title: Sampling of intracellular metabolites for stationary and non-stationary (13)C metabolic flux analysis in Escherichia coli.
abstract: The analysis of metabolic intermediates is a rich source of isotopic information for (13)C metabolic flux analysis ((13)C-MFA) and extends the range of its applications. The sampling of labeled metabolic intermediates is particularly important to obtain reliable isotopic information. The assessment of the different sampling procedures commonly used to generate such data, therefore, is crucial. In this work, we thoroughly evaluated several sampling procedures for stationary and non-stationary (13)C-MFA using Escherichia coli. We first analyzed the efficiency of these procedures for quenching metabolism and found that procedures based on cold or boiling solvents are reliable, in contrast to fast filtration, which is not. We also showed that separating the cells from the broth is not necessary in isotopic stationary state conditions. On the other hand, we demonstrated that the presence of metabolic intermediates outside the cells strongly affects the transient isotopic data monitored during non-stationary (13)C-labeling experiments. Meaningful isotopic data can be obtained by recovering intracellular labeled metabolites from pellets of cells centrifuged in cold solvent. We showed that if the intracellular pools are not separated from the extracellular ones, accurate flux maps can be established provided that the contribution of exogenous compounds is taken into account in the metabolic flux model.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR 5504, F-31400 Toulouse, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Millard
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR 5504, F-31400 Toulouse, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Massou
authors.affiliation: Universität des Saarlande, Systembiotechnologie Campus, D-66123 Saarbrücken, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Wittmann
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR 5504, F-31400 Toulouse, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR 5504, F-31400 Toulouse, France. Electronic address: fabien.letisse@insa-toulouse.fr.
authors.forename: Fabien
authors.initials: F
authors.lastname: Létisse
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D048168:Escherichia coli K12 / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D008954:Models, Biological
chemical_list: D002247:Carbon Isotopes
keywords: (13)C-labeling experiments; Isotopes; Isotopologue; Mass isotopomer; Metabolism; Sampling procedures