scholarly journals Deletion ofPHO13, Encoding Haloacid Dehalogenase Type IIA Phosphatase, Results in Upregulation of the Pentose Phosphate Pathway in Saccharomyces cerevisiae

2014 ◽  
Vol 81 (5) ◽  
pp. 1601-1609 ◽  
Author(s):  
Soo Rin Kim ◽  
Haiqing Xu ◽  
Anastashia Lesmana ◽  
Uros Kuzmanovic ◽  
Matthew Au ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Pentose Phosphate ◽  
Sequencing Analysis ◽  
Xylose Metabolism ◽  
Fermentation Inhibitors ◽  
Haloacid Dehalogenase ◽  
Content Type ◽  
Had Superfamily ◽  
Transcriptomic Level

ABSTRACTThe haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes inSaccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identifiedPHO13to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion ofPHO13(pho13Δ), we investigated the response ofS. cerevisiaetopho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed thatpho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced bypho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus,pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation bypho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function ofS. cerevisiaePho13 and the HAD superfamily enzymes and for developingS. cerevisiaestrains with industrially attractive phenotypes.

scholarly journals Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Dong-Dong Yang ◽  
Gustavo M. de Billerbeck ◽  
Jin-jing Zhang ◽  
Frank Rosenzweig ◽  
Jean-Marie Francois
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Lignin Degradation ◽  
Molecular Mechanisms ◽  
Ancestral State Reconstruction ◽  
Ancestral State ◽  
State Reconstruction ◽  
Content Type ◽  
Aryl Aldehydes ◽  
Aryl Aldehyde

ABSTRACTHomology searches indicate thatSaccharomyces cerevisiaestrain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). YeastAADgenes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes,AAD4andAAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeastAADgenes are undergoing pseudogenization. The 5′ sequence ofAAD15has been deleted from the genome. Repair of anAAD3missense mutation at the catalytically essential Tyr73residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates thatAADgenes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeastAADgenes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeastAADgene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role forAADgenes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications.IMPORTANCEFunctional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the firstSaccharomyces cerevisiaegenome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-memberAADfamily. Here, we demonstrate that proteins encoded by two members of this family exhibit aliphatic and aryl-aldehyde reductase activity, and further that such activity can be recovered from pseudogenizedAADgenes via ancestral-state reconstruction. The phylogeny of yeastAADgenes suggests that these proteins may have played an important ancestral role in detoxifying aromatic aldehydes in ligninolytic fungi. However, in yeast adapted to niches rich in sugars,AADgenes become subject to mutational erosion. Our findings shed new light on the selective pressures and molecular mechanisms by which genes undergo pseudogenization.

scholarly journals The synthetic cannabinoid JWH-018 modulates Saccharomyces cerevisiae energetic metabolism

2019 ◽  
Vol 19 (5) ◽  
Author(s):  
Carla Ferreira ◽  
Joana Couceiro ◽  
Carlos Família ◽  
Carolina Jardim ◽  
Pedro Antas ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Synthetic Cannabinoids ◽  
Cellular Level ◽  
Pentose Phosphate ◽  
Synthetic Cannabinoid ◽  
Glycolytic Flux ◽  
Vast Number ◽  
2D Gel ◽  
The Impact

ABSTRACT Synthetic cannabinoids are a group of novel psychoactive substances with similar properties to Δ9-THC. Among the vast number of synthetic cannabinoids, designed to be tested in clinical trials, JWH-018 was the first novel psychoactive substance found in the recreational drug marketplace. The consumption of JWH-018 shows typical effects of CB1 agonists including sedation, cognitive dysfunction, tachycardia, postural hypotension, dry mouth, ataxia and psychotropic effects, but appeared to be more potent than Δ9-THC. However, studies on human cells have shown that JWH-018 toxicity depends on the cellular line used. Despite these studies, the underlying molecular mechanisms to JWH-018 action has not been clarified yet. To understand the impact of JWH-018 at molecular and cellular level, we used Saccharomyces cerevisiae as a model. The results showed an increase in yeast growth rate in the presence of this synthetic cannabinoid due to an enhancement in the glycolytic flux at expense of a decrease in pentose phosphate pathway, judging by 2D-Gel proteomic analysis, qRT-PCR experiments and ATP measurements. Overall, our results provide insights into molecular mechanisms of JWH-018 action, also indicating that Saccharomyces cerevisiae is a good model to study synthetic cannabinoids.

scholarly journals Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Kathy Parisi ◽  
Stephen R. Doyle ◽  
Eunice Lee ◽  
Rohan G. T. Lowe ◽  
Nicole L. van der Weerden ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Deletion ◽  
Screening Method ◽  
Large Family ◽  
Mechanisms Of Action ◽  
Sequencing Analysis ◽  
Differential Growth ◽  
Content Type ◽  
Plant Defensins ◽  
Nonessential Gene

ABSTRACT Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Saccharomyces cerevisiae. Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both S. cerevisiae and the cereal pathogen Fusarium graminearum. These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.

scholarly journals Mitochondrial Genomic Dysfunction Causes Dephosphorylation of Sch9 in the Yeast Saccharomyces cerevisiae

2011 ◽  
Vol 10 (10) ◽  
pp. 1367-1369 ◽  
Author(s):  
Shigeyuki Kawai ◽  
Jörg Urban ◽  
Manuele Piccolis ◽  
Nicolas Panchaud ◽  
Claudio De Virgilio ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Retrograde Signaling ◽  
Yeast Saccharomyces Cerevisiae ◽  
Content Type ◽  
Insight Into

ABSTRACTTORC1-dependent phosphorylation ofSaccharomyces cerevisiaeSch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ0cells but not in respiration-incompetentpetmutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.

scholarly journals β-Galactomannan and Saccharomyces cerevisiae var. boulardii Modulate the Immune Response against Salmonella enterica Serovar Typhimurium in Porcine Intestinal Epithelial and Dendritic Cells

2012 ◽  
Vol 19 (3) ◽  
pp. 368-376 ◽  
Author(s):  
Roger Badia ◽  
M. Teresa Brufau ◽  
Ana Maria Guerrero-Zamora ◽  
Rosil Lizardo ◽  
Irina Dobrescu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dendritic Cells ◽  
Salmonella Enterica ◽  
Molecular Mechanisms ◽  
Intestinal Epithelial ◽  
Content Type ◽  
Gm Csf ◽  
Tnf Α ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericaserovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed β-galactomannan (βGM) prebiotic compared to the proven probioticSaccharomyces cerevisiaevar.boulardiion porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) coculturedin vitrowithSalmonella. We observed that bothS. cerevisiaevar.boulardiiand βGM inhibited the association ofSalmonellawith IECsin vitro. Our data indicated that βGM has a higher ability thanS. cerevisiaevar.boulardiito inhibitSalmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, βGM andS. cerevisiaevar.boulardiiinduced some effects on DCs that were not observed on IECs: βGM andS. cerevisiaevar.boulardiishowed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of βGM orS. cerevisiaevar.boulardiion DCs cocultured withSalmonellashowed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control withSalmonella. In conclusion, the addition of βGM inhibitsSalmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.

scholarly journals Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

2013 ◽  
Vol 79 (17) ◽  
pp. 5197-5207 ◽  
Author(s):  
D. Rossouw ◽  
E. H. Heyns ◽  
M. E. Setati ◽  
S. Bosch ◽  
F. F. Bauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Modification ◽  
Target Genes ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Systematic Screening ◽  
Content Type ◽  
Genes Encoding ◽  
Ethanol Yields ◽  
Trehalose Biosynthesis

ABSTRACTThe ability ofSaccharomyces cerevisiaeto efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1andACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ andtdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, theTPS1gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of theTPS1gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines.

scholarly journals Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

2014 ◽  
Vol 80 (24) ◽  
pp. 7694-7701 ◽  
Author(s):  
Guo-Chang Zhang ◽  
In Iok Kong ◽  
Heejin Kim ◽  
Jing-Jing Liu ◽  
Jamie H. D. Cate ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Auxotrophic Mutant ◽  
Marker Genes ◽  
Fermentation Inhibitors ◽  
Multiple Alleles ◽  
Content Type ◽  
Auxotrophic Mutants ◽  
Yeast Strains ◽  
Cas9 Nuclease ◽  
Beneficial Traits

ABSTRACTIndustrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain,Saccharomyces cerevisiaeATCC 4124, withura3,trp1,leu2, andhis3auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploidS. cerevisiaestrains that have been widely used in the wine, beer, and fermentation industries.

scholarly journals Harnessing Genetic Diversity in Saccharomyces cerevisiae for Fermentation of Xylose in Hydrolysates of Alkaline Hydrogen Peroxide-Pretreated Biomass

2013 ◽  
Vol 80 (2) ◽  
pp. 540-554 ◽  
Author(s):  
Trey K. Sato ◽  
Tongjun Liu ◽  
Lucas S. Parreiras ◽  
Daniel L. Williams ◽  
Dana J. Wohlbach ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Rapid Evolution ◽  
Biofuel Production ◽  
Ecological Niches ◽  
Alkaline Hydrogen Peroxide ◽  
Xylose Metabolism ◽  
Content Type ◽  
Inhibitory Compounds ◽  
Lignocellulosic Biofuel

ABSTRACTThe fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeastSaccharomyces cerevisiaeis known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverseS. cerevisiaestrains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na+, acetate, andp-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts ofpCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose andpCA or FA with a wildS. cerevisiaestrain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

scholarly journals Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Yixuan Zhu ◽  
Jingtao Zhang ◽  
Lang Zhu ◽  
Zefang Jia ◽  
Qi Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Budding Yeast ◽  
Ectopic Expression ◽  
Redox Balance ◽  
Inducible Promoter ◽  
Pentose Phosphate ◽  
Xylose Utilization ◽  
Xylose Metabolism ◽  
Utilization Pathway

Xylose is the second most abundant sugar in lignocellulose, but it cannot be used as carbon source by budding yeast Saccharomyces cerevisiae. Rational promoter elements engineering approaches were taken for efficient xylose fermentation in budding yeast. Among promoters surveyed, HXT7 exhibited the best performance. The HXT7 promoter is suppressed in the presence of glucose and derepressed by xylose, making it a promising candidate to drive xylose metabolism. However, simple ectopic expression of both key xylose metabolic genes XYL1 and XYL2 by the HXT7 promoter resulted in massive accumulation of the xylose metabolic byproduct xylitol. Through the HXT7-driven expression of a reported redox variant, XYL1-K270R, along with optimized expression of XYL2 and the downstream pentose phosphate pathway genes, a balanced xylose metabolism toward ethanol formation was achieved. Fermented in a culture medium containing 50 g/L xylose as the sole carbon source, xylose is nearly consumed, with less than 3 g/L xylitol, and more than 16 g/L ethanol production. Hence, the combination of an inducible promoter and redox balance of the xylose utilization pathway is an attractive approach to optimizing fuel production from lignocellulose.

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