Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
J Biosci Bioeng. 2012 Apr;113(4):451-5. doi: 10.1016/j.jbiosc.2011.11.017. Epub 2011 Dec 16.
Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains.
木质纤维素生物质是生产生物乙醇的有前途的原料，因为它在全球范围内丰富且用途较少。然而，用弱酸处理木质纤维素生物质以释放纤维素和半纤维素会产生许多副产物，包括糠醛和 5-羟甲基糠醛，它们会抑制酵母发酵，因为它们会在细胞中产生活性氧 (ROS)。为了获得生物乙醇酵母菌株对氧化应激的高耐受性，我们专注于酿酒酵母的转录激活因子 Msn2，它调节许多参与抗氧化应激反应的基因，并构建了组成型过表达 Msn2 的生物乙醇酵母菌株。过表达 Msn2 的生物乙醇菌株表现出对氧化应激的耐受性，这可能是由于各种抗氧化酶基因的高水平表达。出乎意料的是，与对照菌株相比，这些菌株表现出对乙醇的敏感性，这可能是由于 Msn2 和 Msn4 之间的表达水平失衡所致。在糠醛存在下，工程菌株表现出较低的细胞内 ROS 水平，并且与对照菌株相比表现出快速生长。在糠醛存在下的发酵试验表明，过表达转录激活因子 Msn2 的菌株通过降低细胞内 ROS 水平表现出发酵初始速率的提高。我们的结果表明，在生物乙醇酵母菌株中过表达转录激活因子 Msn2 通过降低细胞内 ROS 水平赋予糠醛耐受性，并在存在糠醛时增强发酵的初始速率，这表明这些菌株能够通过诱导 ROS 积累来快速适应抑制发酵的各种化合物。我们的结果不仅有望提高木质纤维素生物质生产生物乙醇的效率，而且为工业酵母菌株的分子育种提供了新的见解。
