Metabolic Pathway Determination and Flux Analysis in Nonmodel Microorganisms Through 13C-Isotope Labeling

  • Xueyang Feng
  • Wei-Qin Zhuang
  • Peter Colletti
  • Yinjie J. Tang
Part of the Methods in Molecular Biology book series (MIMB, volume 881)


C-isotope labeling is a commonly used technique for determining and quantifying pathways in microorganisms under various growth conditions. The experimental protocol consists of feeding the cell with a composition-defined substrate and measuring isotopic labeling patterns in the synthesized metabolites (often the amino acids). Not only can the labeling information be cross-referenced with genomic information to identify the novel pathways, but it can also be used to decipher absolute carbon fluxes through the metabolic network of interest. This technique can be widely used for functional characterization of nonmodel microbial species, and thus we provide a 13C-pathway and flux analysis protocol. The five key procedures are: (1) growing cells using labeled substrates, (2) measuring extracellular metabolite and biomass component, (3) analyzing isotopic labeling patterns in amino acids and central metabolites using gas chromatography-mass spectrometry, (4) tracing 13C carbon transitions in metabolites and discovering new pathways, and (5) estimating flux distributions based on isotopomer constraints. This protocol provides complementary information to the recently published protocol for 13C-based metabolic flux analysis of the model species Escherichia coli (Nat Protoc 4:878–892, 2009).

Key words

Amino acid GC-MS Genomic information Metabolite Constraint Novel pathway 



This study was supported in part by an NSF Career Grant (MCB0954016) and by a DOE bioenergy research grant (DEFG0208ER64694).


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xueyang Feng
    • 1
  • Wei-Qin Zhuang
    • 1
  • Peter Colletti
    • 1
  • Yinjie J. Tang
    • 1
  1. 1.Department of Energy, Environmental and Chemical EngineeringWashington UniversitySt. LouisUSA

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