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13C-based metabolic flux analysis

Abstract

Stable isotope, and in particular 13C-based flux analysis, is the exclusive approach to experimentally quantify the integrated responses of metabolic networks. Here we describe a protocol that is based on growing microbes on 13C-labeled glucose and subsequent gas chromatography mass spectrometric detection of 13C-patterns in protein-bound amino acids. Relying on publicly available software packages, we then describe two complementary mathematical approaches to estimate either local ratios of converging fluxes or absolute fluxes through different pathways. As amino acids in cell protein are abundant and stable, this protocol requires a minimum of equipment and analytical expertise. Most other flux methods are variants of the principles presented here. A true alternative is the analytically more demanding dynamic flux analysis that relies on 13C-pattern in free intracellular metabolites. The presented protocols take 5–10 d, have been used extensively in the past decade and are exemplified here for the central metabolism of Escherichia coli.

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Figure 1: Workflow of a 13C metabolic flux analysis experiment.
Figure 2: Detection of 13C labeling patterns by GC-MS analysis of derivatized amino acids.
Figure 3: Metabolic and isotopic dynamics during 13C labeling experiments in batch and continuous cultivations.
Figure 4: Metabolic flux ratios and absolute fluxes calculated for E. coli grown in glucose-limited continuous cultures at 0.12 h−1.
Figure 5: Difference in flux estimates obtained by iterative fitting with ill-defined networks.

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Acknowledgements

We thank Katharina Nöh and Wolfgang Wiechert for support with 13CFLUX and comments on the respective protocol parts presented in this protocol, as well as Roelco J. Kleijn, Dominik Heer, Julian Schnidder, and Daniel Heine for constructive comments on the script.

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Correspondence to Sarah-Maria Fendt.

Supplementary information

Supplementary Data 1

Raw GC-MS data and physiological parameters for E. coli example (ZIP 4350 kb)

Supplementary Data 2

FiatFlux files for E. coli example (ZIP 1521 kb)

Supplementary Data 3

13CFLUX files for E. coli example (ZIP 11113 kb)

Supplementary Method 1

Step-by-step tutorial for 13CFLUX software (PDF 111 kb)

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Zamboni, N., Fendt, SM., Rühl, M. et al. 13C-based metabolic flux analysis. Nat Protoc 4, 878–892 (2009). https://doi.org/10.1038/nprot.2009.58

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