The Experts below are selected from a list of 38898 Experts worldwide ranked by ideXlab platform
Brigitte Thomasset - One of the best experts on this subject based on the ideXlab platform.
-
13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux analysis.
Analytical biochemistry, 2017Co-Authors: Sébastien Acket, Anthony Degournay, Franck Merlier, Brigitte ThomassetAbstract:Abstract Metabolic Flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define Fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography–high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic Flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].
-
13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux analysis
Analytical Biochemistry, 2017Co-Authors: Sébastien Acket, Anthony Degournay, Franck Merlier, Brigitte ThomassetAbstract:Metabolic Flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define Fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography ehigh resolution mass spectrometry, without derivatized procedure, adapted for Metabolic Flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic en-richments of free sugars compared to our previous methods.
Maciek R. Antoniewicz - One of the best experts on this subject based on the ideXlab platform.
-
A guide to Metabolic Flux analysis in Metabolic engineering: Methods, tools and applications.
Metabolic engineering, 2020Co-Authors: Maciek R. AntoniewiczAbstract:Abstract The field of Metabolic engineering is primarily concerned with improving the biological production of value-added chemicals, fuels and pharmaceuticals through the design, construction and optimization of Metabolic pathways, redirection of intracellular Fluxes, and refinement of cellular properties relevant for industrial bioprocess implementation. Metabolic network models and Metabolic Fluxes are central concepts in Metabolic engineering, as was emphasized in the first paper published in this journal, “Metabolic Fluxes and Metabolic engineering” (Metabolic Engineering, 1: 1–11, 1999). In the past two decades, a wide range of computational, analytical and experimental approaches have been developed to interrogate the capabilities of biological systems through analysis of Metabolic network models using techniques such as Flux balance analysis (FBA), and quantify Metabolic Fluxes using constrained-based modeling approaches such as Metabolic Flux analysis (MFA) and more advanced experimental techniques based on the use of stable-isotope tracers, i.e. 13C-Metabolic Flux analysis (13C-MFA). In this review, we describe the basic principles of Metabolic Flux analysis, discuss current best practices in Flux quantification, highlight potential pitfalls and alternative approaches in the application of these tools, and give a broad overview of pragmatic applications of Flux analysis in Metabolic engineering practice.
-
High-resolution 13C Metabolic Flux analysis.
Nature protocols, 2019Co-Authors: Christopher P. Long, Maciek R. AntoniewiczAbstract:Precise quantification of Metabolic pathway Fluxes in biological systems is of major importance in guiding efforts in Metabolic engineering, biotechnology, microbiology, human health, and cell culture. 13C Metabolic Flux analysis (13C-MFA) is the predominant technique used for determining intracellular Fluxes. Here, we present a protocol for 13C-MFA that incorporates recent advances in parallel labeling experiments, isotopic labeling measurements, and statistical analysis, as well as best practices developed through decades of experience. Experimental design to ensure that Fluxes are estimated with the highest precision is an integral part of the protocol. The protocol is based on growing microbes in two (or more) parallel cultures with 13C-labeled glucose tracers, followed by gas chromatography–mass spectrometry (GC–MS) measurements of isotopic labeling of protein-bound amino acids, glycogen-bound glucose, and RNA-bound ribose. Fluxes are then estimated using software for 13C-MFA, such as Metran, followed by comprehensive statistical analysis to determine the goodness of fit and calculate confidence intervals of Fluxes. The presented protocol can be completed in 4 d and quantifies Metabolic Fluxes with a standard deviation of ≤2%, a substantial improvement over previous implementations. The presented protocol is exemplified using an Escherichia coli ΔtpiA case study with full supporting data, providing a hands-on opportunity to step through a complex troubleshooting scenario. Although applications to prokaryotic microbial systems are emphasized, this protocol can be easily adjusted for application to eukaryotic organisms. Precise quantification of Metabolic pathway Fluxes is needed in many applications, e.g., microbiological engineering. The authors describe a GC–MS method for 13C Metabolic Flux analysis with data analysis using Metran software.
-
(13)C-Metabolic Flux analysis of co-cultures: A novel approach.
Metabolic engineering, 2015Co-Authors: Nikodimos A. Gebreselassie, Maciek R. AntoniewiczAbstract:Abstract In this work, we present a novel approach for performing 13C Metabolic Flux analysis (13C-MFA) of co-culture systems. We demonstrate for the first time that it is possible to determine Metabolic Flux distributions in multiple species simultaneously without the need for physical separation of cells or proteins, or overexpression of species-specific products. Instead, Metabolic Fluxes for each species in a co-culture are estimated directly from isotopic labeling of total biomass obtained using conventional mass spectrometry approaches such as GC–MS. In addition to determining Metabolic Fluxes, this approach estimates the relative population size of each species in a mixed culture and inter-species metabolite exchange. As such, it enables detailed studies of microbial communities including species dynamics and interactions between community members. The methodology is experimentally validated here using a co-culture of two E. coli knockout strains. Taken together, this work greatly extends the scope of 13C-MFA to a large number of multi-cellular systems that are of significant importance in biotechnology and medicine.
-
Methods and advances in Metabolic Flux analysis: a mini-review
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Maciek R. AntoniewiczAbstract:Metabolic Flux analysis (MFA) is one of the pillars of Metabolic engineering. Over the past three decades, it has been widely used to quantify intracellular Metabolic Fluxes in both native (wild type) and engineered biological systems. Through MFA, changes in Metabolic pathway Fluxes are quantified that result from genetic and/or environmental interventions. This information, in turn, provides insights into the regulation of Metabolic pathways and may suggest new targets for further Metabolic engineering of the strains. In this mini-review, we discuss and classify the various methods of MFA that have been developed, which include stoichiometric MFA, ^13C Metabolic Flux analysis, isotopic non-stationary ^13C Metabolic Flux analysis, dynamic Metabolic Flux analysis, and ^13C dynamic Metabolic Flux analysis. For each method, we discuss key advantages and limitations and conclude by highlighting important recent advances in Flux analysis approaches.
-
Towards dynamic Metabolic Flux analysis in CHO cell cultures
Biotechnology journal, 2011Co-Authors: Woo Suk Ahn, Maciek R. AntoniewiczAbstract:Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study Metabolic Flux dynamics in CHO cell cultures using conventional Metabolic Flux analysis techniques that were developed for systems at Metabolic steady state. In this paper we review progress on Flux analysis in CHO cells and techniques for dynamic Metabolic Flux analysis. Application of these new tools may allow identification of intracellular Metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.
Sébastien Acket - One of the best experts on this subject based on the ideXlab platform.
-
13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux analysis.
Analytical biochemistry, 2017Co-Authors: Sébastien Acket, Anthony Degournay, Franck Merlier, Brigitte ThomassetAbstract:Abstract Metabolic Flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define Fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography–high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic Flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].
-
13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux analysis
Analytical Biochemistry, 2017Co-Authors: Sébastien Acket, Anthony Degournay, Franck Merlier, Brigitte ThomassetAbstract:Metabolic Flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define Fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography ehigh resolution mass spectrometry, without derivatized procedure, adapted for Metabolic Flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic en-richments of free sugars compared to our previous methods.
Elmar Heinzle - One of the best experts on this subject based on the ideXlab platform.
-
Investigation of glutamine metabolism in CHO cells by dynamic Metabolic Flux analysis
BMC Proceedings, 2013Co-Authors: Judith Wahrheit, Averina Nicolae, Elmar HeinzleAbstract:Background Glutamine metabolism represents one of the major targets in Metabolic engineering and process optimization due to its importance as cellular energy, carbon and nitrogen source. Metabolic Flux analysis represents a powerful method to investigate the physiology and metabolism of cells [1]. Classical Metabolic Flux analysis methods require steady state conditions. However, industrially relevant cultivation conditions, i.e. batch and fed-batch cultivations, are characterized by changing environmental conditions and Metabolic shifts [2]. We used dynamic Metabolic Flux analysis to study the impact of glutamine availability or limitation on the physiology of CHO K1 cells capturing Metabolic dynamics during batchand fed-batch cultivations.
-
Stable isotope-assisted metabolomics to detect Metabolic Flux changes in mammalian cell cultures.
Current opinion in biotechnology, 2012Co-Authors: Daniel Mueller, Elmar HeinzleAbstract:The determination of Metabolic Fluxes provides detailed information of cellular physiology, and the assessment of Metabolic Flux changes upon a certain perturbation can help to improve biotechnological and pharmaceutical processes. Stable isotope-assisted metabolomics using tracer-labeled substrates is the method of choice to determine the Fluxes. Though well-established for microbial cultures, the application to mammalian cells is generally complex and still limited. However, there have been great achievements in recent years and it is now emerging that stable isotope-assisted Metabolic Flux analysis in mammalian cell cultures will help improving biotechnological production and will also support drug development and discovery.
-
Metabolic Flux analysis in eukaryotes.
Current opinion in biotechnology, 2010Co-Authors: Jens Niklas, Konstantin Schneider, Elmar HeinzleAbstract:Metabolic Flux analysis (MFA) represents a powerful tool for systems biology research on eukaryotic cells. This review describes recent advances, the challenges as well as applications of Metabolic Flux analysis comprising fungi, mammalian cells and plants. While MFA is widely established and applied in microorganisms, it remains still a challenge to adapt these methods to eukaryotic cell systems having a higher complexity particularly concerning compartmentation or media composition. In fungi MFA was used in the past few years to analyze a variety of conditions and factors and their effects on cellular metabolism. In mammalian cells MFA was applied mainly in cell culture technology and in medical and toxicological research. (13)C Metabolic studies on native whole plants are additionally challenging by the fact that CO(2) is usually the only carbon source.
-
Hybrid optimization for 13C Metabolic Flux analysis using systems parametrized by compactification.
BMC systems biology, 2008Co-Authors: Tae Hoon Yang, Oliver Frick, Elmar HeinzleAbstract:Background The importance and power of isotope-based Metabolic Flux analysis and its contribution to understanding the Metabolic network is increasingly recognized. Its application is, however, still limited partly due to computational inefficiency. 13C Metabolic Flux analysis aims to compute in vivo Metabolic Fluxes in terms of metabolite balancing extended by carbon isotopomer balances and involves a nonlinear least-squares problem. To solve the problem more efficiently, improved numerical optimization techniques are necessary.
Anthony Degournay - One of the best experts on this subject based on the ideXlab platform.
-
13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux analysis.
Analytical biochemistry, 2017Co-Authors: Sébastien Acket, Anthony Degournay, Franck Merlier, Brigitte ThomassetAbstract:Abstract Metabolic Flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define Fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography–high resolution mass spectrometry, without derivatized procedure, adapted for Metabolic Flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic enrichments of free sugars compared to our previous methods [5, 6].
-
13C labeling analysis of sugars by high resolution-mass spectrometry for Metabolic Flux analysis
Analytical Biochemistry, 2017Co-Authors: Sébastien Acket, Anthony Degournay, Franck Merlier, Brigitte ThomassetAbstract:Metabolic Flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define Fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography ehigh resolution mass spectrometry, without derivatized procedure, adapted for Metabolic Flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic en-richments of free sugars compared to our previous methods.