title: Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry.
abstract: Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype. Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose. Of particular interest are methods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution. In this paper, we present data validating the use of mass spectrometry (MS) for the quantification of complex metabolic flux networks. In the context of the lysine biosynthesis flux network of Corynebacterium glutamicum (ATCC 21799) under glucose limitation in continuous culture, operating at 0.1 x h(-1) after the introduction of 50% [1-13C]glucose, we deploy a bioreaction network analysis methodology for flux determination from mass isotopomer measurements of biomass hydrolysates, while thoroughly addressing the issues of measurement accuracy, flux observability and data reconciliation. The analysis enabled the resolution of the involved anaplerotic activity of the microorganism using only one labeled substrate, the determination of the range of most of the exchange fluxes and the validation of the flux estimates through satisfaction of redundancies. Specifically, we determined that phosphoenolpyruvate carboxykinase and synthase do not carry flux at these experimental conditions and identified a high futile cycle between oxaloacetate and pyruvate, indicating a highly active in vivo oxaloacetate decarboxylase. Both results validated previous in vitro activity measurements. The flux estimates obtained passed the chi2 statistical test. This is a very important result considering that prior flux analyses of extensive metabolic networks from isotopic measurements have failed criteria of statistical consistency.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Maria I
authors.initials: MI
authors.lastname: Klapa
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Aon
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D018533:Biomass; D002247:Carbon Isotopes; D003352:Corynebacterium / Q000201:enzymology / Q000378:metabolism*; D003470:Culture Media; D004734:Energy Metabolism / Q000502:physiology; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005947:Glucose / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis* / Q000737:chemistry; D008956:Models, Chemical; D062907:Oxaloacetic Acid / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000378:metabolism; D017866:Phosphotransferases (Paired Acceptors) / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D003470:Culture Media; D062907:Oxaloacetic Acid; D019289:Pyruvic Acid; D017866:Phosphotransferases (Paired Acceptors); C022509:pyruvate, water dikinase; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D005947:Glucose; D008239:Lysine