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High-resolution 13C metabolic flux analysis

Abstract

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.

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Fig. 1: Overview of procedure for high-resolution 13C metabolic flux analysis.
Fig. 2: Batch culture sampling to assess changes.
Fig. 3: Overview of procedures.
Fig. 4: Demonstration of the workflow for 13C-MFA for the E. coli ΔtpiA case study.

Data availability

All data relevant to this protocol are included in the Supplementary Information files.

Software availability

The Metran software is freely available for academic research and educational purposes. The technical licensing office at the Massachusetts Institute of Technology can be contacted to request a copy of the Metran software (https://tlo.mit.edu/technologies/metran-software-13c-metabolic-flux-analysis).

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Acknowledgements

This work was supported by grant NSF MCB-1616332.

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Affiliations

Authors

Contributions

C.P.L. performed all experiments. C.P.L. and M.R.A. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Maciek R. Antoniewicz.

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The authors declare no competing interests.

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Related links

Key references using this protocol

Orbán-Németh, Z. et al. Nat. Protoc. 13, 478–494 (2018): https://doi.org/10.1038/nprot.2017.146

Key data used in this protocol

Orbán-Németh, Z. et al. Nat. Protoc. 13, 478–494 (2018): https://doi.org/10.1038/nprot.2017.146

Supplementary information

Supplementary Data 1

Raw GC–MS data files in CDF file format for the E. coli ΔtpiA case study.

Reporting Summary

Supplementary Data 2

Integrated mass isotopomer distributions for the E. coli ΔtpiA case study.

Supplementary Data 3

Metabolic network model used for 13C-MFA for the E. coli ΔtpiA case study.

Supplementary Data 4

Metran file with default E. coli model.

Supplementary Data 5

Metran file for the E. coli ΔtpiA case study.

Supplementary Data 6

Flux analysis results for the E. coli ΔtpiA case study.

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Long, C.P., Antoniewicz, M.R. High-resolution 13C metabolic flux analysis. Nat Protoc 14, 2856–2877 (2019). https://doi.org/10.1038/s41596-019-0204-0

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