Comparative whole genome sequencing and bioinformatic analysis of afreeze-thaw stress-resistant, industrial Saccharomyces cerevisiae strain

Abstract

Yeasts have been around for thousands of years; they have benefited people in many fields such as science, medicine, food and agriculture. In particular, Saccharomyces cerevisiae is used in multi-enzyme pathways for the expression of protein biocatalysts and to synthesize chemicals and small molecular weight compounds important for medicine and nutrition. Due to these advances, S. cerevisiae is currently the primary model organism for the study of eukaryotic biology and human diseases. S. cerevisiae is a unicellular eukaryote. It has 16 chromosomes with subcellular organelles containing and these organelles commonly found in eukaryotes. S. cerevisiae has a classical eukaryotic cell cycle (including G1, S, G2, and M). Different strains of S. cerevisiae have been established to fill the gaps and requirements in genetic, biochemistry and physiology research. The CEN.PK family is frequently used in industrial biotechnology research, while the BY strain family derived from the S288c strain is mainly used in genetic studies. Yeast contains a large number of orthologous genes in the human genome. By examining the expression of some genes in yeast, the mechanism in more complex eukaryotes can be understood. S. cerevisiae has highly developed homologous recombination and contributes to the basic knockout operation of genes. Furthermore, S. cerevisiae is an important model for understanding the role of stress response genes in living organisms. S. cerevisiae cells can experience different environmental stress conditions such as metal toxicity, heat or cold shock during growth, essential nutrient limitations, hyperosmotic or hypoosmotic pressure, and ethanol toxicity. To overcome these stress conditions, S. cerevisiae cells have been developed to detect stress signals and respond to these signals through general or specific stress response and protection programs. Cryopreservation is a long-term storage method of various living cells, and the freeze-thaw tensile strength is important in cryopreservation. However, this method includes freezing and thawing processes that cause fatal damage to cells. Under freeze-thaw stress conditions, cells are exposed to more than one type of stress. These are; cold during freezing, dehydration, osmotic, ice crystal formation and oxidative stress during thawing. Therefore, it is important to obtain freeze-thaw tolerant organisms and to examine all freeze-thaw tolerance mechanisms. Yeasts are organisms that have a high survival rate when rapidly frozen at -80 °C. However, it is usually applied to commercial products at -20 °C and is highly damaging to cells, predominantly lethal to cells. Applications of freeze-thaw stress in S. cerevisiae are concerned with inducing this cross-resistance to overcome the effects of freeze-thaw stress. Additional mechanisms at gene expression levels are thought to be triggered and maintained during freeze-thaw exposure to achieve multiple stress tolerances and freeze-thaw stress tolerances. Metabolic engineering; it is defined as enhanced production of metabolites and cellular activities. It is done with through manipulation of the enzymatic, transport and regulatory functions of the cell by modifications of cellular networks including metabolic, gene regulatory and signaling networks using recombinant DNA technology. Metabolic engineering strategies can be divided into two groups as rational engineering and inverse metabolic engineering. Evolutionary engineering is a common strategy used in biological research to achieve the desired phenotype by improving its properties such as high environmental tolerance and improvement of product yield. Evolutionary engineering differs from metabolic engineering in that it is based on random methods; genetic modifications are not directed. Ploidy is the number of complete sets of chromosomes in a cell, which means the number of possible alleles for autosomal and pseudoautosomal genes. Many eukaryotic creatures have two sets of chromosomes (diploid) or more than two sets of chromosomes (polyploid). During the evolution of plants, animals, and fungi, ancient whole-genome duplication (WGD) or hybridization events frequently result in diploid and polyploid conditions. Increased chromosomal sets, development, cellular stress, disease, and evolution all cause polyploidy. Yeasts, which belong to the kingdom of fungi, can exist in both haploid and diploid forms. Polyploid yeasts, on the other hand, are widespread. Allopolyploid cells are formed when two or more cells from closely related but not identical species fuse together. Euploidy refers to the stance in which cells have a chromosomal number that is an integral multiple of the characteristic circum haploid number. Due to the common occurrence of polyploidy and aneuploidy in yeast, variable chromosome numbers elicit characteristics that may be beneficial in specific circumstances. As a result, the physiology and fitness of cells with different ploidy levels may differ. Bioinformatics is a highly interdisciplinary field that drives knowledge discovery from biological data using computational analysis. Today, bioinformatics is becoming an important part of most life science research. The process by which the DNA sequence of gene expression is copied into a gene product or RNA is explained by the central dogma of molecular biology. Microarray and more recently RNA sequencing; it has been widely used to measure gene expression levels. In this thesis, ploidy and genomic differences between the industrial Saccharomyces cerevisiae strain R625 and the freeze-thaw resistant evolved strain P8 obtained from R625 by evolutionary engineering were analyzed to gain insight into the complex molecular mechanisms of ploidy and freeze-thaw stress resistance.