One‐shot 13C15N‐metabolic flux analysis for simultaneous quantification of carbon and nitrogen flux

Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13 C 15 N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi‐atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA‐based 13 C 15 N‐metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system. Synopsis Bayesian 13 C 15 N‐MFA is an approach for simultaneous quantification of intracellular carbon (C) and nitrogen (N) metabolic fluxes in any living system. It is applied to Mycobacterium bovis BCG to measure the first CN flux map, highlighting glutamate as the central node for nitrogen metabolism. Bayesian 13 C 15 N‐MFA allows for simultaneous quantification of intracellular carbon and nitrogen fluxes in living systems. BMA resolved the most likely operation modes of the anaplerotic node in M. bovis BCG. Glutamate was confirmed as the central node for nitrogen metabolism in M. bovis BCG. Graphical Abstract Bayesian 13 C 15 N‐MFA is an approach for simultaneous quantification of intracellular carbon (C) and nitrogen (N) metabolic fluxes in any living system. It is applied to Mycobacterium bovis BCG to measure the first CN flux map, highlighting glutamate as the central node for nitrogen metabolism.