Chronological lifespan analysis of stress-resistant yeasts

Abstract

Saccharomyces cerevisiae has an important place in human life with its wide usage in various processes like fermentation, brewing, bread and wine production from the oldest times in history. Since its genome sequencing in 1996, it has become one of the most well-known and studied model organisms in many different areas of biology such as cell biology, biotechnology, cancer and aging research. Compared to other model organisms, its ease in genetic manipulation and cultivation conditions made it a convenient host for the production of heterologous proteins and economically valuable products. Yeast shares 30% of homology with many human genes, thus it is a convenient platform to study eukaryotic cell metabolism along with disease models. Aging is a common process all living organisms share and involves numerous metabolic and physiological changes that usually result from accumulated damage and deterioration. Despite the developed technology and improved living conditions through every passage of human life, it is estimated that the aging population will cover one-fifth of the whole population of the world by end of the century and age related diseases will cause a socioeconomic burden to governments. Studying aging in humans is complicated because of the long lifespan and economic and ethical concerns. Although a wide range of organisms share similar aging patterns, using yeast provides a convenient and accurate eukaryotic model with a shorter lifespan and easier growth capability. There are two main approaches to studying aging in yeast: chronological lifespan (CLS) and replicative lifespan (RLS). CLS seeks to analyze the lifespan of undivided yeast cells after they enter the stationary phase, mostly caused by decreased nutrients or toxic metabolite accumulation. RLS defines the number of cell divisions a cell undergoes before its death. CLS analysis is particularly useful for analyzing the response and survival of the cell against certain stress factors and modeling G0 cells that arrest their cell cycle. Metabolic engineering is an effective biotechnological approach for improving metabolic processes and product formation of the organism by altering the existing mechanisms or introducing new ones through the usage of recombinant DNA technology. In classical metabolic engineering, information on metabolic, genetic and physiological data of the strain is gathered, then the manipulations on relevant factors are employed to obtain the desired phenotype. Yet in the inverse metabolic engineering approach, for example when evolutionary engineering is employed, the desired phenotype is achieved using laboratory-based evolutionary settings. In this strategy, yeast strains can become resistant to certain stress factors or produce desired molecules throughout the increased stress treatment during culture. After obtaining the desired phenotype, yeast strains are examined by genomic or transcriptomic analyses to further determine the molecular changes in the genome or transcriptome. With more advanced xix technologies such as CRISPR-Cas9, altered genetic traits can be transferred to wild type strains to evoke the same resistant phenotype. In this study, previously obtained stress-resistant S. cerevisiae strains were analyzed for their CLS and viability performances. Stress factors selected for this purpose were antimycin, boron and freeze-thaw stresses which can affect the production efficiency or viability of yeast strains. In parallel with the general evolutionary engineering strategy, strains were obtained by increasing the stress levels gradually, which is the concentration of the compound in the case of antimycin and boron, and repeat numbers in the case of freeze-thaw stress, in selection cultures until the resistant population is achieved. It was shown previously that the evolved strains could become cross resistant to other stress types or their longevity can be affected by the process. The aim of this study was to determine which type of stress resistances can extend or shorten the CLS of the yeast thus affecting the lifespan of the industrial and laboratory yeast strains. For this aim, both quantitative and semi-quantitative CLS analyses were carried out. In the semi-quantitative CLS experiment, OD600 values of the yeast strains were set to 6 before they were spotted onto agar plates every 2nd day with serial dilutions. The longevity of the resistant strains was compared with their control strains visually, based on the growth on the plates. According to the results, P8 which is the freeze-thaw stress-resistant, industrial polyploid strain had a longer CLS than its industrial reference strain, whereas the antimycin and boron-resistant strains did not have a longer CLS than their reference strain. In the second part of the study, quantitative CLS analysis was done by spreading the long-lived industrial P8 strain along with its industrial reference strain R625 and the laboratory reference strain 905 to agar plates. The longevity was measured by counting colony-forming units (CFUs). The experiment was repeated until the viability of the cultures was reduced to 0.0001% from day 0 of the experiment where the viability was accepted as 100%. In the second part of the study, further validating the results from the semi-quantitative analysis, P8 exhibited longer CLS compared to its industrial reference strain and could live until the 10th day of the experiment. Among the various stress-resistant strains tested in this study, only the freeze-thaw stress-resistant, industrial P8 strain was found to have a longer CLS. However, the antimycin and boron-resistant yeast strains did not show a longer CLS compared to their laboratory reference strain. Since the freeze-thaw response was previously associated with oxidative stress response and nutrient metabolism alterations, the longer CLS of the freeze-thaw stress-resistant industrial strain can be related to changes in respective pathways that originated from the evolutionary engineering process. In the scope of the research done for this study, despite being studied in other organisms, the effect of boron resistance on longevity was studied in yeast for the first time. Similarly, antimycin resistance was examined for its effect on longevity in yeast for the first time, as well. Further studies to analyze genomic and transcriptomic changes that occurred by the acquired resistance can be performed and these changes can be transferred to wild-type or reference strains to assess the viability and CLS profiles.