Fig. 1: Framework for identifying metabolic integration points using HRMAS NMR with labeled substrates and dFBA.

a, HRMAS NMR resolves live-cell anaerobic metabolism of ¹³C-labeled substrates. A defined medium containing a ¹³C-labeled substrate is inoculated with C. difficile cells in an HRMAS rotor insert. Successive ¹H- and ¹³C NMR spectra of the growing cells are acquired throughout log-phase growth to monitor metabolism of the labeled substrate. NMR spectra are processed, and peaks are assigned to metabolites using ¹H-¹³C HSQC spectra and reference data. b, NMR signal trajectories inform dFBA simulations to identify metabolic integration points. Logistic curves for metabolites are fit to integrated ¹³C-signal trajectories and scaled to estimated concentrations using information from standard solutions measured by GC or NMR. Estimated metabolite exchange fluxes are derived from the logistic curves representing multiple NMR runs to constrain dFBA simulations. Metabolic integration points are identified where dFBA solutions predict substrates to exchange electrons or functional groups. c, ¹³C NMR of ¹³C- and ¹⁵N-labeled substrates confirms dFBA predictions of nutrient flow. C. difficile cells are grown in defined media containing ¹³C- and ¹⁵N-labeled substrates under NMR acquisition. ¹⁵N flow to ¹³C backbones is measured by quantifying the relative areas of split ¹³C NMR subpeaks at the ¹³C-alanine alpha carbon.