Evolutionary engineering of freeze-thaw stress-resistant yeasts without using chemical mutagenesis

Abstract

Saccharomyces cerevisiae, also known as budding yeast or baker's yeast is a unicellular microorganism from the fungi kingdom. It has been consistently used in winemaking, brewing and baking bread throughout human history. After 1930s, laboratory studies were conducted to obtain strains with increased product quality. Today, S. cerevisiae is the most popular yeast strain due to its good fermentative abilities. S. cerevisiae with high fermentation performance and tolerance to environmental stresses is preferred for industrial applications. During bread production, yeast cells are exposed to a variety of environmental stresses including freeze–thaw, high sugar concentrations, air-drying and oxidative stress. Stress conditions cause a decline in cell growth rate, product yield and quality. Cells give responses to stress conditions, as environmental stress response (ESR) and stress-specific response. ESR mechanism is not specific to the stress factor and it can be used to explain the cross-resistance of the yeast cells against various stress types. One of the reasons for cross-resistance is the use of the same transcription factors as a response to a variety of different stresses. S. cerevisiae is exposed to freeze-thaw stress during the cryopreservation and frozen dough process. Freeze-thaw stress causes physiological injuries to cells. At high freezing rates, formation of intracellular ice crystals causes cellular damages; while at low freezing rates formation of extracellular ice crystals causes cellular dehydration. The thawing process causes oxidative stress which leads to oxidative damage on proteins, nucleic acids and other biomolecules inside the cell. Studies conducted in S. cerevisiaes' stress-specific response against freeze-thaw stress revealed cells focus on regulating the contents of the cell membrane, protecting cell wall integrity, increasing degradation of damaged proteins from stress and increasing overall protein synthesis under stress conditions. Cryoprotective agents can be added to decrease ice crystal formation under freezing conditions. Alternatively, yeast levels in the product can be increased to increase product yield. However, these methods can decrease product quality and increase cost. Thus, stress-resistant S. cerevisiae strains are preferred for industrial applications. Stress-resistant strains can be obtained by metabolic engineering. Evolutionary engineering is an inverse metabolic engineering method that mimics the natural evolution process. In this approach, the desired phenotype is selected first and the genes responsible for the phenotype are determined later by reverse engineering methods. In this study, freeze-thaw resistant yeast strains were obtained with the evolutionary engineering method. A reference yeast strain was exposed to freeze-thaw stress in the form of pulse stress selection. The evolved strains obtained under stress conditions generally show mutations mainly in their stress-induced genes. This allows ease in reverse engineering studies to determine genes related to the applied stress. Freeze-thaw stress was applied in the form of pulse stress selection to maintain the survival rate of cells with increasing stress levels and to induce selective pressure. In this study, a S. cerevisiae CEN.PK113-7D reference strain was exposed to gradually increasing freeze-thaw stress until the final population was obtained. The final population was obtained after 10 cycles of freeze-thaw stress application. Ten mutant individuals were randomly selected from the final population and their resistance to freeze-thaw stress was tested with the spot assay method. Four evolved strains labeled as FT-1, FT-5, FT-6 and FT-9 that showed the highest freeze-thaw resistance were selected for detailed analysis. Further physiological characterizations of the evolved strains were made by cross resistance analysis. FT-1, FT-6 and FT-9 showed cross-resistance to potassium chloride (KCl) and iron stress. KCl, at high concentrations, causes hyperosmotic stress to the cell. This cross-resistance could be the result of a similar response mechanism activated by the cell to protect itself from dehydration caused by freezing stress. Metals such as iron increase generation of ROS in cell and cause oxidative stress. The cross resistance to iron stress could be the result of activation of similar pathways used by the cell as a response to oxidative stress caused by thawing process. All evolved strains tested showed resistance to boric acid. Boric acid disrupts cell wall synthesis in S. cerevisiae. The freezing process also causes cell wall damage in S. cerevisiae. Inducing cell wall synthesis due to freezing stress may also result with increased resistance to boric acid. The aim of this study was to obtain freeze-thaw stress-resistant S. cerevisiae strains from a reference laboratory strain, without using chemical mutagenesis, by evolutionary engineering. Physiological characterization of the evolved strains was also performed by determining their cross-resistance to selected stress factors. Further genomic, transcriptomic and proteomic analyses could be performed on the selected FT-9 strain to identify the genes, pathways and molecular mechanisms responsible for resistance against freeze-thaw stress and the pathways that cause cross-resistance to selected stress factors.