利用SPT3的定向进化提高工业酿酒酵母乙醇耐受性

Improving ethanol tolerance of Saccharomyces cerevisiae industrial strain by directed evolution of SPT3

摘要:

利用对转录因子的定向进化可对多基因控制的性状进行有效的代谢工程改造。本研究对酿酒酵母负责胁迫相关基因转录的SAGA复合体成分SPT3编码基因进行易错PCR随机突变，并研究了SPT3的定向进化对酿酒酵母乙醇耐性的影响。将SPT3的易错PCR产物连接改造的pYES2.0表达载体并转化酿酒酵母Saccharomy cescerevisiae 4126，构建了突变体文库。通过筛选在高浓度乙醇中耐受性提高的突变株，获得了一株在10％(V/V)乙醇中生长较好的突变株M25。该突变株利用125 g/L的葡萄糖进行乙醇发酵时，终点乙醇产量比对照菌株提高了11.7％。由此表明，SPT3是对酿酒酵母乙醇耐性进行代谢工程改造的一个重要的转录因子。

Abstract:

Directed evolution of transcription factors can be employed to effectively improve the phenotypes which are controlled by multiple genetic loci. In this study, we used error-prone PCR for the directed evolution of SPT3, which is the component of yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex responsible for the transcription of stress-related genes, and studied its effect on the improvement of ethanol tolerance. Mutant library was constructed by ligating the error-prone PCR products with a modified pYES2.0 plasmid, and the expression plasmids were subsequently transformed to yeast industrial strain Saccharomyces cerevisiae 4126. One mutant strain M25 showing superior growth in presence of 10% ethanol was selected. M25 produced 11.7% more ethanol than the control strain harboring the empty vector when 125 g/L glucose was used as substrate. This study revealed that SPT3 is an important transcription factor for the metabolic engineering of yeast ethanol tolerance.