Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution

2019 ◽  
Vol 189 (3) ◽  
pp. 810-821 ◽  
Author(s):  
Ryosuke Mitsui ◽  
Ryosuke Yamada ◽  
Hiroyasu Ogino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Tolerance ◽  
Genome Evolution

scholarly journals Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 219
Author(s):  
Il-Sup Kim ◽  
Woong Choi ◽  
Jonghyeon Son ◽  
Jun Hyuck Lee ◽  
Hyoungseok Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Stress Tolerance ◽  
Genetic Network ◽  
Yeast Cells ◽  
Enzymatic Antioxidants ◽  
Freezing And Thawing ◽  
Metal Ion Homeostasis ◽  
Transgenic Yeast

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

scholarly journals Proteomic Analysis of the Increased Stress Tolerance of Saccharomyces cerevisiae Encapsulated in Liquid Core Alginate-Chitosan Capsules

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e49335 ◽  
Author(s):  
Johan O. Westman ◽  
Mohammad J. Taherzadeh ◽  
Carl Johan Franzén
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Tolerance ◽  
Proteomic Analysis ◽  
Liquid Core

scholarly journals Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains

2020 ◽  
Vol 8 (12) ◽  
pp. 1914
Author(s):  
Elizabeth L. I. Wightman ◽  
Heinrich Kroukamp ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen ◽  
Helena K. M. Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Evolution ◽  
Colorimetric Detection ◽  
Cre Recombinase ◽  
Control Measures ◽  
Genomic Rearrangements ◽  
Industrial Yeast ◽  
Poor Growth ◽  
Yeast Strains ◽  
Industrial Strains

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

scholarly journals Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation

AMB Express ◽  
2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yasin Kitichantaropas ◽  
Chuenchit Boonchird ◽  
Minetaka Sugiyama ◽  
Yoshinobu Kaneko ◽  
Satoshi Harashima ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Temperature ◽  
Stress Tolerance ◽  
Ethanol Fermentation ◽  
Cellular Mechanisms ◽  
Multiple Stress ◽  
Potential Use ◽  
Multiple Stress Tolerance

scholarly journals Mpt5p, a Stress Tolerance- and Lifespan-Promoting PUF Protein in Saccharomyces cerevisiae, Acts Upstream of the Cell Wall Integrity Pathway

2006 ◽  
Vol 6 (2) ◽  
pp. 262-270 ◽  
Author(s):  
Mark S. Stewart ◽  
Sue Ann Krause ◽  
Josephine McGhie ◽  
Joseph V. Gray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Life Span ◽  
Stress Tolerance ◽  
Synthetic Medium ◽  
Cell Wall Integrity ◽  
Puf Proteins ◽  
Downstream Target ◽  
Cell Wall Integrity Pathway ◽  
Puf Protein

ABSTRACT Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Δ mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Δ mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3′ untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Δ mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

2007 ◽  
Vol 131 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Takashi Hirasawa ◽  
Katsunori Yoshikawa ◽  
Yuki Nakakura ◽  
Keisuke Nagahisa ◽  
Chikara Furusawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Data Analysis ◽  
Stress Tolerance ◽  
Dna Microarray ◽  
Microarray Data ◽  
Target Genes ◽  
Microarray Data Analysis ◽  
Ethanol Stress ◽  
Dna Microarray Data

scholarly journals Role of HSP90 in Salt Stress Tolerance via Stabilization and Regulation of Calcineurin

2000 ◽  
Vol 20 (24) ◽  
pp. 9262-9270 ◽  
Author(s):  
Jun Imai ◽  
Ichiro Yahara
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Salt Stress ◽  
Stress Tolerance ◽  
Normal Level ◽  
Immune Complexes ◽  
Salt Stress Tolerance ◽  
Stressed Cells ◽  
Calcineurin Activity

ABSTRACT The role of HSP90 in stress tolerance was investigated inSaccharomyces cerevisiae. Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses. Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression. Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity. Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells. In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses. Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not. Treatment of extracts from Hsc82-overexpressing cells with Ca2+-calmodulin increased the calcineurin activity associated with Cna2 immune complexes. Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin. When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress. In these cells, the amount of Cna2 was reduced, likely as a result of degradation. The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

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