Exploiting Natural Variation inSaccharomyces cerevisiaeto Identify Genes for Increased Ethanol Resistance

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.

Genetic Architecture of Ethanol-Responsive Transcriptome Variation in Saccharomyces cerevisiae Strains

“…A significant fraction of hotspot loci, including additive and epi-hotspots, include candidate causal regulators and transcription factors; others implicate genes that influence physiology via enzymatic or transporter functions. Given that we previously showed that many of the transcript differences affect ethanol tolerance (Lewis et al 2010), these results suggest insights into ethanol-stress defense. A recurring theme among affected genes links to respiration and carbon metabolism (Table 3 and Figure 4); this is consistent with a prior screen of the yeast deletion library that found that strains lacking respiratory components showed increased ethanol sensitivity (Yoshikawa et al 2009).…”

“…Over half of the genome (3287/6532 genes) exhibited differential ethanol-responsive expression (FDR , 0.01) in at least one strain (File S5). This fraction is significantly higher than that of our previous study (Lewis et al 2010), likely a reflection of increased statistical power from additional replicates and improved array technology (Brem et al 2002;Smith and Kruglyak 2008). …”

“…Deletions in the BY4741 background were obtained from Open Biosystems and verified by diagnostic PCR. MKT1 was deleted from a haploid derivative of YPS163 (YPS163-1 hoD::HygMX; Lewis et al 2010) by homologous recombination with the mkt1::KanMX cassette amplified from the yeast knockout strain (Winzeler et al 1999) and subsequently verified by diagnostic PCR.…”

“…For ethanol shock experiments, cells were grown four generations to an optical density (OD 600 ) of 0.3. A sample of unstressed cells was collected, and then ethanol was added to a final concentration of 5% (v/v) for 30 min, which comprises the peak expression response for each strain (Lewis et al 2010). …”

“…A prior study performed eQTL mapping of steady-state growth on ethanol as a carbon source, but did not interrogate the dynamic response to ethanol stress at high concentrations (Smith and Kruglyak 2008). We previously showed extensive natural variation in the response to acute ethanol stress in three yeast strains: a lab strain derived from the commonly used S288c, vineyard isolate M22, and oak-soil strain YPS163 (Lewis et al 2010). Across these strains, we identified thousands of gene expression differences in response to ethanol.…”