关键基因过表达提高酿酒酵母抑制剂耐受性及乙醇发酵性能

Improvement of inhibitor stress tolerance and ethanol fermentation of Saccharomyces cerevisiae by overexpression of novel key genes

木质纤维素预处理过程中会产生多种抑制物,抑制酿酒酵母细胞生长及乙醇发酵性能,为挖掘耐性基因、构建新的菌株,进一步提高酿酒酵母对这些抑制物的胁迫耐受性,研究在硫酸锌添加条件下转录组学分析过程中筛选到的可能关键基因ADE17、SSZ1、SET5、PPR1、OGG1和YKL222C过表达对酿酒酵母环境胁迫耐受性的影响.结果显示,不同基因过表达对酿酒酵母在多种抑制物胁迫条件下生长性能的影响不同,其中ADE17过表达对菌株在乙酸、糠醛、苯酚、丙酸和氯化钠胁迫条件下的生长提升最显著,而OGG1和SSZ1过表达对菌株生长的影响相对较弱.进一步对菌株进行驯化,在混合抑制物条件下驯化得到的BADE17-2和BADE17-4菌株延滞期比对照菌株BADE17缩短23 h.上述研究表明,硫酸锌添加从多方面影响了酿酒酵母耐受性,且关键基因过表达对不同环境胁迫条件具有多样性的影响,并且通过基因过表达和驯化方式结合可进一步提高酿酒酵母环境胁迫耐受性,提高纤维素乙醇发酵效率.

Various toxic inhibitors are released during the pretreatment of lignocellulosic biomass, and inhibit cell growth and ethanol production of Saccharomyces cerevisiae. Therefore, development of yeast strains with improved stress tolerance is of importance to enhance ethanol production efficiency from cellulosic biomass. Multiple genes are involved in stress tolerance, but novel genes remain to be explored from omics analysis. In our previous studies, zinc sulfate has been shown to improve the acetic acid tolerance of S. cerevisiae, but key genes involved in stress tolerance by zinc sulfate addition are still not clear.Six key genes, including ADE17, SSZ1, SET5, PPR1, OGG1, and YKL222 C, were identified from the transcriptomic analysis of zinc sulfate addition, and these genes were overexpressed in S. cerevisiae. Stress tolerance of the engineered strains was compared, and adaptive evolution was performed to further improve ethanol production. Overexpression of the six genes improved growth ability under acetic acid conditions as well as tolerance to other inhibitory conditions. The six selected genes showed various effects under different stress conditions, and ADE17 showed the strongest stimulation of cell growth under acetic acid, furfural, phenol, propionic acid, and Na Cl stresses, whereas OGG1 and SSZ1 showed the weakest effects. Furthermore, evolutionary engineering was performed with the BADE17 strain overexpressing ADE17, and the results showed that the adapted strains BADE17-2 and BADE17-4 exhibited a significantly shortened lag phase, which was 23 h shorter under multiple stress conditions than that of the control strain carrying the empty plasmid. The results demonstrated that zinc sulfate exerts various influences on yeast stress tolerance, and different key genes are impacted differently. A combination of gene overexpression and evolutionary engineering is an efficient method to improve stress tolerance and cellulosic ethanol production in S. cerevisiae.