Microbial Resources Research Center, College of Natural Sciences, Ewha Womans,University, Seoul, Korea.
Biotechnol Bioeng. 2011 Aug;108(8):1776-87. doi: 10.1002/bit.23141. Epub 2011 Apr 3.
Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565-1568), seems to a powerful tool for creating ethanol-tolerant strains. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1-5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by using gTME.
由于高浓度乙醇是乙醇发酵过程中的主要应激因素，因此具有乙醇耐受性的酵母菌株对于工业规模的乙醇生产非常理想。一种名为全局转录机制工程（gTME）的技术利用了酿酒酵母 SPT15 编码 TATA 结合蛋白的突变文库（Alper 等人，2006；Science 314：1565-1568），似乎是一种创建乙醇耐受菌株的强大工具。然而，所创建的菌株在富培养基中耐受高乙醇的能力尚未得到证实。在这项研究中，使用类似的策略获得了五株酿酒酵母的乙醇耐受性增强株（ETS1-5）。将两个选定菌株 ETS2 和 ETS3 的全局转录谱与对照进行比较，发现了 42 个以两倍变化共同调节的基因。在一个基因敲除文库中可获得的 34 个缺失突变体中，有 18 个对乙醇敏感，这表明这些基因与乙醇耐受性密切相关。其中 8 个是新的，大多数功能未知。为了为未来的工业应用建立基础，通过将 ETS2 和 ETS3 的 SPT15 突变等位基因整合到染色体中，创建了菌株 iETS2 和 iETS3，它们在富培养基上的乙醇冲击下也表现出增强的乙醇耐受性和存活能力。在生物反应器中进行 20%葡萄糖 24 小时发酵表明，iETS2 和 iETS3 的生长情况更好，比对照菌株多产生约 25%的乙醇。乙醇得率和生产率也得到了显著提高：对照为 0.31g/g 和 2.6g/L/h，iETS2 和 iETS3 分别为 0.39g/g 和 3.2g/L/h。因此，我们的研究表明 gTME 在生成具有增强的乙醇耐受性的菌株方面具有实用性，从而提高了乙醇产量。通过使用 gTME，可能会进一步创建对其他应激因素（如热、发酵抑制剂、渗透压等）具有增强耐受性的菌株。
