Genomic analysis of freeze-thaw stress-resistant Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae is a well-known unicellular eukaryotic organism widely used in industry for making bread and for ethanol production. In addition, it is highly used as a eukaryotic model organism for research purposes in molecular biology and genetics. Since its genome sequence is well-known for several years and it has homologous genes with other eukaryotes including humans, S. cerevisiae is used as model organism for studying higher eukaryotic organisms. Additional advantages of S. cerevisiae are its easy manipulation and well-known growth conditions. Freeze-thaw stress is a physical stress type, and freeze-thaw stress resistance is highly desired in S. cerevisiae. Freeze-thaw stress causes physiological damage to cell. Freezing step causes ice crystal formation and cellular dehydration and damages the cell. Thawing step creates damage as a result of ROS formation. S. cerevisiae is faced with this stress type while used in bakery industry. First, the dough prepared with yeast is frozen and stored. Then, the dough is thawed for the baking process. This would cause the drop of gassing power of the yeast. The drop in rising power is due to cyclic Adenosine Monophosphate (cAMP)-Protein Kinase A (PKA) pathway. There is a glucose-induced increase of cAMP which results in the activation of PKA. Then, trehalase breaks down the trehalose, which is an important component in yeast stress resistance, including resistant to freeze- thaw stress. Thus, freeze-thaw stress resistance is important for improving the efficiency of bread making process and other yeast bioprocesses. In this study, genomic and physiological analysis of previously obtained freeze-thaw stress-resistant S. cerevisiae strains were performed. The freeze-thaw stress-resistant mutant strains were obtained by an inverse metabolic engineering strategy, evolutionary engineering, without using any chemical mutagenesis. After batch selection, genetic stability of the mutants was determined to understand if the resistance to freeze-thaw stress was an adaptation, or it was caused by a permanent genomic change. The selected FT1, FT5, FT6 and FT9 mutant strains were found to be genetically stable. Determination of the cell wall integrity was done using lyticase susceptibility assay. The assay was performed with the genetically stable, freeze-thaw stress-resistant mutant FT9. Results of the lyticase susceptibility assay demonstrated that the freeze-thaw stress-resistant mutant FT9 was resistant to lyticase degradation more than the reference strain (905), under both stress and nonstress conditions. Comparative whole genome sequencing analysis of the freeze-thaw stress-resistant mutant (FT9) revealed only one single nucleotide variation (SNV). The SNV was located on CDC25 gene, and it was a missense SNV. As a result of the missense SNV on this gene encoding a cell division cycle (Cdc) protein, threonine was replaced by lysine (T1415K). In conclusion, a previously obtained freeze-thaw stress-resistant S. cerevisiae evolved strain was characterized in this study at genomic and physiological levels. The results revealed that cAMP/PKA pathway-related changes occurred in the freeze-thaw stress-resistant mutant strain in order to protect it against damage. However, in order to fully comprehend the molecular basis of freeze-thaw stress resistance of this strain, its comparative transcriptomic and metabolic analyses would be necessary as future studies.