Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae

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, α-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.

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Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion

Bioprocess and Biosystems Engineering ◽

10.1007/s00449-012-0734-0 ◽

2012 ◽

Vol 35 (9) ◽

pp. 1455-1465 ◽

Cited By ~ 14

Author(s):

Min Zhang ◽

Yu Xiao ◽

Rongrong Zhu ◽

Qin Zhang ◽

Shi-Long Wang

Keyword(s):

Saccharomyces Cerevisiae ◽

Electron Beam ◽

Protoplast Fusion ◽

Ethanol Tolerance ◽

High Energy ◽

Pulse Electron Beam ◽

Pulse Electron ◽

Energy Pulse

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

Eukaryotic Cell ◽

10.1128/ec.00053-15 ◽

2015 ◽

Vol 14 (9) ◽

pp. 884-897 ◽

Cited By ~ 13

Author(s):

Steve Swinnen ◽

Annelies Goovaerts ◽

Kristien Schaerlaekens ◽

Françoise Dumortier ◽

Pieter Verdyck ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Tolerance ◽

Stress Factors ◽

Causative Mutation ◽

Ethanol Stress ◽

Limiting Factor ◽

Ethanol Sensitivity ◽

Content Type ◽

High Ethanol ◽

Very High

ABSTRACTVery high ethanol tolerance is a distinctive trait of the yeastSaccharomyces cerevisiaewith 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 theura3Δ0mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage forLYS2,LEU2,HIS3, andMET15. 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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Membrane Fluidity of Saccharomyces cerevisiae from Huangjiu (Chinese Rice Wine) Is Variably Regulated by OLE1 To Offset the Disruptive Effect of Ethanol

Applied and Environmental Microbiology ◽

10.1128/aem.01620-19 ◽

2019 ◽

Vol 85 (23) ◽

Author(s):

Yijin Yang ◽

Yongjun Xia ◽

Wuyao Hu ◽

Leren Tao ◽

Li Ni ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fatty Acid ◽

Membrane Fluidity ◽

Ethanol Tolerance ◽

Acid Metabolism ◽

Ethanol Stress ◽

Disruptive Effect ◽

Rice Wine ◽

Chinese Rice Wine ◽

High Ethanol

ABSTRACT An evolution and resequencing strategy was used to research the genetic basis of Saccharomyces cerevisiae BR20 (with 18 vol% ethanol tolerance) and the evolved strain F23 (with 25 vol% ethanol tolerance). Whole-genome sequencing and RNA sequencing (RNA-seq) indicated that the enhanced ethanol tolerance under 10 vol% ethanol could be attributed to amino acid metabolism, whereas 18 vol% ethanol tolerance was due to fatty acid metabolism. Ultrastructural analysis indicated that F23 exhibited better membrane integrity than did BR20 under ethanol stress. At low concentrations (<5 vol%), the partition of ethanol into the membrane increased the membrane fluidity, which had little effect on cell growth. However, the toxic effects of medium and high ethanol concentrations (5 to 20 vol%) tended to decrease the membrane fluidity. Under high ethanol stress (>10 vol%), the highly tolerant strain was able to maintain a relatively constant fluidity by increasing the content of unsaturated fatty acid (UFA), whereas less-tolerant strains show a continuous decrease in fluidity and UFA content. OLE1, which was identified as the only gene with a differential single-nucleotide polymorphism (SNP) mutation site related to fatty acid metabolism, was significantly changed in response to ethanol. The role of OLE1 in membrane fluidity was positively validated in its overexpressed transformants. Therefore, OLE1 lowered the rate of decline in membrane fluidity and thus enabled the yeast to better fight the deleterious effects of ethanol. IMPORTANCE Yeasts with superior ethanol tolerance are desirable for winemakers and wine industries. In our previous work, strain F23 was evolved with superior ethanol tolerance and fermentation activity to improve the flavor profiles of Chinese rice wine. Therefore, exploring the genomic variations and ethanol tolerance mechanism of strain F23 could contribute to an understanding of its effect on the flavor characteristics in the resulting Chinese rice wine. The cellular membrane plays a vital role in the ethanol tolerance of yeasts; however, how the membrane is regulated to fight the toxic effect of ethanol remains to be elucidated. This study suggests that the membrane fluidity is variably regulated by OLE1 to offset the disruptive effect of ethanol. Current work will help develop more ethanol-tolerant yeast strains for wine industries and contribute to a deep understanding of its high flavor-producing ability.

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Segregation of altered parental properties in fusions between Saccharomyces cerevisiae and the D-xylose fermenting yeasts Candida shehatae and Pichia stipitis

Canadian Journal of Microbiology ◽

10.1139/m92-203 ◽

1992 ◽

Vol 38 (12) ◽

pp. 1233-1237 ◽

Cited By ~ 12

Author(s):

Abindra S. Gupthar

Keyword(s):

Saccharomyces Cerevisiae ◽

Polyethylene Glycol ◽

Gene Transfer ◽

Key Words ◽

Protoplast Fusion ◽

Ethanol Tolerance ◽

Pichia Stipitis ◽

Candida Shehatae ◽

Auxotrophic Mutants ◽

Parental Type

A prototrophic strain of Saccharomyces cerevisiae CSIR Y190 MATa xyl−, resistant to high levels of ethanol, was hybridized with xylose-fermenting, auxotrophic mutants of Candida shehatae and Pichia stipitis through polyethylene glycol-induced protoplast fusion in an attempt to produce ethanol-tolerant, xylose-fermenting hybrids. Mononucleate fusants were obtained, but these dissociated into a mixture of parental-type segregants. Purified Candida- and Pichia-resembling segregants failed to acquire improved ethanol tolerance but expressed other novel properties of S. cerevisiae, suggesting that karyogamy was impaired after internuclear gene transfer. Key words: Pichia, Candida, Saccharomyces protoplast fusion.

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Traditional Norwegian Kveik Yeasts: Underexplored Domesticated Saccharomyces cerevisiae Yeasts

10.1101/194969 ◽

2017 ◽

Cited By ~ 1

Author(s):

Richard Preiss ◽

Caroline Tyrawa ◽

George van der Merwe

Keyword(s):

Saccharomyces Cerevisiae ◽

Human Activity ◽

Ethanol Tolerance ◽

High Rate ◽

Metabolite Analysis ◽

Genetic Fingerprinting ◽

Traditional Practice ◽

Western Norway ◽

Beer Production ◽

Beer Yeast

AbstractHuman activity has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a home-brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavour negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are highly temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>30 °C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, phenotypic screens, lab-scale fermentations, and flavour metabolite analysis using HS-SPME-GC-MS. Genetic fingerprinting via interdelta PCR suggests that kveik yeasts form a lineage distinct from other domesticated yeasts. Our analyses confirm that kveik yeasts display hallmarks of domestication such as loss of 4-vinylguaiacol production and high flocculation, and show superior thermotolerance, ethanol tolerance, fermentation rate, and unique flavour metabolite production profiles in comparison to other ale strains, suggesting a broad industrial potential for this group of yeasts.

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Oenological Potential of Autochthonous Saccharomyces cerevisiae Yeast Strains from the Greek Varieties of Agiorgitiko and Moschofilero

Beverages ◽

10.3390/beverages7020027 ◽

2021 ◽

Vol 7 (2) ◽

pp. 27

Author(s):

Dimitrios Kontogiannatos ◽

Vicky Troianou ◽

Maria Dimopoulou ◽

Polydefkis Hatzopoulos ◽

Yorgos Kotseridis

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcoholic Fermentation ◽

Ethanol Tolerance ◽

Random Amplified Polymorphic Dna ◽

Yeast Strains ◽

Rapd Pcr ◽

Autochthonous Yeast ◽

Polymerase Chain ◽

Grape Varieties ◽

H2s Production

Nemea and Mantinia are famous wine regions in Greece known for two indigenous grape varieties, Agiorgitiko and Moschofilero, which produce high quality PDO wines. In the present study, indigenous Saccharomyces cerevisiae yeast strains were isolated and identified from spontaneous alcoholic fermentation of Agiorgitiko and Moschofilero musts in order to evaluate their oenological potential. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) recovered the presence of five distinct profiles from a total of 430 yeast isolates. The five obtained strains were evaluated at microvinifications trials and tested for basic oenological and biochemical parameters including sulphur dioxide and ethanol tolerance as well as H2S production in sterile grape must. The selected autochthonous yeast strains named, Soi2 (Agiorgitiko wine) and L2M (Moschofilero wine), were evaluated also in industrial (4000L) fermentations to assess their sensorial and oenological characteristics. The volatile compounds of the produced wines were determined by GC-FID. Our results demonstrated the feasibility of using Soi2 and L2M strains in industrial fermentations for Agiorgitiko and Moschofilero grape musts, respectively.

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