scholarly journals A CRISPRi screen of essential genes reveals that proteasome regulation dictates acetic acid tolerance in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Vaskar Mukherjee ◽  
Ulrika Lind ◽  
Robert P St. Onge ◽  
Anders Blomberg ◽  
Yvonne Nygård
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
High Resolution ◽  
Acid Stress ◽  
Transport Processes ◽  
Essential Genes ◽  
26S Proteasome ◽  
Organelle Transport ◽  
Biomass Hydrolysis ◽  
Cell Factories

CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions and this technology provides a means for screening changed expression of essential genes. In this study, a Saccharomyces cerevisiae CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for industrial conversion of lignocellulosic biomasses. Tolerance towards acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of >9,000 strains, where >98% of all essential and respiratory growth-essential genes were targeted with multiple gRNAs. The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP-salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where a high-resolution CRISPRi library screening paves the way to understand and bioengineer the robustness of yeast against acetic acid stress.

scholarly journals Role of Essential Genes in Mitochondrial Morphogenesis inSaccharomyces cerevisiae

2005 ◽  
Vol 16 (11) ◽  
pp. 5410-5417 ◽  
Author(s):  
Katrin Altmann ◽  
Benedikt Westermann
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Transport Processes ◽  
Essential Genes ◽  
26S Proteasome ◽  
Organelle Transport ◽  
Mitochondrial Morphology ◽  
Strain Collection ◽  
Mitochondrial Morphogenesis ◽  
Yeast Genes

Mitochondria are essential organelles of eukaryotic cells. Inheritance and maintenance of mitochondrial structure depend on cytoskeleton-mediated organelle transport and continuous membrane fusion and fission events. However, in Saccharomyces cerevisiae most of the known components involved in these processes are encoded by genes that are not essential for viability. Here we asked which essential genes are required for mitochondrial distribution and morphology. To address this question, we performed a systematic screen of a yeast strain collection harboring essential genes under control of a regulatable promoter. This library contains 768 yeast mutants and covers approximately two thirds of all essential yeast genes. A total of 119 essential genes were found to be required for maintenance of mitochondrial morphology. Among these, genes were highly enriched that encode proteins involved in ergosterol biosynthesis, mitochondrial protein import, actin-dependent transport processes, vesicular trafficking, and ubiquitin/26S proteasome-dependent protein degradation. We conclude that these cellular pathways play an important role in mitochondrial morphogenesis and inheritance.

scholarly journals Endogenous lycopene improves ethanol production under acetic acid stress in Saccharomyces cerevisiae

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuo Pan ◽  
Bin Jia ◽  
Hong Liu ◽  
Zhen Wang ◽  
Meng-Zhe Chai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Ethanol Production ◽  
Acid Stress

scholarly journals Acid stress adaptation protects Saccharomyces cerevisiae from acetic acid-induced programmed cell death

Gene ◽  
2005 ◽  
Vol 354 ◽  
pp. 93-98 ◽  
Author(s):  
Sergio Giannattasio ◽  
Nicoletta Guaragnella ◽  
Manuela Corte-Real ◽  
Salvatore Passarella ◽  
Ersilia Marra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Acetic Acid ◽  
Programmed Cell Death ◽  
Acid Stress ◽  
Stress Adaptation

scholarly journals Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress

2011 ◽  
Vol 39 (16) ◽  
pp. 6896-6907 ◽  
Author(s):  
Nuno P. Mira ◽  
Sílvia F. Henriques ◽  
Greg Keller ◽  
Miguel C. Teixeira ◽  
Rute G. Matos ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Acetic Acid ◽  
Dna Binding ◽  
Binding Site ◽  
Acid Stress ◽  
Dna Binding Site

scholarly journals Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

2016 ◽  
Vol 82 (7) ◽  
pp. 2156-2166 ◽  
Author(s):  
Yingying Chen ◽  
Lisa Stabryla ◽  
Na Wei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Metabolic Engineering ◽  
Acid Stress ◽  
Acid Resistance ◽  
Biofuel Production ◽  
Gene Target ◽  
Content Type ◽  
Inverse Metabolic Engineering ◽  
Acetic Acid Resistance

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

scholarly journals A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors

PLoS Genetics ◽  
2021 ◽  
Vol 17 (10) ◽  
pp. e1009826
Author(s):  
Gert Vanmarcke ◽  
Quinten Deparis ◽  
Ward Vanthienen ◽  
Arne Peetermans ◽  
Maria R. Foulquié-Moreno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lignocellulosic Biomass ◽  
Causative Mutation ◽  
Small Scale ◽  
Whole Genome ◽  
Biomass Hydrolysis ◽  
Inhibitor Tolerance ◽  
Cell Factories ◽  
Biomass Hydrolysates

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.

scholarly journals Effect of Dissolved Oxygen and Acid on the Yield of 2,3-butanediol by Saccharomyces Cerevisiae W141

2020 ◽  
Author(s):  
ZiLiang Yin ◽  
DeAn Liu ◽  
ZeMing Ye ◽  
Jingping Ge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Acetic Acid ◽  
Dissolved Oxygen ◽  
Oxygen Content ◽  
Acid Stress ◽  
Redox Balance ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Chain Fatty Acids

Abstract Background: The yeast Saccharomyces cerevisiae is a promising host cell to produce 2,3-butanediol (2,3-BDO). However, the fermentation environment restricts 2,3‑BDO yield, productivity, and titre from engineered yeast. In the present study, we propose a strategy in which a suitable dissolved oxygen content and acid stress level can improve the 23-BDO yield of S. cerevisiae W141. Five different concentrations of short-chain fatty acids were evaluated and noxE overexpression was performed to disrupt the intracellular redox balance and alter the NADH content associated with 2,3‑BDO synthesis, which can significantly increase or inhibit 2,3‑BDO yield.Results: The five assayed short-chain fatty acids have different effects on the fermentation characteristics of yeast, were formic, butyric and valeric acids can inhibit the synthesis of 2,3‑BDO. Only low concentrations of acetic and propionic acids could significantly increase the yield of 2,3‑BDO, especially when 1 g/L acetic acid was added, which stimulated the expression of acid stress-related genes in S. cerevisiae W141 (haa1p and hog1p) and increase the 2,3-BDO yield by 29.74%. To further verify that acid stress primarily disrupts the intracellular redox balance by altering the NADH content, we constructed a S. cerevisiae strain, W141-E, which overexpresses the noxE gene of Lactobacillus. After adding 1 g/L acetic acid, the 2,3‑BDO yield from in S. cerevisiae W141-E increased by 43.64%, confirming the validity of our strategy. When the optimized fermentation oxygen content was 0.6 vvm, the 2,3‑BDO yield from S. cerevisiae was greatly improved after the addition of acetic acide.Conclusions: In the present study, we demonstrated that a suitable dissolved oxygen and acid stress are highly effective for increasing the 2,3-BDO yield from S. cerevisiae W141. 2,3-BDO biosynthesis was heavily dependent on the intracellular NADH content, which is closely associated with glycolysis and the TCA cycle and is likely important for the production of 2,3-BDO by S. cerevisiae.

scholarly journals A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae

mSystems ◽  
2021 ◽  
Author(s):  
Vaskar Mukherjee ◽  
Ulrika Lind ◽  
Robert P. St. Onge ◽  
Anders Blomberg ◽  
Yvonne Nygård
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Acid Tolerance ◽  
Essential Genes ◽  
Industrial Yeast ◽  
Acetic Acid Tolerance ◽  
Content Type ◽  
Fuels And Chemicals ◽  
Proteasome Regulation ◽  
And Oxidative Stress

Acetic acid is inhibitory to the growth of the yeast Saccharomyces cerevisiae , causing ATP starvation and oxidative stress, which leads to the suboptimal production of fuels and chemicals from lignocellulosic biomass. In this study, where each strain of a CRISPRi library was characterized individually, many essential and respiratory growth-essential genes that regulate tolerance to acetic acid were identified, providing a new understanding of the stress response of yeast and new targets for the bioengineering of industrial yeast.

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