scholarly journals Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Jeroen G. Nijland ◽  
Hyun Yong Shin ◽  
Leonie G. M. Boender ◽  
Paul P. de Waal ◽  
Paul Klaassen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Second Generation ◽  
Xylose Metabolism ◽  
Altered Expression ◽  
Content Type ◽  
Xylose Transport ◽  
Second Generation Bioethanol ◽  
A Genome ◽  
Hexose Transporters

ABSTRACT Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16. Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (V max) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell. IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

scholarly journals Global Analysis of Furfural-Induced Genomic Instability Using a Yeast Model

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Lei Qi ◽  
Ke Zhang ◽  
Yu-Ting Wang ◽  
Jian-Kun Wu ◽  
Yang Sui ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Global Analysis ◽  
Chromosomal Rearrangements ◽  
Point Mutations ◽  
Mitotic Recombination ◽  
Whole Genome ◽  
Wild Type ◽  
Content Type ◽  
A Genome

ABSTRACT Furfural is an important renewable precursor for multiple commercial chemicals and fuels; a main inhibitor existing in cellulosic hydrolysate, which is used for bioethanol fermentation; and a potential carcinogen, as well. Using a genetic system in Saccharomyces cerevisiae that allows detection of crossover events, we observed that the frequency of mitotic recombination was elevated by 1.5- to 40-fold when cells were treated with 0.1 g/liter to 20 g/liter furfural. Analysis of the gene conversion tracts associated with crossover events suggested that most furfural-induced recombination resulted from repair of DNA double-strand breaks (DSBs) that occurred in the G1 phase. Furfural was incapable of breaking DNA directly in vitro but could trigger DSBs in vivo related to reactive oxygen species accumulation. By whole-genome single nucleotide polymorphism (SNP) microarray and sequencing, furfural-induced genomic alterations that range from single base substitutions, loss of heterozygosity, and chromosomal rearrangements to aneuploidy were explored. At the whole-genome level, furfural-induced events were evenly distributed across 16 chromosomes but were enriched in high-GC-content regions. Point mutations, particularly the C-to-T/G-to-A transitions, were significantly elevated in furfural-treated cells compared to wild-type cells. This study provided multiple novel insights into the global effects of furfural on genomic stability. IMPORTANCE Whether and how furfural affects genome integrity have not been clarified. Using a Saccharomyces cerevisiae model, we found that furfural exposure leads to in vivo DSBs and elevation in mitotic recombination by orders of magnitude. Gross chromosomal rearrangements and aneuploidy events also occurred at a higher frequency in furfural-treated cells. In a genome-wide analysis, we show that the patterns of mitotic recombination and point mutations differed dramatically in furfural-treated cells and wild-type cells.

scholarly journals Novel Propagation Strategy of Saccharomyces cerevisiae for Enhanced Xylose Metabolism during Fermentation on Softwood Hydrolysate

Fermentation ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 288
Author(s):  
Andreea Cristina Dobrescu ◽  
Henrique César Teixeira Veras ◽  
Cristiano Varrone ◽  
Jan Dines Knudsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Second Generation ◽  
Xylose Fermentation ◽  
Carbon Sources ◽  
Fed Batch ◽  
Xylose Metabolism ◽  
Second Generation Bioethanol ◽  
Utilisation Rate ◽  
Shake Flasks ◽  
Production Strategy

An economically viable production of second-generation bioethanol by recombinant xylose-fermenting Saccharomyces cerevisiae requires higher xylose fermentation rates and improved glucose–xylose co-consumption. Moreover, xylose-fermenting S. cerevisiae recognises xylose as a non-fermentable rather than a fermentable carbon source, which might partly explain why xylose is not fermented into ethanol as efficiently as glucose. This study proposes propagating S. cerevisiae on non-fermentable carbon sources to enhance xylose metabolism during fermentation. When compared to yeast grown on sucrose, cells propagated on a mix of ethanol and glycerol in shake flasks showed up to 50% higher xylose utilisation rate (in a defined xylose medium) and a double maximum fermentation rate, together with an improved C5/C6 co-consumption (on an industrial softwood hydrolysate). Based on these results, an automated propagation protocol was developed, using a fed-batch approach and the respiratory quotient to guide the ethanol and glycerol-containing feed. This successfully produced 71.29 ± 0.91 g/L yeast with an average productivity of 1.03 ± 0.05 g/L/h. These empirical findings provide the basis for the design of a simple, yet effective yeast production strategy to be used in the second-generation bioethanol industry for increased fermentation efficiency.

scholarly journals Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production

2014 ◽  
Vol 80 (22) ◽  
pp. 6908-6918 ◽  
Author(s):  
Johan O. Westman ◽  
Valeria Mapelli ◽  
Mohammad J. Taherzadeh ◽  
Carl Johan Franzén
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Second Generation ◽  
Raw Materials ◽  
Defined Medium ◽  
Bioethanol Production ◽  
Dilute Acid ◽  
Fermentation Inhibitors ◽  
Inhibitor Tolerance ◽  
Content Type ◽  
Second Generation Bioethanol

ABSTRACTYeast has long been considered the microorganism of choice for second-generation bioethanol production due to its fermentative capacity and ethanol tolerance. However, tolerance toward inhibitors derived from lignocellulosic materials is still an issue. Flocculating yeast strains often perform relatively well in inhibitory media, but inhibitor tolerance has never been clearly linked to the actual flocculation abilityper se. In this study, variants of the flocculation geneFLO1were transformed into the genome of the nonflocculating laboratory yeast strainSaccharomyces cerevisiaeCEN.PK 113-7D. Three mutants with distinct differences in flocculation properties were isolated and characterized. The degree of flocculation and hydrophobicity of the cells were correlated to the length of the gene variant. The effect of different strength of flocculation on the fermentation performance of the strains was studied in defined medium with or without fermentation inhibitors, as well as in media based on dilute acid spruce hydrolysate. Strong flocculation aided against the readily convertible inhibitor furfural but not against less convertible inhibitors such as carboxylic acids. During fermentation of dilute acid spruce hydrolysate, the most strongly flocculating mutant with dense cell flocs showed significantly faster sugar consumption. The modified strain with the weakest flocculation showed a hexose consumption profile similar to the untransformed strain. These findings may explain why flocculation has evolved as a stress response and can find application in fermentation-based biorefinery processes on lignocellulosic raw materials.

scholarly journals Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production

2013 ◽  
Vol 6 (1) ◽  
pp. 168 ◽  
Author(s):  
Lorenzo Favaro ◽  
Marina Basaglia ◽  
Alberto Trento ◽  
Eugéne Van Rensburg ◽  
Maria García-Aparicio ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Second Generation ◽  
Bioethanol Production ◽  
Grape Marc ◽  
Second Generation Bioethanol

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Global Analysis of Genes Essential forFrancisella tularensisSchu S4 GrowthIn Vitroand for Fitness during Competitive Infection of Fischer 344 Rats

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Philip M. Ireland ◽  
Helen L. Bullifent ◽  
Nicola J. Senior ◽  
Stephanie J. Southern ◽  
Zheng Rong Yang ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Insertion Site ◽  
Therapeutic Drug ◽  
Content Type ◽  
Fischer 344 ◽  
A Genome ◽  
Fischer 344 Rat ◽  
Schu S4

ABSTRACTThe highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

scholarly journals CovRS-Regulated Transcriptome Analysis of a Hypervirulent M23 Strain of Group A Streptococcus pyogenes Provides New Insights into Virulence Determinants

2015 ◽  
Vol 197 (19) ◽  
pp. 3191-3205 ◽  
Author(s):  
Yun-Juan Bao ◽  
Zhong Liang ◽  
Jeffrey A. Mayfield ◽  
Shaun W. Lee ◽  
Victoria A. Ploplis ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Broad Spectrum ◽  
Virulence Genes ◽  
Expression Profiles ◽  
Altered Expression ◽  
Content Type ◽  
Metabolic Genes ◽  
A Genome ◽  
Group A ◽  
Regulated Gene Expression

ABSTRACTThe two-componentcontrolofvirulence (Cov) regulator (R)-sensor (S) (CovRS) regulates the virulence ofStreptococcus pyogenes(group AStreptococcus[GAS]). Inactivation of CovS during infection switches the pathogenicity of GAS to a more invasive form by regulating transcription of diverse virulence genes via CovR. However, the manner in which CovRS controls virulence through expression of extended gene families has not been fully determined. In the current study, the CovS-regulated gene expression profiles of a hypervirulentemm23GAS strain (M23ND/CovS negative [M23ND/CovS−]) and a noninvasive isogenic strain (M23ND/CovS+), under different growth conditions, were investigated. RNA sequencing identified altered expression of ∼349 genes (18% of the chromosome). The data demonstrated that M23ND/CovS−achieved hypervirulence by allowing enhanced expression of genes responsible for antiphagocytosis (e.g.,hasABC), by abrogating expression of toxin genes (e.g.,speB), and by compromising gene products with dispensable functions (e.g.,sfb1). Among these genes, several (e.g.,parEandparC) were not previously reported to be regulated by CovRS. Furthermore, the study revealed that CovS also modulated the expression of a broad spectrum of metabolic genes that maximized nutrient utilization and energy metabolism during growth and dissemination, where the bacteria encounter large variations in available nutrients, thus restructuring metabolism of GAS for adaption to diverse growth environments. From constructing a genome-scale metabolic model, we identified 16 nonredundant metabolic gene modules that constitute unique nutrient sources. These genes were proposed to be essential for pathogen growth and are likely associated with GAS virulence. The genome-wide prediction of genes associated with virulence identifies new candidate genes that potentially contribute to GAS virulence.IMPORTANCEThe CovRS system modulates transcription of ∼18% of the genes in theStreptococcus pyogenesgenome. Mutations that inactivate CovR or CovS enhance the virulence of this bacterium. We determined complete transcriptomes of a naturally CovS-inactivated invasive deep tissue isolate of anemm23strain ofS. pyogenes(M23ND) and its complemented avirulent variant (CovS+). We identified diverse virulence genes whose altered expression revealed a genetic switching of a nonvirulent form of M23ND to a highly virulent strain. Furthermore, we also systematically uncovered for the first time the comparative levels of expression of a broad spectrum of metabolic genes, which reflected different metabolic needs of the bacterium as it invaded deeper tissue of the human host.

scholarly journals Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae

2017 ◽  
Vol 37 (11) ◽  
Author(s):  
Jayasankar Mohanakrishnan Kaimal ◽  
Ganapathi Kandasamy ◽  
Fabian Gasser ◽  
Claes Andréasson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Protein Aggregation ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Content Type ◽  
Dependent Protein ◽  
And Function ◽  
Cellular Dysfunction

ABSTRACT Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

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