scholarly journals D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain

2020 ◽  
Author(s):  
Jeroen G Nijland ◽  
Hyun Yong Shin ◽  
Eleonora Dore ◽  
Donny Rudinatha ◽  
Paul P de Waal ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Performance ◽  
Glucose Consumption ◽  
Glycolytic Flux ◽  
Overflow Metabolism ◽  
Evolutionary Engineering ◽  
Xylose Transport ◽  
Conversion Rates ◽  
Cost Efficient ◽  
Glucose Recycling

Abstract Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

scholarly journals Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain

2019 ◽  
Vol 19 (8) ◽  
Author(s):  
Jeroen G Nijland ◽  
Xiang Li ◽  
Hyun Yong Shin ◽  
Paul P de Waal ◽  
Arnold J M Driessen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Morphological Analysis ◽  
Membrane Transporters ◽  
Evolutionary Engineering ◽  
Xylose Consumption ◽  
Xylose Transport ◽  
Xylose Uptake ◽  
Cost Efficient ◽  
Engineered Saccharomyces Cerevisiae ◽  
Selection Of

ABSTRACT Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113–7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

scholarly journals Gcn5p and Ubp8p Affect Protein Ubiquitylation and Cell Proliferation by Altering the Fermentative/Respiratory Flux Balance in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Antonella De Palma ◽  
Giulia Fanelli ◽  
Elisabetta Cretella ◽  
Veronica De Luca ◽  
Raffaele Antonio Palladino ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Consumption ◽  
Redox Balance ◽  
Glycolytic Enzymes ◽  
Glycolytic Flux ◽  
Direct Role ◽  
Content Type ◽  
Proteomic Approach ◽  
Altered Glucose

ABSTRACT Protein ubiquitylation regulates not only endocellular trafficking and proteasomal degradation but also the catalytic activity of enzymes. In Saccharomyces cerevisiae, we analyzed the composition of the ubiquitylated proteomes in strains lacking acetyltransferase Gcn5p, Ub-protease Ubp8p, or both to understand their involvement in the regulation of protein ubiquitylation. We analyzed His6Ub proteins with a proteomic approach coupling micro-liquid chromatography and tandem mass spectrometry (μLC-MS/MS) in gcn5Δ, ubp8Δ and ubp8Δ gcn5Δ strains. The Ub-proteome altered in the absence of Gcn5p, Ubp8p, or both was characterized, showing that 43% of the proteins was shared in all strains, suggesting their functional relationship. Remarkably, all major glycolytic enzymes showed increased ubiquitylation. Phosphofructokinase 1, the key enzyme of glycolytic flux, showed a higher and altered pattern of ubiquitylation in gcn5Δ and ubp8Δ strains. Severe defects of growth in poor sugar and altered glucose consumption confirmed a direct role of Gcn5p and Ubp8p in affecting the REDOX balance of the cell. IMPORTANCE We propose a study showing a novel role of Gcn5p and Ubp8p in the process of ubiquitylation of the yeast proteome which includes main glycolytic enzymes. Interestingly, in the absence of Gcn5p and Ubp8p glucose consumption and redox balance were altered in yeast. We believe that these results and the role of Gcn5p and Ubp8p in sugar metabolism might open new perspectives of research leading to novel protocols for counteracting the enhanced glycolysis in tumors.

scholarly journals Codon-Optimized Bacterial Genes Improve l-Arabinose Fermentation in Recombinant Saccharomyces cerevisiae

2008 ◽  
Vol 74 (7) ◽  
pp. 2043-2050 ◽  
Author(s):  
Beate Wiedemann ◽  
Eckhard Boles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Dry Weight ◽  
Rational Approach ◽  
Lag Phase ◽  
Evolutionary Engineering ◽  
Conversion Rates ◽  
Starting Point ◽  
Arabinose Isomerase

ABSTRACT Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like l-arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial l-arabinose utilization pathway consisting of l-arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. l-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on l-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the l-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved l-arabinose conversion rates. With this rational approach, the ethanol production rate from l-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h−1 (g dry weight)−1 to 0.036 g ethanol h−1 (g dry weight)−1 and the ethanol yield could be increased from 0.24 g ethanol (g consumed l-arabinose)−1 to 0.39 g ethanol (g consumed l-arabinose)−1. These improvements make up a new starting point for the construction of more-efficient industrial l-arabinose-fermenting yeast strains by evolutionary engineering.

scholarly journals Moderate Pulsed Electric Field Enhances Fermentation Capacity and Induces Stress Responses in Saccharomyces cerevisiae

2021 ◽  
Vol 25 (01) ◽  
pp. 160-164
Author(s):  
Nanjiao Ying
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Electric Field ◽  
Stress Responses ◽  
Ethanol Yield ◽  
Glucose Consumption ◽  
Pulsed Electric Field ◽  
Yeast Cells ◽  
Glycolytic Flux ◽  
Physiological Level ◽  
Fermentation Capacity

Saccharomyces cerevisiae has always gained a huge amount of attention due to its extensive and enormous application value. Various methods are used to optimize the fermentation characteristics of S. cerevisiae. In this work, we investigated the fermentation capacity and stress responses on a physiological level of S. cerevisiae which affected by moderate pulsed electric field (PEF, 1 to 3 kV·cm-1). The fermentation capacity was illustrated by glucose consumption and metabolites yield (ethanol and glycerol). The expressions of ten glycolysis-associated genes were studied to elucidate the fermentative processes. The results showed that fermentation capacity of S. cerevisiae was promoted by moderate PEF treatments, with higher glucose consumption and ethanol yield. Yeast cells also gained a faster growth rate under constant PEF stimulation. Analysis of gene expression involved in glycolytic pathway showed that moderate PEF treatments induced a higher glycolytic flux, especially in terms of synthesis of ethanol. It would be a promising technique for PEF to enhance fermentation capacity and growth of S. cerevisiae. © 2021 Friends Science Publishers

scholarly journals High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Buprenorphine Evaluation in Plasma—Application to Pharmacokinetic Studies in Rabbits

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 437
Author(s):  
Marta Tikhomirov ◽  
Błażej Poźniak ◽  
Tomasz Śniegocki
Keyword(s):  
High Performance ◽  
Pharmacokinetic Profile ◽  
Pharmacokinetic Studies ◽  
Limit Of Quantification ◽  
Reliable Determination ◽  
Main Challenge ◽  
Analytical Protocol ◽  
Liquid Chromatography Tandem Mass ◽  
Cost Efficient

The precise and reliable determination of buprenorphine concentration is fundamental in certain medical or research applications, particularly in pharmacokinetic studies of this opioid. The main challenge is, however, the development of an analytical method that is sensitive enough, as the detected in vivo concentrations often fall in very low ranges. Thus, in this study we aimed at developing a sensitive, repeatable, cost-efficient, and easy HPLC analytical protocol for buprenorphine in rabbit plasma. In order to obtain this, the HPLC-MS2 system was used to elaborate and validate the method for samples purified with liquid-liquid extraction. Fragment ions 468.6→396.2 and 468.6→414.2 were monitored, and the method resulted in a high repeatability and reproducibility and a limit of quantification of 0.25 µg/L with a recovery of 98.7–109.0%. The method was linear in a range of 0.25–2000 µg/L. The suitability of the analytical procedure was tested in rabbits in a pilot pharmacokinetic study, and it was revealed that the method was suitable for comprehensively describing the pharmacokinetic profile after buprenorphine intravenous administration at a dose of 300 µg/kg. Thus, the method suitability for pharmacokinetic application was confirmed by both the good validation results of the method and successful in vivo tests in rabbits.

scholarly journals Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

2007 ◽  
Vol 73 (8) ◽  
pp. 2432-2439 ◽  
Author(s):  
Carole Guillaume ◽  
Pierre Delobel ◽  
Jean-Marie Sablayrolles ◽  
Bruno Blondin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Alcoholic Fermentation ◽  
Wine Yeast ◽  
Glycolytic Flux ◽  
Hexose Transporter ◽  
Wine Yeasts ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Fructose ◽  
Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

scholarly journals A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Omid Oftadeh ◽  
Pierre Salvy ◽  
Maria Masid ◽  
Maxime Curvat ◽  
Ljubisa Miskovic ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Biomass Composition ◽  
Industrial Biotechnology ◽  
Overflow Metabolism ◽  
Cellular Expression ◽  
A Genome ◽  
Wide Range ◽  
Eukaryotic Organisms ◽  
Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

Reliability evaluation of wearable radio frequency identification tags: Design and fabrication of a two-part textile antenna

2018 ◽  
Vol 89 (4) ◽  
pp. 560-571 ◽  
Author(s):  
Xiaochen Chen ◽  
Leena Ukkonen ◽  
Johanna Virkki
Keyword(s):  
Radio Frequency ◽  
Radio Frequency Identification ◽  
Integrated Circuit ◽  
High Performance ◽  
Cyclic Strain ◽  
Moist Environment ◽  
Frequency Identification ◽  
Identification Tags ◽  
Cost Efficient ◽  
The One

Passive radio frequency identification-based technology is a convincing approach to the achievement of versatile energy- and cost-efficient wireless platforms for future wearable applications. By using two-part antenna structures, the antenna-electronics interconnections can remain non-stressed, which can significantly improve the reliability of the textile-embedded wireless components. In this article, we describe fabrication of two-part stretchable and non-stretchable passive ultra-high frequency radio frequency identification textile tags using electro-textile and embroidered antennas, and test their reliability when immersed as well as under cyclic strain. The results are compared to tags with traditional one-part dipole antennas fabricated from electro-textiles and by embroidery. Based on the results achieved, the initial read ranges of the two-part antenna tags, around 5 m, were only slightly shorter than those of the one-part antenna tags. In addition, the tag with two-part antennas can maintain high performance in a moist environment and during continuous stretching, unlike the one-part antenna tag where the antenna-integrated circuit attachment is under stress.

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