School of Chemical Engineering and Technology, Hebei University of Technology, No. 8 Guangrong Road, Hongqiao District, Tianjin 300130, PR China.
FEMS Yeast Res. 2021 Mar 18;21(2). doi: 10.1093/femsyr/foab012.
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.
提高酵母对异丁醇的耐受性是实现高浓度工业生产的关键。在这里，我们使用 EMS 诱变筛选出对异丁醇耐受性更高的酿酒酵母。通过这种方法，我们获得了在含 16 g/L 异丁醇的培养基中具有高存活率的 EMS39。然后，我们对 EMS39 中的异丁醇合成进行了代谢工程改造。使用携带 PGK1p-ILV2、PGK1p-ILV3 和 TDH3p-cox4-ARO10 的约 2μ 质粒分别过表达 ILV2、ILV3 和 ARO10 基因，将构建的质粒转入 EMS39 和野生型 W303-1A 中，得到的菌株分别命名为 EMS39-20 和 W303-1A-20。结果表明，与 W303-1A-20 相比，EMS39-20 提高了 49.9%的异丁醇产量。EMS39 的全基因组重测序分析表明，其 59 多个基因的开放阅读框或调控区发生了突变。这些基因主要富集在细胞生长、基础转录因子、细胞完整性信号转导、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，还对 EMS39-20 进行了转录组分析。最后，通过反向工程测试表明，过表达 CWP2 和 SRP4039 可以提高酵母对异丁醇的耐受性。总之，EMS 诱变可用于提高酵母对异丁醇的耐受性。本研究为酵母耐受异丁醇的机制和提高酿酒酵母异丁醇产量提供了新的见解。
