Identification of novel causative genes determining the complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis

Swinnen, Steve; Schaerlaekens, Kristien; Pais, Thiago M.; Claesen, Jürgen; Hubmann, Georg; Yang, Yudi; Demeke, Mekonnen M.; Foulquié-Moreno, María R.; Goovaerts, Annelies; Souvereyns, Kris; Clement, Lieven; Dumortier, Françoise; Thevelein, Johan M.

doi:10.1101/gr.131698.111

Cited by 164 publications

(210 citation statements)

References 43 publications

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“…Natural variation in MKT1 has been implicated in a large number of phenotypic differences between yeast strains (Nickoloff et al 1986;Steinmetz et al 2002;Deutschbauer and Davis 2005;Sinha et al 2006;Zhu et al 2008;Ehrenreich et al 2010), including tolerance to ethanol (Swinnen et al 2012) and other stresses (Steinmetz et al 2002;Demogines et al 2008), though the mechanism of its effect remains obscure. Our results strongly suggest that the pleiotropic and condition-specific effects of Mkt1p are linked to widespread differences in transcriptional regulation and stress-activated signaling.…”

Section: Discussionmentioning

confidence: 99%

“…Natural variation in MKT1 has been implicated in a large number of phenotypic differences, ranging from mitochondrial stability (Nickoloff et al 1986), drug resistance ), thermotolerance (Steinmetz et al 2002;Sinha et al 2006), sporulation (Deutschbauer and Davis 2005), and DNA-damage sensitivity (Ehrenreich et al 2010). Importantly, natural variation in MKT1 has been linked to high ethanol tolerance in a Brazilian bioethanol production strain compared to a lab strain (Swinnen et al 2012). Our observation that MKT1 affects a large number of transcripts responding to ethanol suggests a potential link between Mkt1p-affected transcriptional responses and ethanol defense (see below and Discussion).…”

Section: Eqtl Mapping Of the Ethanol Responsementioning

confidence: 99%

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

et al. 2014

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Natural variation in gene expression is pervasive within and between species, and it likely explains a significant fraction of phenotypic variation between individuals. Phenotypic variation in acute systemic responses can also be leveraged to reveal physiological differences in how individuals perceive and respond to environmental perturbations. We previously found extensive variation in the transcriptomic response to acute ethanol exposure in two wild isolates and a common laboratory strain of Saccharomyces cerevisiae. Many expression differences persisted across several modules of coregulated genes, implicating trans-acting systemic differences in ethanol sensing and/or response. Here, we conducted expression QTL mapping of the ethanol response in two strain crosses to identify the genetic basis for these differences. To understand systemic differences, we focused on "hotspot" loci that affect many transcripts in trans. Candidate causal regulators contained within hotspots implicate upstream regulators as well as downstream effectors of the ethanol response. Overlap in hotspot targets revealed additive genetic effects of trans-acting loci as well as "epihotspots," in which epistatic interactions between two loci affected the same suites of downstream targets. One epi-hotspot implicated interactions between Mkt1p and proteins linked to translational regulation, prompting us to show that Mkt1p localizes to P bodies upon ethanol stress in a strain-specific manner. Our results provide a glimpse into the genetic architecture underlying natural variation in a stress response and present new details on how yeast respond to ethanol stress. N ATURAL variation in gene expression is hypothesized to be a major source of phenotypic variation between individuals (King and Wilson 1975;Oleksiak et al. 2002). Gene expression variation underlies differences in susceptibility to infectious disease (Li et al. 2010;Barreiro et al. 2012), drug sensitivity (Fay et al. 2004;Kvitek et al. 2008;Maranville et al. 2011;Hodgins-Davis et al. 2012;Chang et al. 2013), inflammation (Gargalovic et al. 2006Orozco et al. 2012), cardiovascular disease (Romanoski et al. 2010), metabolism (Fraser et al. 2010Rossouw et al. 2012;Skelly et al. 2013), morphology (Yvert et al. 2003Chin et al. 2012;Skelly et al. 2013), and even behavior (Ziebarth et al. 2012). Still, identifying the genetic and molecular mechanisms that underlie the expression variation is a major challenge.Expression quantitative trait loci (eQTL) mapping (reviewed in Gilad et al. 2008) is a powerful approach to dissect the genetic basis of expression differences. Transcript abundance is treated as a quantitative trait whose genetic determinants can be implicated by well-established linkage mapping techniques. The first eQTL studies in yeast illuminated the genetic landscape of gene expression-variation determinants under standard growth conditions (Brem et al. 2002;Yvert et al. 2003). Subsequent eQTL studies surveyed the genetic control of transcript abundance in Arabidopsi...

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Section: Discussionmentioning

confidence: 99%

Section: Eqtl Mapping Of the Ethanol Responsementioning

confidence: 99%

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

et al. 2014

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“…This powerful approach, which relies on crossing two parents, one inferior and the other superior for a trait of interest, followed by whole-genome sequencing of a large set of recombination segregants, allowed identification of potential genetic loci linked to high ethanol tolerance and, thereby, determination of causative genes. In particular, MKT1, SWS2, and AJP1 (14) were isolated by this approach, as well as the VPS16, VPS28, and VPS70 genes, which encode components of the vacuole protein sorting system (15,16). However, the causative genes identified are apparently dependent on several criteria, including the origin of the parental strains, the culture conditions, the size of the segregant sample, and the performance of the algorithm used to analyze the data.…”

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confidence: 99%

Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress

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Formosa-Dague

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et al. 2016

Appl Environ Microbiol

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A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae. However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucosegrown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3-to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells. IMPORTANCEEthanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties. T he yeast Saccharomyces cerevisiae is a remarkable ethanol producer that is also very sensitive to its main fermentative product. At low to moderate concentrations (5 to 7%), ethanol mainly affects the growth rate, and at higher concentrations (Ͼ10%), it can strongly impair cell integrity, eventually leading to cell death with features of apoptosis (1). These inhibitory and toxic effects are ascribed to the fact that ethanol alters cell membrane fluidity and dissipates the transmembrane electrochemical potential, thereby creating permeability to ionic species and causing leakage of metabolites (2). Recent works using lipidomic methodologies confirmed the relationship between the composition of lipids, notably ergosterol and unsaturated fatty acids, and ethanol tolerance (3, 4). Moreover, as it diffuses freely into cells, ethanol at high concen...

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“…We identified a stable haploid segregant from VR1 with an even higher ethanol tolerance (referred to as VR1-5B) ( Fig. 1) (34). The difference in ethanol tolerance between VR1 and VR1-5B may be due to the presence of recessive mutations in the diploid strain and/or to the difference in ploidy between the two strains.…”

Section: Resultsmentioning

confidence: 97%

“…VR1 is a diploid strain, and thus, we first isolated a segregant with similarly high ethanol tolerance in order to be able to perform quantitative trait locus (QTL) mapping by crossing (34). We sporulated VR1 and isolated segregants using a dissection microscope.…”

Section: Resultsmentioning

confidence: 99%

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

et al. 2015

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Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3⌬0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.

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Identification of novel causative genes determining the complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis

Cited by 164 publications

References 43 publications

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

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

Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

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