sulphuric acid treatment induce the general stress response pathway, fail to activate the protein kinase A signalling cascade and requires the mechanisms of cell wall integrity and high osmolarity glycerol pathways in order to survive in this stressful condition. In the present work, we used transcriptome-wide analysis as well as physiological assays to identify the transient metabolic responses of S. cerevisiae under sulphuric acid treatment. The results presented herein indicate that survival depends on a metabolic reprogramming of the yeast cells in order to assure the yeast cell viability by preventing cell growth under this harmful condition. It involves the differential expression of a subset of genes related to cell wall composition and integrity, oxidation-reduction processes, carbohydrate metabolism, ATP synthesis and iron uptake. These results open prospects for application of this knowledge in the improvement of industrial processes based on metabolic engineering to select yeasts resistant to acid treatment.

BackgroundPolyhexamethylene biguanide (PHMB) is an antiseptic polymer that is mainly used for cleaning hospitals and pools and combating Acantamoeba infection. Its fungicide activity was recently shown by its lethal effect on yeasts that contaminate the industrial ethanol process, and on the PE-2 strain of Saccharomyces cerevisiae, one of the main fermenting yeasts in Brazil. This pointed to the need to know the molecular mechanism that lay behind the cell resistance to this compound. In this study, we examined the factors involved in PHMB-cell interaction and the mechanisms that respond to the damage caused by this interaction. To achieve this, two research strategies were employed: the expression of some genes by RT-qPCR and the analysis of mutant strains.ResultsCell Wall integrity (CWI) genes were induced in the PHMB-resistant Saccharomyces cerevisiae strain JP-1, although they are poorly expressed in the PHMB-sensitive Saccharomyces cerevisiae PE2 strain. This suggested that PHMB damages the glucan structure on the yeast cell wall. It was also confirmed by the observed sensitivity of the yeast deletion strains, Δslg1, Δrom2, Δmkk2, Δslt2, Δknr4, Δswi4 and Δswi4, which showed that the protein kinase C (PKC) regulatory mechanism is involved in the response and resistance to PHMB. The sensitivity of the Δhog1 mutant was also observed. Furthermore, the cytotoxicity assay and gene expression analysis showed that the part played by YAP1 and CTT1 genes in cell resistance to PHMB is unrelated to oxidative stress response. Thus, we suggested that Yap1p can play a role in cell wall maintenance by controlling the expression of the CWI genes.ConclusionThe PHMB treatment of the yeast cells activates the PKC1/Slt2 (CWI) pathway. In addition, it is suggested that HOG1 and YAP1 can play a role in the regulation of CWI genes.

Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sulphuric acid to control the bacterial population. This treatment can lead to loss of cell viability, with consequences on the fermentation yield. Thus, the aim of this study was to define the functional cellular responses to inorganic acid stress. Saccharomyces cerevisiae strains with mutation in several signalling pathways, as well as cells expressing pH-sensitive GFP derivative ratiometric pHluorin, were tested for cell survival and cytosolic pH (pHc) variation during exposure to low external pH (pHex). Mutants in calcium signalling and proton extrusion were transiently sensitive to low pHex, while the CWI slt2Δ mutant lost viability. Rescue of this mutant was observed when cells were exposed to extreme low pHex or glucose starvation and was dependent on the induced reduction of pHc. Therefore, a lowered pHc leads to a complete growth arrest, which protects the cells from lethal stress and keeps cells alive. Cytosolic pH is thus a signal that directs the growth stress-tolerance trade-off in yeast. A regulatory model was proposed to explain this mechanism, indicating the impairment of glucan synthesis as the primary cause of low pHex sensitivity.