Department of Life Science, College of Natural Sciences, Ewha Womans University, 11-1, Daehyun-dong, Seodaemun-gu, Seoul 120-750, South Korea.
Appl Microbiol Biotechnol. 2011 Aug;91(4):1159-72. doi: 10.1007/s00253-011-3298-z. Epub 2011 May 10.
Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1-5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 °C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 °C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 °C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains.
耐乙醇和热应激的酿酒酵母菌株对于工业乙醇生产至关重要。在本研究中，通过筛选转座子介导的突变体文库，分离出了五株耐受高达 15%乙醇的菌株（Tn1-5）。其中两株菌株表现出对热（42°C）的耐受性。转座子插入位点的确定和 Northern blot 分析鉴定了七个可能的基因（CMP2、IMD4、SSK2、PPG1、DLD3、PAM1 和 MSN2），并揭示了 CMP2 和 IMD4、SSK2 和 PPG1 的同时下调，DLD3 的下调以及 PAM1 和 MSN2 的开放阅读框中断，表明可以赋予乙醇和/或耐热性。这些七个单个基因的敲除突变体具有乙醇耐受性，其中三个（SSK2、PPG1 和 PAM1）对热具有耐受性。通过同源或过表达每个基因，这些耐受表型恢复为敏感表型。五个转座子突变体在含有 30%葡萄糖和初始 6%乙醇的丰富培养基中，在 30°C 下培养时，比对照菌株具有更高的乙醇产量和更快的生长速度。其中，两个耐热转座子突变体（Tn2 和 Tn3）在 42°C 时与对照相比，表现出明显增强的生长和乙醇产量。本研究中鉴定的基因可能为开发工业酵母菌株提供应用基础。
