Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyse five representatives with varying ethanol tolerances. The most tolerant strain, AJ4, was dominant in co-culture at 0% and 10% ethanol. Unexpectedly, although it does not have the highest NIC or MIC, MY29 was the dominant strain in co-culture at 6% ethanol, which may be linked to differences in its basal lipidome. Whilst relatively few lipidomic differences were observed between strains, a significantly higher PE concentration was observed in the least tolerant strain, MY26, at 0% and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggests a limited set of membrane compositions that diverse yeast species use to achieve this. Importance Many microbial processes are not implemented at the industrial level because the product yield is poorer and more expensive than can be achieved by chemical synthesis. It is well established that microbes show stress responses during bioprocessing, and one reason for poor product output from cell factories is production conditions that are ultimately toxic to the cells. During fermentative processes, yeast cells encounter culture media with high sugar content, which is later transformed into high ethanol concentrations. Thus, ethanol toxicity is one of the major stresses in traditional and more recent biotechnological processes. We have performed a multilayer phenotypic and lipidomic characterization of a large number of industrial and environmental strains of Saccharomyces to identify key resistant and non-resistant isolates for future applications.

Biological membranes form the boundaries to cells. They are integral to cellular function, retaining the valuable components inside and preventing access of unwanted molecules. Many different classes of molecules demonstrate disruptive properties to the plasma membrane. These include alcohols, detergents and antimicrobial agents. Understanding this disruption and the mechanisms by which it can be mitigated is vital for improved therapeutics as well as enhanced industrial processes where the compounds produced can be toxic to the membrane. This mini-review describes the most common molecules that disrupt cell membranes along with a range of in vitro liposome-based techniques that can be used to monitor and delineate these disruptive processes.

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During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII–XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms.