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

ABSTRACT: 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.

SUBMITTER: Swinnen S

PROVIDER: S-EPMC4551594 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Project description:BackgroundSaccharomyces cerevisiae has been widely used for bio-ethanol production and development of rational genetic engineering strategies leading both to the improvement of productivity and ethanol tolerance is very important for cost-effective bio-ethanol production. Studies on the identification of the genes that are up- or down-regulated in the presence of ethanol indicated that the genes may be involved to protect the cells against ethanol stress, but not necessarily required for ethanol tolerance.ResultsIn the present study, a novel network based approach was developed to identify candidate genes involved in ethanol tolerance. Protein-protein interaction (PPI) network associated with ethanol tolerance (tETN) was reconstructed by integrating PPI data with Gene Ontology (GO) terms. Modular analysis of the constructed networks revealed genes with no previously reported experimental evidence related to ethanol tolerance and resulted in the identification of 17 genes with previously unknown biological functions. We have randomly selected four of these genes and deletion strains of two genes (YDR307W and YHL042W) were found to exhibit improved tolerance to ethanol when compared to wild type strain. The genome-wide transcriptomic response of yeast cells to the deletions of YDR307W and YHL042W in the absence of ethanol revealed that the deletion of YDR307W and YHL042W genes resulted in the transcriptional re-programming of the metabolism resulting from a mis-perception of the nutritional environment. Yeast cells perceived an excess amount of glucose and a deficiency of methionine or sulfur in the absence of YDR307W and YHL042W, respectively, possibly resulting from a defect in the nutritional sensing and signaling or transport mechanisms. Mutations leading to an increase in ribosome biogenesis were found to be important for the improvement of ethanol tolerance. Modulations of chronological life span were also identified to contribute to ethanol tolerance in yeast.ConclusionsThe system based network approach developed allows the identification of novel gene targets for improved ethanol tolerance and supports the highly complex nature of ethanol tolerance in yeast.

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

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