Genomic analysis of sulfur dioxide-stress-resistant Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae, a single-celled eukaryotic microorganism, is a budding yeast species. It is widely used as a preferred microorganism in many industrial processes such as baking, alcohol fermentation, biofuel and recombinant protein production, and in basic research to understand the complex biological processes of higher eukaryotic organisms, including humans, as it is a eukaryotic model organism. Sulfite (SO-23) is a form of SO2 in the cell and is a natural intermediate produced in the sulfate assimilation pathway. Molecular SO2 and HSO-3 (bisulfite) are other forms of SO2 that cause different pH changes in the cell. SO2 can inhibit the activity of enzymes such as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ATPase, which catalyze ATP production, or can cause disulfide bonds to break due to its reducing nature. Due to its effects such as pH decrease and ATPase inhibition, SO2 affects viability by activating acid and oxidative stress mechanisms. For this reason, developing SO2 resistance is important for S. cerevisiae strains used in industry. Despite its stress-inducing properties, SO2 is a molecule preferred in the food industry for protective purposes, due to its antioxidant and antimicrobial properties. Yeast cells, which have an important role in wine production, have been observed to gain resistance to this molecule through natural selection in different industrial areas to date. S. cerevisiae is a yeast the entire genome of which has been sequenced. S.cerevisiae is used safely due to its advantageous features. In addition, it can easily adapt to diverse stressful environments when used in the industry or food industry, with the resistance mechanisms it can develop against different stress conditions. For this reason, S. cerevisiae yeast strains that are resistant to SO2 stress can be used safely in different industrial applications such as wine production and baking, and will help meet the nutritional needs of the customers. A S. cerevisiae strain resistant to SO2 stress was obtained previously by evolutionary engineering. There are previous studies in the literature investigating SO2 stress in S. cerevisiae yeast strains. In one of these, the chromosomal rearrangement required for the expression of SSU1-R (sulfide resistance gene) in the SO2-resistant S. cerevisiae strain was investigated and the effect of Fzf1 transcription factor on this gene was examined. The aim of this study was to identify the genetic changes underlying the SO2-resistance of an evolved S. cerevisiae strain previously obtained by random mutagenesis by the chemical mutagen ethyl methanesulfonate (EMS) followed by evolutionary engineering. A comparative genomic analysis was conducted to elucidate the genetic factors contributing to the acquired SO2-resistance. Lyticase sensitivity assay was also employed to determine whether the acquisition of SO2 resistance changed the cell wall integrity, by comparing the results with that of the reference strain. The results revealed that the mutations identified in the ATG8, SSU1 and FZF1 genes that are related to sulfite transport and autophagy may play a key role in the SO2-resistance of S. cerevisiae. In addition, the mutation observed in the DCW1 gene that is involved in cell wall biosynthesis and the higher cell wall integrity of the evolved strain than the reference strain indicated that the increased cell wall integrity may also play a key role in the SO2-resistance of S. cerevisiae. The potential role of the identified gene mutations in SO2- resistance should be verified with reverse engineering and genome editing studies as a future work.