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

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

Abstract

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 

Notes

Acknowledgments

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

References

  1. 1.
    Zamboni N, Sauer U (2009) Novel biological insights through metabolomics and 13C-flux analysis. Curr Opin Microbiol 12:553–558PubMedCrossRefGoogle Scholar
  2. 2.
    Tang YJ, Martin HG, Myers S, Rodriguez S, Baidoo EK, Keasling JD (2009) Advances in analysis of microbial metabolic fluxes via 13C isotopic labeling. Mass Spectrom Rev 28:362–375PubMedCrossRefGoogle Scholar
  3. 3.
    Tang YJ, Pingitore F, Mukhopadhyay A, Phan R, Hazen TC, Keasling JD (2007) Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using GC-MS and FT-ICR mass spectrometry. J Bacteriol 189:940–949PubMedCrossRefGoogle Scholar
  4. 4.
    Feng X, Mouttaki H, Lin L, Huang R, Wu B, Hemme CL, He Z, Zhang B, Hicks LM, Xu J, Zhou J, Tang YJ (2009) Characterization of the central metabolic pathways in Thermoanaerobacter sp. X514 via isotopomer-assisted metabolite analysis. Appl Environ Microbiol 75(15):5001–5008PubMedCrossRefGoogle Scholar
  5. 5.
    Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) C-13-based metabolic flux analysis. Nat Protoc 4:878–892PubMedCrossRefGoogle Scholar
  6. 6.
    Tang YJ, Meadows AL, Kirby J, Keasling JD (2007) Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling. J Bacteriol 189:894–901PubMedCrossRefGoogle Scholar
  7. 7.
    Wahl SA, Dauner M, Wiechert W (2004) New tools for mass isotopomer data evaluation in 13C flux analysis: mass isotope correction, data consistency checking, and precursor relationships. Biotechnol Bioeng 85:259–268PubMedCrossRefGoogle Scholar
  8. 8.
    Tang YJ, Martin HG, Deutschbauer A, Feng X, Huang R, Llora X, Arkin A, Keasling JD (2009) Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbations. Biotechnol Prog 25:1254–1259PubMedCrossRefGoogle Scholar
  9. 9.
    Tang YJ, Yi S, Zhuang W, Zinder SH, Keasling JD, Alvarez-Cohen L (2009) Investigation of carbon metabolism in “Dehalococcoides ethenogenes” strain 195 via isotopic and transcriptomic analysis. J Bacteriol 191:5224–5231PubMedCrossRefGoogle Scholar
  10. 10.
    Pingitore F, Tang YJ, Kruppa GH, Keasling JD (2007) Analysis of amino acid isotopomers using FT-ICR MS. Anal Chem 79:2483–2490PubMedCrossRefGoogle Scholar
  11. 11.
    Tang YJ, Hwang JS, Wemmer D, Keasling JD (2007) The Shewanella oneidensis MR-1 fluxome under various oxygen conditions. Appl Environ Microbiol 73:718–729PubMedCrossRefGoogle Scholar
  12. 12.
    Tang YJ, Martin HG, Dehal PS, Deutschbauer A, Llora X, Meadows A, Arkin A, Keasling JD (2009) Metabolic flux analysis of Shewanella spp. reveals evolutionary robustness in central carbon metabolism. Biotechnol Bioeng 102:1161–1169PubMedCrossRefGoogle Scholar
  13. 13.
    Marshal J (2004) Production of secondary metabolites from acetyl Co-A precursors in bacterial and fungal hosts. PhD thesis, In Chemical engineering. University of California, BerkeleyGoogle Scholar
  14. 14.
    Garcia DE, Baidoo EE, Benke PI, Pingitore F, Tang YJ, Villa S, Keasling JD (2008) Separation and mass spectrometry in microbial metabolomics. Curr Opin Microbiol 11:233–239PubMedCrossRefGoogle Scholar
  15. 15.
    Iwatani S, Van Dien S, Shimbo K, Kubota K, Kageyama N, Iwahata D, Miyano H, Hirayama K, Usuda Y, Shimizu K, Matsui K (2007) Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS. J Biotechnol 128:93–111PubMedCrossRefGoogle Scholar
  16. 16.
    Tang YJ, Shui WQ, Myers S, Feng X, Bertozzi C, Keasling JD (2009) Isotopomer analysis of both free metabolites and proteinogenic amino acids to investigate aerobic metabolism and hypoxic response of Mycobacterium smegmatis. Biotechnol Lett 31:1233–1240PubMedCrossRefGoogle Scholar
  17. 17.
    Dauner M, Sauer U (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol Prog 16:642–649PubMedCrossRefGoogle Scholar
  18. 18.
    Antoniewicz MR, Kelleher JK, Stephanopoulos G (2007) Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis. Anal Chem 79:7554–7559PubMedCrossRefGoogle Scholar
  19. 19.
    Green ML, Karp PD (2005) Genome annotation errors in pathway databases due to semantic ambiguity in partial EC numbers. Nucleic Acids Res 33:4035–4039PubMedCrossRefGoogle Scholar
  20. 20.
    Wu B, Zhang B, Feng X, Rubens JR, Huang R, Hicks LM, Pakrasi HB, Tang YJ (2010) An alternate isoleucine biosynthesis pathway involves citramalate synthase in Cyanothece sp. ATCC 51142. Microbiology 156:596–602PubMedCrossRefGoogle Scholar
  21. 21.
    Zupte C, Stephanopoulos G (1994) Modeling of isotope distributions and intracellular fluxes in metabolic networks using atom mapping matrices. Biotechnol Prog 10:489–498CrossRefGoogle Scholar
  22. 22.
    Stephanopoulos GN, Aristidou AA, Nielsen J (1998) Metabolic engineering principles and methodologies. Academic, San DiegoGoogle Scholar
  23. 23.
    Schmidt K, Carlsen M, Nielsen J, Villadsen J (1997) Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices. Biotechnol Bioeng 55:831–840PubMedCrossRefGoogle Scholar
  24. 24.
    Varma A, Palsson BO (1994) Metabolic flux balancing: basic concepts, scientific and practical use. BioTechnology 12:994–998CrossRefGoogle Scholar
  25. 25.
    Suthers PF, Burgard AP, Dasika MS, Nowroozi F, Van Dien S, Keasling JD, Maranas CD (2007) Metabolic flux elucidation for large-scale models using 13C labeled isotopes. Metab Eng 9:387–405PubMedCrossRefGoogle Scholar
  26. 26.
    Zhao J, Shimizu K (2003) Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method. J Biotechnol 101:101–117PubMedCrossRefGoogle Scholar
  27. 27.
    Moxley JF, Jewett MC, Antoniewicz MR, Villas-Boas SG, Alper H, Wheeler RT, Tong L, Hinnebusch AG, Ideker T, Nielsen J, Stephanopoulos G (2009) Linking high resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p. Proc Natl Acad Sci U S A 106:6477–6482PubMedCrossRefGoogle Scholar
  28. 28.
    Tang YJ, Sapra R, Joyner D, Hazen TC, Myers S, Reichmuth D, Blanch H, Keasling JD (2009) Analysis of metabolic pathways and fluxes in a newly discovered thermophilic and ethanol-tolerant Geobacillus strain. Biotechnol Bioeng 102:1377–1386PubMedCrossRefGoogle Scholar
  29. 29.
    Hua Q, Joyce AR, Palsson BO, Fong SS (2007) Metabolic characterization of Escherichia coli adapted to growth on lactate. Appl Environ Microbiol 73:4639–4647PubMedCrossRefGoogle Scholar
  30. 30.
    Mahadevan R, Edwards JS, Doyle FJ (2002) Dynamic flux balance analysis of diauxic growth in Escherichia coli. Biophys J 83:1331–1340PubMedCrossRefGoogle Scholar
  31. 31.
    Ruhl M, Zamboni N, Sauer U (2009) Dynamic flux responses in riboflavin overproducing Bacillus subtilis to increasing glucose limitation in fed-batch culture. Biotechnol Bioeng 105:795–804Google Scholar
  32. 32.
    Shastri AA, Morgan JA (2007) A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms. Phytochemistry 68:2302–2312PubMedCrossRefGoogle Scholar
  33. 33.
    Nöh K, Grönke K, Luo B, Takors R, Oldiges M, Wiechert W (2007) Metabolic flux analysis at ultra short time scale: isotopically non-stationary 13C labeling experiments. J Biotechnol 129:249–267PubMedCrossRefGoogle Scholar
  34. 34.
    Bennett BD, Yuan J, Kimball EH, Rabinowitz JD (2008) Absolute quantitation of intracellular metabolite concentrations by an isotope ratiobased approach. Nat Protoc 3:1299–1311PubMedCrossRefGoogle Scholar
  35. 35.
    Malaisse WJ, Zhang Y, Jijakli H, Courtois P, Sener A (2004) Enzyme-to-enzyme channelling in the early steps of glycolysis in rat pancreatic islets. Int J Biochem Cell Biol 36:1510–1520PubMedCrossRefGoogle Scholar
  36. 36.
    Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129PubMedCrossRefGoogle Scholar
  37. 37.
    Stolyar S, Van Dien S, Hillesland KL, Pinel N, Lie TJ, Leigh JA, Stahl DA (2007) Metabolic modeling of a mutualistic microbial community. Mol Syst Biol 3:92PubMedCrossRefGoogle Scholar
  38. 38.
    Madsen EL (2008) Environmental microbiology: from genomes to biogeochemistry. Blackwell, CarltonGoogle Scholar
  39. 39.
    Sauer U (2004) High-throughput phenomics: experimental methods for mapping fluxomes. Curr Opin Biotechnol 15:58–63PubMedCrossRefGoogle Scholar
  40. 40.
    Wiechert W, Möllney M, Petersen S, de Graaf AA (2001) A universal framework for 13C metabolic flux analysis. Metab Eng 3:265–283PubMedCrossRefGoogle Scholar
  41. 41.
    Quek LE, Wittmann C, Nielsen LK, Krömer JO (2009) OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis. Microb Cell Fact 8:25PubMedCrossRefGoogle Scholar
  42. 42.
    Antoniewicz MR, Kelleher JK, Stephanopoulos G (2007) Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. Metab Eng 9:68–86PubMedCrossRefGoogle Scholar
  43. 43.
    Tang K-H, Feng X, Tang YJ, Blankenship RE (2009) Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114. PLoS One 4:e7233PubMedCrossRefGoogle Scholar
  44. 44.
    Tang K-H, Feng X, Zhuang W-Q, Alvarez-Cohen L, Blankenship RE, Tang YJ (2010) Carbon flow of Heliobacterium modesticaldum is more related to firmicutes than to the green sulfur bacteria. J Biol Chem 285:35104–35112PubMedCrossRefGoogle Scholar
  45. 45.
    Tang YJ, Chakraborty R, Martin HG, Chu J, Hazen TC, Keasling JD (2007) Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-NTA. Appl Environ Microbiol 73:3859–3864PubMedCrossRefGoogle Scholar
  46. 46.
    Feng X, Banerjee A, Berla B, Page L, Wu B, Pakrasi HB, Tang YJ (2010) Mixotrophic and photoheterotrophic metabolisms in Cyanothece sp. ATCC 51142 under continuous light. Microbiology 156:2566–2574PubMedCrossRefGoogle Scholar
  47. 47.
    Tang KH, Tang YJ, Blankenship RE (2011) Carbon metabolic pathways in phototrophic bacteria and their broader evolutionary implications. Frontier in Microbiology 2:165Google Scholar

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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