¹⁵N‑NMR-Based Approach for Amino Acids-Based ¹³C‑Metabolic Flux Analysis of Metabolism

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 by ¹H and/or ¹³C NMR, we present here novel experiments that exploit the ¹⁵N 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 containing ¹⁵N-labeled metabolites with fully predictable ¹³C-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 the ¹⁵N 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 of ¹³C-fluxomics studies