Dataset Information

Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae.

ABSTRACT: Saccharomyces cerevisiae is a typical fermentation yeast in beer production. Improving ethanol tolerance of S. cerevisiae will increase fermentation efficiency, thereby reducing capital costs. Here, we found that S. cerevisiae strain L exhibited a higher ethanol tolerance (14%, v/v) than the fermentative strain Q (10%, v/v). In order to enhance the strain Q ethanol tolerance but preserve its fermentation property, protoplast fusion was performed with haploids from strain Q and L. The fusant Q/L-f2 with 14% ethanol tolerance was obtained. Meanwhile, the fermentation properties (flocculability, SO2 production, ?-N assimilation rate, GSH production, etc.) of Q/L-f2 were similar to those of strain Q. Therefore, our works established a series of high ethanol-tolerant strains in beer production. Moreover, this demonstration of inactivated protoplast fusion in industrial S. cerevisiae strain opens many doors for yeast-based biotechnological applications.

SUBMITTER: Xin Y

PROVIDER: S-EPMC7201837 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae.

Xin Yi Y Yang Mei M Yin Hua H Yang Jianming J

BioMed research international 20200120

Saccharomyces cerevisiae is a typical fermentation yeast in beer production. Improving ethanol tolerance of S. cerevisiae will increase fermentation efficiency, thereby reducing capital costs. Here, we found that S. cerevisiae strain L exhibited a higher ethanol tolerance (14%, v/v) than the fermentative strain Q (10%, v/v). In order to enhance the strain Q ethanol tolerance but preserve its fermentation property, protoplast fusion was performed with haploids from strain Q a ...[more]

PMID: 32420325

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Project description:BACKGROUND: Saccharomyces 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. RESULTS: In 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. CONCLUSIONS: The 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.

Project description:1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0.8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7.0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13-20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C(16:0), C(16:1) and C(18:1) acids. Whole cells contained a slightly greater proportion of C(16:0) and a somewhat smaller proportion of C(18:1) acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas-liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Delta(5,7,22,24(28))-ergostatetraen-3beta-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C(29) di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes.

Project description:BackgroundDerived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain.ResultsA highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p.ConclusionEnriched background of transcription abundance and enhanced expressions of ethanol-tolerance genes associated with heat shock proteins, trehalose-glycolysis-pentose phosphate pathways and PDR gene family are accountable for the tolerant yeast to withstand the ethanol stress, maintain active metabolisms, and complete ethanol fermentation under the ethanol stress. Transcription factor Msn4p appeared to be a key regulator of gene interactions for ethanol-tolerance in the tolerant yeast Y-50316.

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Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae.

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Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in <i>Saccharomyces cerevisiae</i>.

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