Academic journal article Genetics

Exploiting Natural Variation in Saccharomyces Cerevisiae to Identify Genes for Increased Ethanol Resistance

Academic journal article Genetics

Exploiting Natural Variation in Saccharomyces Cerevisiae to Identify Genes for Increased Ethanol Resistance

Article excerpt

ABSTRACT

Ethanol production from lignocellulosic biomass holds promise as an alternative fuel. However, industrial stresses, including ethanol stress, limit microbial fermentation and thus prevent cost competitiveness with fossil fuels. To identify novel engineering targets for increased ethanol tolerance, we took advantage of natural diversity in wild Saccharomyces cerevisiae strains. We previously showed that an S288c-derived lab strain cannot acquire higher ethanol tolerance after a mild ethanol pretreatment, which is distinct from other stresses. Here, we measured acquired ethanol tolerance in a large panel of wild strains and show that most strains can acquire higher tolerance after pretreatment. We exploited this major phenotypic difference to address the mechanism of acquired ethanol tolerance, by comparing the global gene expression response to 5% ethanol in S288c and two wild strains. Hundreds of genes showed variation in ethanol-dependent gene expression across strains. Computational analysis identified several transcription factor modules and known coregulated genes as differentially expressed, implicating genetic variation in the ethanol signaling pathway. We used this information to identify genes required for acquisition of ethanol tolerance in wild strains, including new genes and processes not previously linked to ethanol tolerance, and four genes that increase ethanol tolerance when overexpressed. Our approach shows that comparative genomics across natural isolates can quickly identify genes for industrial engineering while expanding our understanding of natural diversity.

CELLULOSIC materials are an attractive source for biofuel production, given the availability of agricultural residues that do not directly compete with food sources (Solomon 2010). However, fermentation of cellulosic biomass is problematic. Stressful byproducts generated during preprocessing, coupled with the unique composition of pentose and hexose sugars, limit microbial ethanol production. Significant attention is therefore being dedicated toward engineering stress-tolerance microbes for cellulosic fermentation.

Saccharomyces cerevisiae has been the organism of choice for ethanol production, because of its inherent ethanol tolerance. However, high ethanol levels can still inhibit viability and fermentation, and engineering greater ethanol resistance has led to improved bioethanol production (Alper et al. 2006). Ethanol affects many cellular processes, including membrane fluidity, protein stability, and energy status (reviewed recently in Stanley et al. 2010). Recent genetic screens have implicated additional genes important for ethanol tolerance, including those involved in vacuolar, peroxisomal, and vesicular transport, mitochondrial function, protein sorting, and aromatic amino acid metabolism (Kubota et al. 2004; Fujita et al. 2006; Van Voorst et al. 2006; Teixeira et al. 2009; Yoshikawa et al. 2009). Yet despite the attention to the mechanism of ethanol tolerance, significant gaps in our knowledge remain.

Several studies have also investigated the global gene expression response to ethanol (Alexandre et al. 2001; Chandler et al. 2004; Fujita et al. 2004; Hirasawa et al. 2007). However, mutational analysis shows that most genes upregulated by ethanol are not required for ethanol tolerance (Yoshikawa et al. 2009). Thus, gene expression responses in a single strain are poor predictors of genes important for tolerance of the initial stressor. Instead, we have argued that the role of stressdependent gene expression changes is not to survive the initial stress, but rather to protect cells against impending stress in a phenomenon known as acquired stress resistance (Berry and Gasch 2008). When cells are pretreated with a mild stress, they often acquire tolerance to what would otherwise be a lethal dose of the same or other stresses. Consistently, the gene expression response triggered by a single stress treatment has no impact on surviving the initial stress, but instead is critical for the increased resistance to subsequent stress (Berry and Gasch 2008). …

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