Home LiteratureArticle Details
PMID: 19478804 Published · ppublish English Journal Article

(13)C-based metabolic flux analysis.

Nature protocols ·Vol. 4 ·No. 6 ·2009-00-00 ·Pages 878-92

Zamboni N, Fendt SM, Rühl M, Sauer U

Abstract

Stable isotope, and in particular (13)C-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 (13)C-labeled glucose and subsequent gas chromatography mass spectrometric detection of (13)C-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 (13)C-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.

MeSH Terms
Amino Acids/metabolism Carbon Isotopes Escherichia coli/metabolism Escherichia coli Proteins/metabolism Gas Chromatography-Mass Spectrometry Glucose/metabolism Metabolic Networks and Pathways Metabolomics/methods Models, Biological
Chemicals
Amino Acids Carbon Isotopes Escherichia coli Proteins Glucose
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Zamboni Nicola
Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
Fendt Sarah-Maria
Rühl Martin
Sauer Uwe
References (67)
67 references, click to expand
  1. An analytic and systematic framework for estimating metabolic flux ratios from 13C tracer experiments.
    BMC Bioinformatics. 2008 Jun 06;9:266 PMID: 18534038
  2. A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology.
    Nat Biotechnol. 2008 Oct;26(10):1155-60 PMID: 18846089
  3. Bidirectional reaction steps in metabolic networks: IV. Optimal design of isotopomer labeling experiments.
    Biotechnol Bioeng. 1999;66(2):86-103 PMID: 10567067
  4. Transient expression and flux changes during a shift from high to low riboflavin production in continuous cultures of Bacillus subtilis.
    Biotechnol Bioeng. 2005 Jan 20;89(2):219-32 PMID: 15584023
  5. Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures.
    Appl Environ Microbiol. 2002 Apr;68(4):1760-71 PMID: 11916694
  6. Genome-scale reconstruction of metabolic network in Bacillus subtilis based on high-throughput phenotyping and gene essentiality data.
    J Biol Chem. 2007 Sep 28;282(39):28791-28799 PMID: 17573341
  7. Citric acid cycle redux.
    Trends Biochem Sci. 1990 Nov;15(11):411-2 PMID: 2278096
  8. Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast.
    Genome Biol. 2005;6(6):R49 PMID: 15960801
  9. Systematic evaluation of objective functions for predicting intracellular fluxes in Escherichia coli.
    Mol Syst Biol. 2007;3:119 PMID: 17625511
  10. Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism.
    Nat Genet. 2005 Jun;37(6):636-40 PMID: 15880104
  11. Planning optimal measurements of isotopomer distributions for estimation of metabolic fluxes.
    Bioinformatics. 2006 May 15;22(10):1198-206 PMID: 16504982
  12. Metabolic flux analysis using mass spectrometry.
    Adv Biochem Eng Biotechnol. 2002;74:39-64 PMID: 11991183
  13. Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS.
    Eur J Biochem. 2003 Mar;270(5):880-91 PMID: 12603321
  14. Multiple high-throughput analyses monitor the response of E. coli to perturbations.
    Science. 2007 Apr 27;316(5824):593-7 PMID: 17379776
  15. Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data.
    Biotechnol Bioeng. 2008 Apr 1;99(5):1170-85 PMID: 17972325
  16. 13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum.
    Appl Environ Microbiol. 2006 Jul;72(7):4743-54 PMID: 16820467
  17. ReMatch: a web-based tool to construct, store and share stoichiometric metabolic models with carbon maps for metabolic flux analysis.
    J Integr Bioinform. 2008 Aug 25;5(2): PMID: 20134058
  18. New stable isotope-mass spectrometric techniques for measuring fluxes through intact metabolic pathways in mammalian systems: introduction of moving pictures into functional genomics and biochemical phenotyping.
    Metab Eng. 2004 Jan;6(1):85-100 PMID: 14734258
  19. A novel metabolic cycle catalyzes glucose oxidation and anaplerosis in hungry Escherichia coli.
    J Biol Chem. 2003 Nov 21;278(47):46446-51 PMID: 12963713
  20. A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information.
    Mol Syst Biol. 2007;3:121 PMID: 17593909
  21. Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of (13)C-labeled primary metabolites.
    FEMS Yeast Res. 2005 Apr;5(6-7):559-68 PMID: 15780655
  22. Integrated sampling procedure for metabolome analysis.
    Biotechnol Prog. 2006 Sep-Oct;22(5):1434-42 PMID: 17022684
  23. Analysis of flux estimates based on (13)C-labelling experiments.
    Eur J Biochem. 2002 Jun;269(11):2795-800 PMID: 12047390
  24. Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol.
    Metab Eng. 2007 May;9(3):277-92 PMID: 17400499
  25. Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments.
    Biotechnol Bioeng. 1997 Jul 5;55(1):101-17 PMID: 18636449
  26. Systematic assignment of thermodynamic constraints in metabolic network models.
    BMC Bioinformatics. 2006 Nov 23;7:512 PMID: 17123434
  27. A universal framework for 13C metabolic flux analysis.
    Metab Eng. 2001 Jul;3(3):265-83 PMID: 11461148
  28. Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry.
    Metab Eng. 2004 Oct;6(4):340-51 PMID: 15491863
  29. High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints.
    Anal Biochem. 2004 Feb 15;325(2):308-16 PMID: 14751266
  30. Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions.
    Metab Eng. 2007 Jan;9(1):68-86 PMID: 17088092
  31. Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source.
    Appl Environ Microbiol. 2004 Dec;70(12):7277-87 PMID: 15574927
  32. Identification of the Entner-Doudoroff pathway in an antibiotic-producing actinomycete species.
    Mol Microbiol. 2004 May;52(3):895-902 PMID: 15101992
  33. Metabolic flux analysis as a tool in metabolic engineering of plants.
    Curr Opin Biotechnol. 2008 Apr;19(2):131-7 PMID: 18378441
  34. Metabolic isotopomer labeling systems. Part II: structural flux identifiability analysis.
    Math Biosci. 2003 Jun;183(2):175-214 PMID: 12711410
  35. New tools for mass isotopomer data evaluation in (13)C flux analysis: mass isotope correction, data consistency checking, and precursor relationships.
    Biotechnol Bioeng. 2004 Feb 5;85(3):259-68 PMID: 14748080
  36. Modeling Lactococcus lactis using a genome-scale flux model.
    BMC Microbiol. 2005 Jun 27;5:39 PMID: 15982422
  37. Genome-scale metabolic model of Helicobacter pylori 26695.
    J Bacteriol. 2002 Aug;184(16):4582-93 PMID: 12142428
  38. Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis.
    Anal Chem. 2007 Oct 1;79(19):7554-9 PMID: 17822305
  39. Effect of Escherichia coli biomass composition on central metabolic fluxes predicted by a stoichiometric model.
    Biotechnol Bioeng. 1998 Oct 20;60(2):230-8 PMID: 10099424
  40. The growing scope of applications of genome-scale metabolic reconstructions using Escherichia coli.
    Nat Biotechnol. 2008 Jun;26(6):659-67 PMID: 18536691
  41. Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing.
    Biotechnol Bioeng. 1996 Jan 20;49(2):111-29 PMID: 18623562
  42. FiatFlux--a software for metabolic flux analysis from 13C-glucose experiments.
    BMC Bioinformatics. 2005 Aug 25;6:209 PMID: 16122385
  43. High-throughput phenomics: experimental methods for mapping fluxomes.
    Curr Opin Biotechnol. 2004 Feb;15(1):58-63 PMID: 15102468
  44. Kinetic flux profiling of nitrogen assimilation in Escherichia coli.
    Nat Chem Biol. 2006 Oct;2(10):529-30 PMID: 16936719
  45. Determination of metabolic flux ratios from 13C-experiments and gas chromatography-mass spectrometry data: protocol and principles.
    Methods Mol Biol. 2007;358:177-97 PMID: 17035687
  46. Experimental identification and quantification of glucose metabolism in seven bacterial species.
    J Bacteriol. 2005 Mar;187(5):1581-90 PMID: 15716428
  47. Cumulative bondomers: a new concept in flux analysis from 2D [13C,1H] COSY NMR data.
    Biotechnol Bioeng. 2002 Dec 30;80(7):731-45 PMID: 12402319
  48. Metabolic networks in motion: 13C-based flux analysis.
    Mol Syst Biol. 2006;2:62 PMID: 17102807
  49. Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts.
    FEMS Yeast Res. 2005 Apr;5(6-7):545-58 PMID: 15780654
  50. Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism.
    Eur J Biochem. 1995 Sep 1;232(2):433-48 PMID: 7556192
  51. In silico atomic tracing by substrate-product relationships in Escherichia coli intermediary metabolism.
    Genome Res. 2003 Nov;13(11):2455-66 PMID: 14559781
  52. Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis.
    Biotechnol Bioeng. 2001 Sep;76(2):144-56 PMID: 11505384
  53. In vivo measurement of fluxes through metabolic pathways: the missing link in functional genomics and pharmaceutical research.
    Annu Rev Nutr. 2003;23:379-402 PMID: 12704218
  54. 13C metabolic flux analysis.
    Metab Eng. 2001 Jul;3(3):195-206 PMID: 11461141
  55. Latent pathway activation and increased pathway capacity enable Escherichia coli adaptation to loss of key metabolic enzymes.
    J Biol Chem. 2006 Mar 24;281(12):8024-33 PMID: 16319065
  56. In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum.
    J Biol Chem. 2000 Nov 17;275(46):35932-41 PMID: 10946002
  57. Metabolic flux analysis at ultra short time scale: isotopically non-stationary 13C labeling experiments.
    J Biotechnol. 2007 Apr 30;129(2):249-67 PMID: 17207877
  58. Photosynthetic Fractionation of the Stable Isotopes of Oxygen and Carbon.
    Plant Physiol. 1993 Jan;101(1):37-47 PMID: 12231663
  59. Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts.
    J Bacteriol. 2003 Dec;185(24):7053-67 PMID: 14645264
  60. Bidirectional reaction steps in metabolic networks: III. Explicit solution and analysis of isotopomer labeling systems.
    Biotechnol Bioeng. 1999;66(2):69-85 PMID: 10567066
  61. Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis.
    Biotechnol Bioeng. 1997 Jul 5;55(1):118-35 PMID: 18636450
  62. GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing.
    Biotechnol Prog. 2000 Jul-Aug;16(4):642-9 PMID: 10933840
  63. Metabolic fluxes and metabolic engineering.
    Metab Eng. 1999 Jan;1(1):1-11 PMID: 10935750
  64. 13C-NMR, MS and metabolic flux balancing in biotechnology research.
    Q Rev Biophys. 1998 Feb;31(1):41-106 PMID: 9717198
  65. Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices.
    Biotechnol Bioeng. 1997 Sep 20;55(6):831-40 PMID: 18636594
  66. Metabolic flux responses to pyruvate kinase knockout in Escherichia coli.
    J Bacteriol. 2002 Jan;184(1):152-64 PMID: 11741855
  67. Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived C-labelling data.
    FEMS Yeast Res. 2007 Mar;7(2):216-31 PMID: 17132142
Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2009.58
Published
2009-00-00
Epub
2009-00-21
Pages
878-92
Language
English
Region
England
NLM ID
101284307
Subset
IM
External Links
PubMed source DOI: 10.1038/nprot.2009.58 View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2