Anaerobic microbial metabolism drives critical functions within global ecosystems, host–microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-resolution magic angle spinning nuclear magnetic resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems.

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