Abstract
Metabolic flux analysis represents an essential perspective to understand cellular physiology and offers quantitative information to guide pathway engineering. A valuable approach for experimental elucidation of metabolic flux is dynamic flux analysis, which estimates the relative or absolute flow rates through a series of metabolic intermediates in a given pathway. It is based on kinetic isotope labeling experiments, liquid chromatography-mass spectrometry (LC-MS), and computational analysis that relate kinetic isotope trajectories of metabolites to pathway activity. Herein, we illustrate the mathematic principles underlying the dynamic flux analysis and mainly focus on describing the experimental procedures for data generation. This protocol is exemplified using cyanobacterial metabolism as an example, for which reliable labeling data for central carbon metabolites can be acquired quantitatively. This protocol is applicable to other microbial systems as well and can be readily adapted to address different metabolic processes.
Original language | American English |
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Title of host publication | Metabolic Pathway Engineering |
Subtitle of host publication | Methods in Molecular Biology, Volume 2096 |
Editors | M. E. Himmel, Y. J. Bomble |
Publisher | Humana Press Inc. |
Pages | 179-196 |
Number of pages | 18 |
DOIs | |
State | Published - 2020 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2096 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Bibliographical note
Publisher Copyright:© Springer Science+Business Media, LLC, part of Springer Nature 2020.
NREL Publication Number
- NREL/CH-2700-77590
Keywords
- Cell harvesting
- Dynamic flux analysis
- Experimental metabolomics
- Isotope tracer
- LC-MS
- Metabolic flux
- Quenching