ABSTRACT

Improving yeast tolerance toward isobutanol is a critical issue enabling high-titer industrial production. Here, we used EMS mutagenesis to screen Saccharomyces cerevisiae with greater tolerance toward isobutanol. By this method, we obtained EMS39 with high-viability in medium containing 16 g/L isobutanol. Then, we metabolically engineered isobutanol synthesis in EMS39. About 2μ plasmids carrying PGK1p-ILV2, PGK1p-ILV3 and TDH3p-cox4-ARO10 were used to over-express ILV2, ILV3 and ARO10 genes, respectively, in EMS39 and wild type W303-1A. And the resulting strains were designated as EMS39-20 and W303-1A-20. Our results showed that EMS39-20 increased isobutanol titers by 49.9% compared to W303-1A-20. Whole genome resequencing analysis of EMS39 showed that more than 59 genes had mutations in their open reading frames or regulatory regions. These 59 genes are enriched mainly into cell growth, basal transcription factors, cell integrity signaling, translation initiation and elongation, ribosome assembly and function, oxidative stress response, etc. Additionally, transcriptomic analysis of EMS39-20 was carried out. Finally, reverse engineering tests showed that overexpression of CWP2 and SRP4039 could improve tolerance of S.cerevisiae toward isobutanol. In conclusion, EMS mutagenesis could be used to increase yeast tolerance toward isobutanol. Our study supplied new insights into mechanisms of tolerance toward isobutanol and enhancing isobutanol production in S. cerevisiae.

中文翻译：

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通过 EMS 诱变提高酿酒酵母中的异丁醇耐受性和滴度

摘要

提高酵母对异丁醇的耐受性是实现高滴度工业生产的关键问题。在这里，我们使用 EMS 诱变来筛选对异丁醇具有更大耐受性的酿酒酵母。通过这种方法，我们获得了在含有 16 g/L 异丁醇的培养基中具有高活力的 EMS39。然后，我们在 EMS39 中代谢工程化异丁醇合成。关于2μ质粒携带PGK1p-ILV2，PGK1p-ILV3和TDH3P-COX4 - ARO10使用过度表达ILV2，ILV3和ARO10基因分别在 EMS39 和野生型 W303-1A 中。所得菌株命名为EMS39-20和W303-1A-20。我们的结果表明，与 W303-1A-20 相比，EMS39-20 将异丁醇滴度提高了 49.9%。EMS39 的全基因组重测序分析表明，超过 59 个基因在其开放阅读框或调控区域中发生突变。这59个基因主要富集在细胞生长、基础转录因子、细胞完整性信号、翻译起始和延伸、核糖体组装和功能、氧化应激反应等方面。此外，还进行了EMS39-20的转录组学分析。最后，逆向工程测试表明CWP2和SRP4039 的过表达可以提高酿酒酵母的耐受性对异丁醇。总之，EMS诱变可用于增加酵母对异丁醇的耐受性。我们的研究为酿酒酵母耐受异丁醇和提高异丁醇产量的机制提供了新的见解。