Saccharomyces cerevisiae is widely used in traditional brewing and modern fermentation industries to produce biofuels, chemicals and other bioproducts, but challenged by various harsh industrial conditions, such as hyperosmotic, thermal and ethanol stresses. Thus, its stress tolerance enhancement has been attracting broad interests. Recently, CRISPR/Cas-based genome editing technology offers unprecedented tools to explore genetic modifications and performance improvement of S. cerevisiae. Here, we presented that the Target-AID (activation-induced cytidine deaminase) base editor of enabling C-to-T substitutions could be harnessed to generate in situ nucleotide changes on the S. cerevisiae genome, thereby introducing protein point mutations in cells. The general transcription factor gene SPT15 was targeted, and total 36 mutants with diversified stress tolerances were obtained. Among them, the 18 tolerant mutants against hyperosmotic, thermal and ethanol stresses showed more than 1.5-fold increases of fermentation capacities. These mutations were mainly enriched at the N-terminal region and the convex surface of the saddle-shaped structure of Spt15. Comparative transcriptome analysis of three most stress-tolerant (A140G, P169A and R238K) and two most stress-sensitive (S118L and L214V) mutants revealed common and distinctive impacted global transcription reprogramming and transcriptional regulatory hubs in response to stresses, and these five amino acid changes had different effects on the interactions of Spt15 with DNA and other proteins in the RNA Polymerase II transcription machinery according to protein structure alignment analysis. Taken together, our results demonstrated that the Target-AID base editor provided a powerful tool for targeted in situ mutagenesis in S. cerevisiae and more potential targets of Spt15 residues for enhancing yeast stress tolerance.

中文翻译：

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在酿酒酵母中通过SPT15碱基编辑增强压力耐受性

酿酒酵母广泛用于传统酿造和现代发酵工业，以生产生物燃料、化学品和其他生物产品，但受到各种恶劣工业条件的挑战，如高渗、热和乙醇胁迫。因此，其抗逆性增强引起了广泛的兴趣。最近，基于 CRISPR/Cas 的基因组编辑技术为探索酿酒酵母的遗传修饰和性能改进提供了前所未有的工具。在这里，我们提出了可以利用实现 C 到 T 替换的 Target-AID（激活诱导的胞苷脱氨酶）碱基编辑器来在酿酒酵母基因组上产生原位核苷酸变化，从而在细胞中引入蛋白质点突变。靶向通用转录因子基因SPT15，共获得36个具有多样化逆境耐受性的突变体。其中，18 个耐受高渗、热和乙醇胁迫的突变体的发酵能力提高了 1.5 倍以上。这些突变主要集中在 Spt15 的鞍形结构的 N 端区域和凸面。三个最耐压力（A140G、P169A 和 R238K）和两个最敏感（S118L 和 L214V）突变体的比较转录组分析揭示了响应压力的共同和独特的影响全局转录重编程和转录调节中心，以及这五个氨基酸根据蛋白质结构比对分析，变化对 Spt15 与 RNA 聚合酶 II 转录机制中的 DNA 和其他蛋白质的相互作用有不同的影响。综合起来，