Isotopic Steady-State Flux Analysis

  • Jörg Schwender

Metabolic flux analysis (MFA) provides an integrated view of the function of biochemical pathways within a cell and is an important methodology in systems biology and metabolic engineering [102]. Originally developed to study microbial metabolism, it has also been applied to plants [67, 74, 83, 85, 88, 106]. A key concept in MFA is the biochemical reaction stoichiometry, which is used to mathematically describe a cellular reaction network at metabolic steady state. For example, the theoretical capability of metabolic network can be explored by exhaustive enumeration of all possible distinct routes in a metabolic network [96, 101; see also Chapter 8].


Metabolic Network Fatty Acid Synthesis Flux Balance Analysis Flux Analysis Central Metabolism 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Agrawal PK, Canvin DT (1970) Contribution of pentose phosphate pathway in developing castor bean endosperm. Can J Bot 49:267–272.Google Scholar
  2. 2.
    Allen DK, Shachar-Hill Y, Ohlrogge JB (2007) Compartment-specific labeling information in 13C metabolic flux analysis of plants. Phytochemistry 68:2197–2210.PubMedGoogle Scholar
  3. 3.
    Alonso AP, Vigeolas H, Raymond P, Rolin D, Dieuaide-Noubhani M (2005) A new substrate cycle in plants. Evidence for a high glucosephosphate glucosephosphate-to-glucose turnover from in vivo steady-state and pulse labeling experiments with [13C]glucose and [14C]glucose. Plant Physiol 138:2220–2232.PubMedGoogle Scholar
  4. 4.
    Alonso AP, Raymond P, Rolin D, Dieuaide-Noubhani M (2007) Substrate cycles in the central metabolism of maize root tips under hypoxia. Phytochemistry 68:2222–2231.PubMedGoogle Scholar
  5. 5.
    Alonso AP, Goffman FD, Ohlrogge JB, Shachar-Hill Y (2007) Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos. Plant J 52:296–308.PubMedGoogle Scholar
  6. 6.
    Alonso AP, Raymond P, Hernould M, Rondeau-Mouro C, de Graaf A, Chourey P, Lahaye M, Shachar-Hill Y, Rolin D, Dieuaide-Noubhani M (2007) A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips. Metab Eng 9:419–432.PubMedGoogle Scholar
  7. 7.
    Antoniewicz MR, Stephanopoulos G, Kelleher JK (2006) Evaluation of regression models in metabolic physiology: predicting fluxes from isotopic data without knowledge of the pathway. Metabolomics 2:41–52.PubMedGoogle Scholar
  8. 8.
    Antoniewicz MR, Kelleher JK, Stephanopoulos G (2006) Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab Eng 8: 324–337.PubMedGoogle Scholar
  9. 9.
    Antoniewicz MR, Kelleher JK, Stephanopoulos G. (2007) Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. Metab Eng 9:68–86.PubMedGoogle Scholar
  10. 10.
    Antoniewicz MR, Kraynie DF, Laffend LA, González-Lergier J, Kelleher JK, Stephanopoulos G (2007) Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol. Metab Eng 9:277–292.PubMedGoogle Scholar
  11. 11.
    Ap Rees T, Beevers H (1960) Pathways of Glucose Dissimilation in Carrot Slices. Plant Physiol 35:830–838.Google Scholar
  12. 12.
    Ap Rees T, Beevers H (1960) Pentose phosphate pathway as a major component of induced respiration of carrot and potato slices. Plant Physiol 35:839–847.Google Scholar
  13. 13.
    Araúzo-Bravo MJ, Shimizu K (2003) An improved method for statistical analysis of metabolic flux analysis using isotopomer mapping matrices with analytical expressions. J Biotechnol 105:117–133.PubMedGoogle Scholar
  14. 14.
    Averill RH, Bailey-Serres J, Kruger NJ (1998) Co-operation between cytosolic and plastidic oxidative pentose phosphate pathways revealed by 6-phosphogluconate dehydrogenase-deficient genotypes of maize. Plant J 14:449–457.Google Scholar
  15. 15.
    Bailey JE (1999) Lessons learned from metabolic engineering for finctional genomics and drug discovery. Nature Biotechnol 17: 616–918.Google Scholar
  16. 16.
    Bao XM, Focke M, Pollard M, Ohlrogge J (2000) Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue. Plant J 22:39–50.PubMedGoogle Scholar
  17. 17.
    Beevers H., Gibbs M (1954) The direct oxidation pathway in plant respiration. Plant Physiol 29:322–324.PubMedGoogle Scholar
  18. 18.
    Beckles DM, Smith AM, ap Rees T (2001) A cytosolic ADP-glucose pyrophosphorylase is a feature of graminaceous endosperms, but not of other starch-storing organs. Plant Physiol 125:818–827.PubMedGoogle Scholar
  19. 19.
    Blank LM, Kuepfer L, Sauer U (2005) Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast. Genome Biol 6: R49.PubMedGoogle Scholar
  20. 20.
    Boatright J, Negre F, Chen X, Kish CM, Wood B, Peel G, Orlova I, Gang D, Rhodes D, Dudareva N (2004) Understanding in vivo benzenoid metabolism in petunia petal tissue. Plant Physiol 135:1993–2011.PubMedGoogle Scholar
  21. 21.
    Bonarius HPJ, Schmid G, Tramper J (1997) Flux analysis of underdetermined metabolic networks: the quest for the missing constraints. Trends Biotechnol 15:308–314.Google Scholar
  22. 22.
    Calvin M, Benson AA (1948) The path of carbon in photosynthesis. Science 107:476–480.PubMedGoogle Scholar
  23. 23.
    Calvin M (1962) The path of carbon in photosynthesis. Science 135:879–889.PubMedGoogle Scholar
  24. 24.
    Canvin DT, Beevers H (1961) Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway. J Biol Chem 236: 988–995.PubMedGoogle Scholar
  25. 25.
    Christensen B, Nielsen J. (2002) Reciprocal 13C-labeling: a method for investigating the catabolism of cosubstrates. Biotechnol Prog. 18:163–166.PubMedGoogle Scholar
  26. 26.
    Costenoble R, Muller D, Barl T, van Gulik WM, van Winden WA, Reuss M, Heijnen JJ (2007) 13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiae. FEMS Yeast Res 7:511–526.PubMedGoogle Scholar
  27. 27.
    Dasilva PMFR, Eastmond PJ, Hill LM, Smith AM, Rawsthorne S (1997) Starch metabolism in developing embryos of oilseed rape. Planta 203:480–487.Google Scholar
  28. 28.
    Dauner M, Bailey JE, Sauer U. (2001) Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol Bioeng 76:144–156.PubMedGoogle Scholar
  29. 29.
    Dennis DT (1989) Fatty acid biosynthesis in plastids. In: Physiology, Biochemistry, and Genetics of Nongreen Plastids (Boyer, C. D., Shannon, R. C., and Hardison, R. C., eds) pp. 120–129, American Society of Plant Physiologists, Rockville, MD.Google Scholar
  30. 30.
    Dieuaide-Noubhani M, Raffard G, Canioni P, Pradet A, Raymond P (1995) Quantification of compartmented metabolic fluxes in maize root- tips using isotope distribution from C-13-labeled or C-14-labeled glucose. J Biol Chem 270:13147–13159.PubMedGoogle Scholar
  31. 31.
    Dixon RA, Chen F, Guo D, Parvathi K (2001) The biosynthesis of monolignols: a “metabolic grid”, or independent pathways to guaiacyl and syringyl units? Phytochemistry 57:1069–1084.PubMedGoogle Scholar
  32. 32.
    Domergue F, Cassagne C, Lessire R (1999) Seed acyl-CoA elongases: the other system of fatty acid synthesis. Ocl-Ol. Corps Gras Lipides 6:101–106.Google Scholar
  33. 33.
    Douce R, Neuberger M (1999) Biochemical dissection of photorespiration. Curr Opin Plant Biol 2:214–222.PubMedGoogle Scholar
  34. 34.
    Edwards S, Nguyen BT, Do B, Roberts JKM (1998) Contribution of malic enzyme, pyruvate kinase, phosphoenolpyruvate carboxylase, and the krebs cycle to respiration and biosynthesis and to intracellular pH regulation during hypoxia in maize root tips observed by nuclear magnetic resonance imaging and gas chromatography-mass spectrometry. Plant Physiol 116:1073–1081.PubMedGoogle Scholar
  35. 35.
    Edwards JS, Covert M, Palsson B (2002) Metabolic modelling of microbes: the flux-balance approach. Environ Microbiol 4:133–140.PubMedGoogle Scholar
  36. 36.
    Emmerling M, Dauner M, Ponti A, Fiaux J, Hochuli M, Szyperski T, Wüthrich K, Bailey JE, Sauer U (2002) Metabolic flux responses to pyruvate kinase knockout in Escherichia coli. J Bacteriol 184:152–164.PubMedGoogle Scholar
  37. 37.
    Ettenhuber C, Spielbauer G, Margl L, Hannah LC, Gierl A, Bacher A, Genschel U, Eisenreich W (2005) Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize. Phytochemistry 66:2632–2642.PubMedGoogle Scholar
  38. 38.
    Fait A, Fromm H, Walter D, Galili G, Fernie AR (2008) Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci 13:14–19.PubMedGoogle Scholar
  39. 39.
    Fatland BL, Nikolau BJ, Wurtele ES (2005) Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis. Plant Cell 17:182–203.PubMedGoogle Scholar
  40. 40.
    Fernie AR, Roscher A, Ratcliffe RG, Kruger NJ (2001) Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells. Planta 212:250–263.PubMedGoogle Scholar
  41. 41.
    Fischer E, Sauer U. (2003) Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem 270:880–891.PubMedGoogle Scholar
  42. 42.
    Fischer E, Zamboni N, Sauer U (2004) High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. Anal Biochem 325: 308–316.PubMedGoogle Scholar
  43. 43.
    Forbes NS, Clark DS, Blanch HW (2001) Using isotopomer path tracing to quantify metabolic fluxes in pathway models containing reversible reactions. Biotechnol Bioeng 74:196–211.PubMedGoogle Scholar
  44. 44.
    Geigenberger P, Stitt M (1991). A futile cycle of sucrose synthesis and degradation is involved in regulating partitioning between sucrose, starch and respiration in cotyledons of germinating Ricinus communis L seedlings when phloem transport is inhibited. Planta 185:81–90.Google Scholar
  45. 45.
    Goffman FD, Ruckle M, Ohlrogge JB, Shachar-Hill Y (2004) Carbon dioxide concentrations are very high in developing oil seeds. Plant Physiol Biochem 42:703–708.PubMedGoogle Scholar
  46. 46.
    Graham JW, Williams TC, Morgan M, Fernie AR, Ratcliffe RG, Sweetlove LJ (2007) Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling. Plant Cell 19:3723–3738.PubMedGoogle Scholar
  47. 47.
    Hatzfeld WD, Stitt M (1990) A study of the rate of recycling of triose phosphates in heterotrophic Chenopodium rubrum cells, potato tubers, and maize endosperm. Planta 180:198–204.Google Scholar
  48. 48.
    Heinrich R, Schuster S (1996) The Regulation of Cellular Systems. Chapman & Hall, New York.Google Scholar
  49. 49.
    Heinzle E, Matsuda F, Miyagawa H, Wakasa K, Nishioka T (2007) Estimation of metabolic fluxes, expression levels and metabolite dynamics of a secondary metabolic pathway in potato using label pulse-feeding experiments combined with kinetic network modelling and simulation. Plant J 50:176–187.PubMedGoogle Scholar
  50. 50.
    Herrmann KM, Weaver LM (1999) The shikimate pathway. Annu Rev Plant Physiol Plant Mol Biol 50:473–503.PubMedGoogle Scholar
  51. 51.
    Ho CL, Noij M, Saito K (1999) Plastidic pathway of serine biosynthesis. J Biol Chem 274:11007–11012.PubMedGoogle Scholar
  52. 52.
    Hoppe A, Hoffmann S, Holzhütter HG (2007) Including metabolite concentrations into flux balance analysis: thermodynamic realizability as a constraint on flux distributions in metabolic networks. BMC Syst Biol 1:23.PubMedGoogle Scholar
  53. 53.
    Huege J, Sulpice R, Gibon Y, Lisec J, Koehl K, Kopka J (2007) GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling. Phytochemistry 68:2258–2272.PubMedGoogle Scholar
  54. 54.
    Iyer VV, Sriram G, Fulton DB, Zhou R, Westgate ME, Shanks JV (2008) Metabolic flux maps comparing the effect of temperature on protein and oil biosynthesis in developing soybean cotyledons. Plant Cell Environ 31:506–517.PubMedGoogle Scholar
  55. 55.
    Junker BH, Lonien J, Heady LE, Rogers A, Schwender J (2007) Parallel determination of enzyme activities and in vivo fluxes in Brassica napus embryos grown on organic or inorganic nitrogen source. Phytochemistry 68:2232–2242.PubMedGoogle Scholar
  56. 56.
    Keeling PL, Wood JR, Tyson RH, Bridges IG (1988) Starch biosynthesis in developing wheat grain. Evidence against the direct involvement of triose phosphates in the metabolic pathway. Plant Physiol 87:311–319.PubMedGoogle Scholar
  57. 57.
    Kiefer P, Nicolas C, Letisse F, Portais JC (2007) Determination of carbon labeling distribution of intracellular metabolites from single fragment ions by ion chromatography tandem mass spectrometry. Anal Biochem 360:182–188.PubMedGoogle Scholar
  58. 58.
    Kim HU, Kim TY, Lee SY (2008) Metabolic flux analysis and metabolic engineering of microorganisms. Mol BioSyst 4:113–120.PubMedGoogle Scholar
  59. 59.
    King SP, Badger MR, Furbank RT (1998) CO2 refixation characteristics of developing canola seeds and silique wall. Aust J Plant Physiol 25:377–386.Google Scholar
  60. 60.
    Kleijn RJ, van Winden WA, van Gulik WM, Heijnen JJ (2005) Revisiting the 13C-label distribution of the non-oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence. FEBS J 272:4970–4982.PubMedGoogle Scholar
  61. 61.
    Kleijn RJ, Geertman JM, Nfor BK, Ras C, Schipper D, Pronk JT, Heijnen JJ, van Maris AJ, van Winden WA (2007) 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 7:216–231.PubMedGoogle Scholar
  62. 62.
    Krook J, Vreugdenhill D, Dijkema C, van der Plas LHW (1998) Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13C-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway. J Exp Bot 49:1917–1924.Google Scholar
  63. 63.
    Kruger NJ, Huddleston JE, Le Lay P, Brown ND, Ratcliffe RG (2007) Network flux analysis: impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures. Phytochemistry 68:2176–2188.PubMedGoogle Scholar
  64. 64.
    Kruger NJ, Le Lay P, Ratcliffe RG (2007) Vacuolar compartmentation complicates the steady-state analysis of glucose metabolism and forces reappraisal of sucrose cycling in plants. Phytochemistry 68: 2189–2196.PubMedGoogle Scholar
  65. 65.
    Kümmel A, Panke S, Heinemann M (2006) Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data. Mol Syst Biol 2:2006.0034.PubMedGoogle Scholar
  66. 66.
    Lichtenthaler HK, Schwender J, Disch A, Rohmer M (1997) Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate independent pathway. FEBS Lett 400:271–274.PubMedGoogle Scholar
  67. 67.
    Libourel IGL, Gehan JP, Shachar-Hill Y (2007) Design of substrate label for steady state flux measurements in plant systems using the metabolic network of Brassica napus embryos. Phytochemistry 68:2211–2221.PubMedGoogle Scholar
  68. 68.
    Ma H, Zeng AP (2003) Reconstruction of metabolic networks from genome data and analysis of their global structure for various organisms. Bioinformatics 19:270–277.PubMedGoogle Scholar
  69. 69.
    Malloy CR, Sherry AD, Jeffrey FMH. (1988) Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy. J Biol Chem 263:6964–6971.PubMedGoogle Scholar
  70. 70.
    Matsuda F, Wakasa K, Miyagawa H (2007) Metabolic flux analysis in plants using dynamic labeling technique: Application to tryptophan biosynthesis in cultured rice cells. Phytochemistry 68:2290–2301.PubMedGoogle Scholar
  71. 71.
    McClung CR, Hsu M, Painter JE, Gagne JM, Karlsberg SD, Salome PA (2000) Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase. Plant Physiol 123:381–392.PubMedGoogle Scholar
  72. 72.
    McNeil SD, Rhodes D, Russell BL, Nuccio ML, Shachar-Hill Y, Hanson AD (2000) Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco. Plant Physiol 124:153–162.PubMedGoogle Scholar
  73. 73.
    Möllney M, Wiechert W, Kownatzki D, de Graaf AA (1999) Bidirectional Reaction Steps in Metabolic Networks: IV. Optimal Design of Isotopomer Labeling Experiments. Biotechnol Bioeng 66:86–103.PubMedGoogle Scholar
  74. 74.
    Morgan JA, Rhodes D (2002) Mathematical modeling of plant metabolism. Metabol. Eng. 4:80–89.Google Scholar
  75. 75.
    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 129:249–267.PubMedGoogle Scholar
  76. 76.
    Ohta D, Fujimori K, Mizutani M, Nakayama Y, Kunpaisal-Hashimoto R, Münzer S, Kozaki A (2000) Molecular cloning and characterization of ATP-phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway. Plant Physiol 122: 907–914.PubMedGoogle Scholar
  77. 77.
    Orlova I, Marshall-Colón A, Schnepp J, Wood B, Varbanova M, Fridman E, Blakeslee JJ, Peer WA, Murphy AS, Rhodes D, Pichersky E, Dudareva N (2006) Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport. Plant Cell 18:3458–3475.PubMedGoogle Scholar
  78. 78.
    Ohlrogge JB, Kuhn DN, Stumpf PK (1979) Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci USA 76:1194–1198.PubMedGoogle Scholar
  79. 79.
    Plaxton WC, Podesta FE (2006) The functional organization and control of plant metabolism. Crit Rev Plant Sci 25:159–198.Google Scholar
  80. 80.
    Pleite R, Pike MJ, Garces R, Martinez-Force E, Rawsthorne S (2005) The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos. J Exp Bot 56:1297–1303.PubMedGoogle Scholar
  81. 81.
    Rantanen A, Rousu J, Kokkonen JT, Tarkiainen V, Ketola RA (2002) Computing positional isotopomer distributions from tandem mass spectrometric data. Met Eng 4:285–94.Google Scholar
  82. 82.
    Rantanen A, Rousu J, Jouhten P, Zamboni N, Maaheimo H, Ukkonen E (2008) An analytic and systematic framework for estimating metabolic flux ratios from 13C tracer experiments. BMC Bioinformatics 9:266.PubMedGoogle Scholar
  83. 83.
    Ratcliffe RG, Shachar-Hill, Y (2006) Measuring multiple fluxes through plant metabolic networks. Plant J 45:490–511.PubMedGoogle Scholar
  84. 84.
    Rawsthorne S (2002) Carbon flux and fatty acid synthesis in plants. Progr Lipid Res 41: 182–196.Google Scholar
  85. 85.
    Rios-Estepa R, Lange BM: Experimental and mathematical approaches to modeling plant metabolic networks. Phytochemistry 68:2351–2374.Google Scholar
  86. 86.
    Romisch-Margl W, Schramek N, Radykewicz T, Ettenhuber C, Eylert E, Huber C, Romisch-Margl L, Schwarz C, Dobner M, Demmel N, Winzenhorlein B, Bacher A, Eisenreich W (2007) 13CO2 as a universal metabolic tracer in isotopologue perturbation experiments. Phytochemistry 68:2273–2289.PubMedGoogle Scholar
  87. 87.
    Rontein D, Dieuaide-Noubhani M, Dufourc EJ, Raymond P, Rolin D (2002) The metabolic architecture of plant cells. Stability of central carbon metabolism and flexibility of anabolic pathways during the growth cycle of tomato cells. J Biol Chem 46: 43948–43960.Google Scholar
  88. 88.
    Roscher A, Kruger NJ, Ratcliffe RG (2000) Strategies for metabolic flux analysis in plants using isotope labelling. J Biotechnol 77:81–102.PubMedGoogle Scholar
  89. 89.
    Roughan PG, Ohlrogge JB (1984) On the assay of acetyl-CoA synthethase activity in chloroplasts and leaf extract. Anal Biochem 216:77–82.Google Scholar
  90. 90.
    Rousu J, Rantanen A, Ketola RA, Kokkonen JT (2005) Isotopomer distribution computation from tandem mass spectrometric data with overlapping fragment spectra. Spectroscopy 19:53–67.Google Scholar
  91. 91.
    Ruuska SA, Schwender J, Ohlrogge JB (2004) The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processes. Plant Physiol 136:2700–2709.PubMedGoogle Scholar
  92. 92.
    Salon C, Raymond P, Pradet A (1988) Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos. J Biol Chem 263:12278–12287.PubMedGoogle Scholar
  93. 93.
    Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Sys Biol 2: 62.Google Scholar
  94. 94.
    Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wuthrich K, Bailey JE (1999) Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol 181:6679–6688.PubMedGoogle Scholar
  95. 95.
    Schaefer J, Stejskal EO, Beard CF (1975) Carbon-13 nuclear magnetic resonance analysis of metabolism in soybean labeled by 13CO2. Plant Physiol 55:1048–1053.PubMedGoogle Scholar
  96. 96.
    Schilling CH, Letscher D, Palsson BO (2000) Theory for the systemic definition of metabolic pathways and their use in interpreting metabolic function from a pathway-oriented perspective. J Theor Biol 203:229–248.PubMedGoogle Scholar
  97. 97.
    Schmidt K, Carlsen M, Nielsen J, Villadsen J. (1997) Modelling isotopomer distribution in biochemical networks using isotopomer mapping matrices. Biotechnol Bioeng 55:831–840.PubMedGoogle Scholar
  98. 98.
    Schmidt K, Marx A, de Graaf AA, Wiechert W, Sahm H, Nielsen J, Villadsen J (1998) 13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches. Biotechnol Bioeng 58:254–257.PubMedGoogle Scholar
  99. 99.
    Schmidt K, Nørregaard LC, Pedersen B, Meissner A, Duus JO, Nielsen JO, Villadsen J (1999) Quantification of intracellular metabolic fluxes from fractional enrichment and 13C-13C coupling constraints on the isotopomer distribution in labeled biomass components. Metab Eng 1:166–179.PubMedGoogle Scholar
  100. 100.
    Schultz CJ, Coruzzi GM (1995) The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments. Plant J 7:61–75.PubMedGoogle Scholar
  101. 101.
    Schuster S, Dandekar T, Fell DA (1999) Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering. Trends Biotechnol 17:53–60.PubMedGoogle Scholar
  102. 102.
    Schwender J (2008) Metabolic flux analysis as a tool in metabolic engineering of plants. Curr Opin Biotechnol 19:131–137.PubMedGoogle Scholar
  103. 103.
    Schwender J, Ohlrogge JB (2002) Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos. Plant Physiol 130: 347–361.PubMedGoogle Scholar
  104. 104.
    Schwender J, Ohlrogge JB, Shachar-Hill Y (2003) A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos. J Biol Chem 278:29442–29453.PubMedGoogle Scholar
  105. 105.
    Schwender J, Goffman F, Ohlrogge JB, Shachar-Hill Y (2004) Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature 432:779–782.PubMedGoogle Scholar
  106. 106.
    Schwender J, Ohlrogge J, Shachar-Hill, Y (2004) Understanding flux in plant metabolic networks. Curr Opin Plant Biol 7:309–317.PubMedGoogle Scholar
  107. 107.
    Schwender J, Shachar-Hill Y, Ohlrogge JB (2006) Mitochondrial metabolism in developing embryos of Brassica napus. J Biol Chem 281:34040–34047.PubMedGoogle Scholar
  108. 108.
    Shastri AA, Morgan JA (2007) A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms. Phytochemistry 68:2302–2312.PubMedGoogle Scholar
  109. 109.
    Singh BK (1999) in Plant Amino Acids: Biochemistry and Biotechnology (Singh, B. K., ed.) pp. 227–247, Marcel Dekker, New York.Google Scholar
  110. 110.
    Spielbauer G, Margl L, Hannah LC, Romisch W, Ettenhuber C, Bacher A, Gierl A, Eisenreich W, Genschel U (2006) Robustness of central carbohydrate metabolism in developing maize kernels. Phytochemistry 67:1460–1475.PubMedGoogle Scholar
  111. 111.
    Sriram G, Fulton B, Iyer VV, Peterson JM, Zhou R, Westgate ME, Spalding MH, Shanks JV (2004) Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional 13C labeling, two-dimensional [13C,1H] nuclear magnetic resonance, and comprehensive isotopomer balancing. Plant Physiol 136:3043–3057.PubMedGoogle Scholar
  112. 112.
    Sriram G, Shanks JV (2004) Improvements in metabolic flux analysis using carbon bond labeling experiments: bondomer balancing and Boolean function mapping. Metab Eng 6:116–132.PubMedGoogle Scholar
  113. 113.
    Sriram G, Fulton DB, Shanks JV (2007) Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing. Phytochemistry 68:2243–2257.PubMedGoogle Scholar
  114. 114.
    Sriram G, Iyera VV, Fulton DB, Shanks JV (2007) Identification of hexose hydrolysis products in metabolic flux analytes: A case study of levulinic acid in plant protein hydrolysate. Metab Eng 9:442–451.PubMedGoogle Scholar
  115. 115.
    Stelling J, Sauer U, Szallasi Z, Doyle FJ 3rd, Doyle J (2004) Robustness of cellular functions. Cell 118:675–685.PubMedGoogle Scholar
  116. 116.
    Stephanopoulos G, Vallino JJ (1991) Network rigidity and metabolic engineering in metabolite overproduction. Science 252:1675–1681.PubMedGoogle Scholar
  117. 117.
    Stephanopoulos GN, Aristidou AA, Nielsen J (1998) Metabolic Engineering: Principles and Methodologies. Academic Press, San Diego.Google Scholar
  118. 118.
    Stitt M, ap Rees T (1980) Estimation of the activity of the oxidative pentose phosphate pathway in pea chloroplasts. Phytochemistry 19:1583–1585.Google Scholar
  119. 119.
    Studart-Guimarães C, Fait A, Nunes-Nesi A, Carrari F, Usadel B, Fernie AR (2007) Reduced expression of succinyl-coenzyme A ligase can be compensated for by up-regulation of the gamma-aminobutyrate shunt in illuminated tomato leaves. Plant Physiol 145: 626–639.PubMedGoogle Scholar
  120. 120.
    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 9:387–405.PubMedGoogle Scholar
  121. 121.
    Szyperski T (1995) Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism. Eur J Biochem 232:433–448.PubMedGoogle Scholar
  122. 122.
    Szyperski T, Glaser RW, Hochuli M, Fiaux J, Sauer U, Bailey JE, Wuthrich K (1999) Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy. Metab Eng 1:189–197.PubMedGoogle Scholar
  123. 123.
    Troufflard S, Roscher A, Thomasset B, Barbotin JN, Rawsthorne S, Portais JC (2007) In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos. Phytochemistry 68:2341–2350.PubMedGoogle Scholar
  124. 124.
    Vandewalle I, Olsson R (1983) The gamma-aminobutyric acid shunt in germinating Sinapsis alba seeds. Plant Sci Lett 31:269–273.Google Scholar
  125. 125.
    van Winden WA, Heijnen JJ, Verheijen PJ, Grievink J (2001) A priori analysis of metabolic flux identifiability from (13)C-labeling data. Biotechnol Bioeng 74:505–516.PubMedGoogle Scholar
  126. 126.
    van Winden W, Verheijen P, Heijnen S (2001) Possible pitfalls of flux calculations based on (13)C-labeling. Metab Eng 3:151–162.PubMedGoogle Scholar
  127. 127.
    Van Winden WA, Heijnen JJ, Verheijen PJT (2002) Cumulative bondomers: a new concept in flux analysis from 2D [13C, 1H] COSY NMR data. Biotechnol Bioeng 80:731–745.PubMedGoogle Scholar
  128. 128.
    van Winden WA, van Dam JC, Ras C, Kleijn RJ, Vinke JL, van Gulik WM, Heijnen JJ (2005) Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113–7D based on mass isotopomer measurements of (13)C-labeled primary metabolites. FEMS Yeast Res 5: 559–568.PubMedGoogle Scholar
  129. 129.
    Varma A, Palsson BO (1994) Metabolic flux balancing: Basic concepts, scientific, and practical use. Nature Biotechnol 12:994–998.Google Scholar
  130. 130.
    Viola R, Davies HV, Chudeck AR (1991) Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Vicia faba L.). Elucidation by 13C-nuclear-magnetic-resonance spectroscopy. Planta 183: 202–208.Google Scholar
  131. 131.
    Weitzel M, Wiechert W, Nöh K (2007) The topology of metabolic isotope labeling networks. BMC Bioinf 8:315.Google Scholar
  132. 132.
    Whitfield HV, Murphy DJ, Hills MJ (1993) Subcellular-localization of fatty-acid elongase in developing seeds of Lunaria annua and Brassica napus. Phytochemistry 32:255–258.Google Scholar
  133. 133.
    Wiechert W (2001) 13C-Metabolic flux analysis. Metabol Eng 3:195–206.Google Scholar
  134. 134.
    Wiechert W. (2002) An introduction to 13C metabolic flux analysis. Genet Eng (NY) 24:215–238.Google Scholar
  135. 135.
    Wiechert W (2007) The thermodynamic meaning of metabolic exchange fluxes. Biophys J 93:2255–2264.PubMedGoogle Scholar
  136. 136.
    Wiechert W, De Graaf AA (1997) Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments. Biotechnol Bioeng 55:101–117.PubMedGoogle Scholar
  137. 137.
    Wiechert W, Siefke C, de Graaf A, Marx A (1997) Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis. Biotechnol Bioeng 55:118–135.PubMedGoogle Scholar
  138. 138.
    Wiechert W, Möllney M, Isermann N, Wurzel M, de Graaf AA (1999) Bidirectional reaction steps in metabolic networks: III. Explicit solution and analysis of isotopomer labeling systems. Biotechnol Bioeng. 66:69–85.PubMedGoogle Scholar
  139. 139.
    Wiechert W, Mollney M, Petersen S, deGraaf AA (2001) A universal framework for 13C-Metabolic flux analysis. Metabol Eng 3:265–283.Google Scholar
  140. 140.
    Wilkie SE, Warren MJ (1998) Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana. Protein Expression Purif 12:381–389.Google Scholar
  141. 141.
    Williams TCR, Miguet L, Masakapalli SK, Kruger NJ, Sweetlove LJ, Ratcliffe RG (2008) Metabolic network fluxes in heterotrophic Arabidopsis cells: stability of the flux distribution under different oxygenation conditions. Plant Physiol, doi:10.1104/pp.108.125195.Google Scholar
  142. 142.
    Wittmann C, Heinzle E (2002) Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria. Appl Environ Microbiol 68:5843–5859.PubMedGoogle Scholar
  143. 143.
    Yang C, Hua Q, Shimizu K (2002) Metabolic flux analysis in Synechocystis using isotope distribution from C-13-labeled glucose. Metab Eng 4:202–216.PubMedGoogle Scholar
  144. 144.
    Yang C, Hua Q, Shimizu K (2002) Quantitative analysis of intracellular metabolic fluxes using GC-MS and two-dimensional NMR spectroscopy. J Bioscience Bioeng 93: 78–87.Google Scholar
  145. 145.
    Yang TH, Frick O, Heinzle E (2008) Hybrid optimization for 13C metabolic flux analysis using systems parametrized by compactification. BMC Syst Biol 2:29.PubMedGoogle Scholar
  146. 146.
    Young JD, Walther JL, Antoniewicz MR, Yoo H, Stephanopoulos G (2008) An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis. Biotechnol Bioeng 99:686–699.PubMedGoogle Scholar
  147. 147.
    Zamboni N, Sauer U (2004) Model-independent fluxome profiling from 2H and 13C experiments for metabolic variant discrimination. Genome Biol 5:R99.PubMedGoogle Scholar
  148. 148.
    Zamboni N, Sauer U (2005) Fluxome profiling in microbes. In: Metabolome Analysis: Strategies for Systems Biology (Vaidayanathan, S., Harrigan. G. G., and Goodacre, R., eds) pp. 307–322, Springer, New York.Google Scholar
  149. 149.
    Zamboni N, Fischer E, Sauer U (2005) FiatFlux – a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6:209.PubMedGoogle Scholar
  150. 150.
    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 101:101–117.PubMedGoogle Scholar

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© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Brookhaven National LaboratoryBiology DepartmentUptonUSA

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