title: 15N-NMR-Based Approach for Amino Acids-Based13C-Metabolic Flux Analysis of Metabolism.
abstract: NMR analysis of the isotope incorporation in amino acids can be used to derive information about the topology and operation of cellular metabolism. Although traditionally performed by1H and/or13C NMR, we present here novel experiments that exploit the15N nucleus to derive the same information with increased efficiency. Combined with a novel Hα-13CO experiment, we increase the coverage of the isotopic space that can be probed by obtaining the complete distribution of isotopic species for the first two carbons of amino acids in cellular biomass hydrolysates. Our approach was evaluated using as reference material a biologically produced sample containing15N-labeled metabolites with fully predictable13C-labeling patterns. Results show excellent agreement between measured and expected isotopomer abundances for the different NMR experiments, with an accuracy and precision within 1%. We also demonstrate how these experiments can give detailed information about metabolic fluxes depending on the expression level of a critical enzyme. Hence, exploiting the15N labeling of a cellular sample accelerates subsequent analysis of the hydrolyzed biomass and increases the coverage of isotopomers that can be quantified, making it a promising tool to increase the throughput and the resolution of13C-fluxomics studies.
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077, Toulouse, France.
authors.forename: Pierre
authors.identifier: 0000-0002-8136-9963
authors.initials: P
authors.lastname: Millard
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077, Toulouse, France.
authors.forename: Edern
authors.initials: E
authors.lastname: Cahoreau
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077, Toulouse, France.
authors.forename: Maud
authors.initials: M
authors.lastname: Heuillet
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077, Toulouse, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.affiliation: LISBP, Université de Toulouse, CNRS, INRA, INSA, 31077, Toulouse, France.
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D018533:Biomass; D002250:Carbon Radioisotopes / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry*; D010427:Pentose Phosphate Pathway; D000075082:Proof of Concept Study; D015203:Reproducibility of Results
chemical_list: D000596:Amino Acids; D002250:Carbon Radioisotopes; D009587:Nitrogen Isotopes