Table_3_Physiological and Molecular Characterization of an Oxidative Stress-Resistant Saccharomyces cerevisiae Strain Obtained by Evolutionary Engineering.DOCX

Oxidative stress is a major stress type observed in yeast bioprocesses, resulting in a decrease in yeast growth, viability, and productivity. Thus, robust yeast strains with increased resistance to oxidative stress are in highly demand by the industry. In addition, oxidative stress is also associated with aging and age-related complex conditions such as cancer and neurodegenerative diseases. Saccharomyces cerevisiae, as a model eukaryote, has been used to study these complex eukaryotic processes. However, the molecular mechanisms underlying oxidative stress responses and resistance are unclear. In this study, we have employed evolutionary engineering (also known as adaptive laboratory evolution – ALE) strategies to obtain an oxidative stress-resistant and genetically stable S. cerevisiae strain. Comparative physiological, transcriptomic, and genomic analyses of the evolved strain were then performed with respect to the reference strain. The results show that the oxidative stress-resistant evolved strain was also cross-resistant against other types of stressors, including heat, freeze-thaw, ethanol, cobalt, iron, and salt. It was also found to have higher levels of trehalose and glycogen production. Further, comparative transcriptomic analysis showed an upregulation of many genes associated with the stress response, transport, carbohydrate, lipid and cofactor metabolic processes, protein phosphorylation, cell wall organization, and biogenesis. Genes that were downregulated included those related to ribosome and RNA processing, nuclear transport, tRNA, and cell cycle. Whole genome re-sequencing analysis of the evolved strain identified mutations in genes related to the stress response, cell wall organization, carbohydrate metabolism/transport, which are in line with the physiological and transcriptomic results, and may give insight toward the complex molecular mechanisms of oxidative stress resistance.氧化应激是酵母生物过程中常见的主要胁迫类型，会导致酵母生长、存活能力与生产性能下降。因此，工业界对具备氧化应激抗性的健壮酵母菌株需求极高。此外，氧化应激还与衰老及癌症、神经退行性疾病等年龄相关复杂病症密切相关。作为真核生物模型的酿酒酵母（Saccharomyces cerevisiae）已被用于研究这类复杂真核过程，但目前氧化应激响应与抗性的分子机制仍不明确。本研究采用进化工程技术（又称适应性实验室进化（adaptive laboratory evolution，ALE）），获得了一株具备氧化应激抗性且遗传稳定的酿酒酵母菌株。随后以参考菌株为对照，对该进化菌株开展比较生理学、转录组学与基因组学分析。结果显示，该进化获得的氧化应激抗性菌株同时对热、冻融、乙醇、钴离子、铁离子及盐类等多种其他胁迫因子具备交叉抗性；其海藻糖与糖原合成水平也有所提升。进一步的转录组分析表明，诸多与胁迫响应、物质转运、碳水化合物、脂质及辅因子代谢过程、蛋白质磷酸化、细胞壁组织与生物发生相关的基因出现上调；而下调基因则主要涉及核糖体与RNA加工、核转运、tRNA及细胞周期相关功能。对该进化菌株的全基因组重测序分析，鉴定出了与胁迫响应、细胞壁组织、碳水化合物代谢/转运相关的基因突变，这与生理学及转录组学结果相符，或可为解析氧化应激抗性的复杂分子机制提供新的见解。