• Energy Research

Abstract Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production.

Chemogenomics, the study of genomic responses to chemical compounds, has the potential to elucidate the basis of cellular resistance to those chemicals. This knowledge can be applied to improve the performance of strains of industrial interest. In this study, a collection of approximately 5,000 haploid single deletion mutants of Saccharomyces cerevisiae in which each nonessential yeast gene was individually deleted, was screened for strains with increased susceptibility toward stress induced by high-glucose concentration (30% w/v), one of the main stresses occurring during industrial alcoholic fermentation processes aiming the production of alcoholic beverages or bio-ethanol. Forty-four determinants of resistance to high-glucose stress were identified. The most significant Gene Ontology (GO) terms enriched in this dataset are vacuolar organization, late endosome to vacuole transport, and regulation of transcription. Clustering the identified resistance determinants by their known physical and genetic interactions further highlighted the importance of nutrient metabolism control in this context. A concentration of 30% (w/v) of glucose was found to perturb vacuolar function, by reducing cell ability to maintain the physiological acidification of the vacuolar lumen. This stress also affects the active rate of proton efflux through the plasma membrane. Based on results of published studies, the present work revealed shared determinants of yeast resistance to high-glucose and ethanol stresses, including genes involved in vacuolar function, cell wall biogenesis (ANP1), and in the transcriptional control of nutrient metabolism (GCN4 and GCR1), with possible impact on the design of more robust strains to be used in industrial alcoholic fermentation processes.

A metabolomic analysis using high resolution 1H NMR spectroscopy coupled with multivariate statistical analysis was used to characterize the alterations in the endo- and exo-metabolome of S. cerevisiae BY4741 during the exponential phase of growth in minimal medium supplemented with different ethanol concentrations (0, 2, 4 and 6% v/v). This study provides evidence that supports the notion that ethanol stress induces reductive stress in yeast cells, which, in turn, appears to be counteracted by the increase in the rate of NAD+ regenerating bioreactions. Metabolomics data also shows increased intra- and extra-cellular accumulation of most amino acids and TCA cycle intermediates in yeast cells growing under ethanol stress suggesting a state of overflow metabolism in turn of the pyruvate branch-point. Given its previous implication in ethanol stress resistance in yeast, this study also focused on the effect of the expression of the aquaglyceroporin encoded by FPS1 in the yeast metabolome, in the absence or presence of ethanol stress. The metabolomics data collected herein shows that the deletion of the FPS1 gene in the absence of ethanol stress partially mimics the effect of ethanol stress in the parental strain. Moreover, the results obtained suggest that the reported action of Fps1 in mediating the passive diffusion of glycerol is a key factor in the maintenance of redox balance, an important feature for ethanol stress resistance, and may interfere with the ability of the yeast cell to accumulate trehalose. Overall, the obtained results corroborate the idea that metabolomic approaches may be crucial tools to understand the function and/or the effect of membrane transporters/porins, such as Fps1, and may be an important tool for the clear-cut design of improved process conditions and more robust yeast strains aiming to optimize industrial fermentation performance.

ABSTRACT The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H + -ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme β-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [ 3 H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

Abstract The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.