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Dynamic 13C Labeling of Fast Turnover Metabolites for Analysis of Metabolic Fluxes and Metabolite Channeling

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Microbial Metabolomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1859))

Abstract

Dynamic or isotopically nonstationary 13C labeling experiments are a powerful tool not only for precise carbon flux quantification (e.g., metabolic flux analysis of photoautotrophic organisms) but also for theĀ investigation of pathway bottlenecks, a cellā€™s phenotype, and metabolite channeling. In general, isotopically nonstationary metabolic flux analysis requires three main components: (1) transient isotopic labeling experiments; (2) metabolite quenching and isotopomer analysis using LC-MS; (3) metabolic network construction and flux quantification. Labeling dynamics of key metabolites from 13C-pulse experiments allow flux estimation of key central pathways by solving ordinary differential equations to fit time-dependent isotopomer distribution data. Additionally, it is important to provide biomass requirements, carbon uptake rates, specific growth rates, and carbon excretion rates to properly and precisely balance the metabolic network. Labeling dynamics through cascade metabolites may also identify channeling phenomena in which metabolites are passed between enzymes without mixing with the bulk phase. In this chapter, we outline experimental protocols to probe metabolic pathways through dynamic labeling. We describe protocols for labeling experiments, metabolite quenching and extraction, LC-MS analysis, computational flux quantification, and metabolite channeling observations.

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Acknowledgements

This work was supported by NSF (CBET 1438125).

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Authors and Affiliations

  1. Department of Environmental, Energy and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA

    Mary Abernathy,Ā Ni WanĀ &Ā Yinjie J. Tang

  2. iHuman Institute, ShanghaiTech University, Shanghai, China

    Wenqing Shui

Authors
  1. Mary Abernathy
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  2. Ni Wan
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  3. Wenqing Shui
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  4. Yinjie J. Tang
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Corresponding author

Correspondence to Yinjie J. Tang .

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Editors and Affiliations

  1. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA

    Edward E.K. Baidoo

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Abernathy, M., Wan, N., Shui, W., Tang, Y.J. (2019). Dynamic 13C Labeling of Fast Turnover Metabolites for Analysis of Metabolic Fluxes and Metabolite Channeling. In: Baidoo, E. (eds) Microbial Metabolomics. Methods in Molecular Biology, vol 1859. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8757-3_18

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  • DOI: https://doi.org/10.1007/978-1-4939-8757-3_18

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  • Print ISBN: 978-1-4939-8756-6

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