The Sensitivity of 13C-Metabolic Flux Analysis to Biomass Composition Underscores the Need for Accurate Biomass Formulation in Escherichia coli

Quantifying intracellular fluxes through 13C-metabolic flux analysis (13C-MFA) provides a detailed view of cellular metabolism. While model formulation, including biomass synthesis reactions, is known to influence flux estimations, the degree to which biomass composition affects 13C-MFA outcomes in Escherichia coli remains debated. Here, we reconstructed a genome-scale 13C-MFA model of E. coli incorporating explicit nucleotide biosynthesis and a modular biomass formulation that separates protein, RNA, and other components. Using isotopic labeling experiments and systematic perturbations of biomass composition, we demonstrate that 13C-MFA can robustly differentiate subtle shifts in the protein/RNA ratio, with deviations as small as 2-3% producing statistically significant but biologically interpretable changes in model fit. Reaction-level analyses revealed consistent flux adjustments across amino acid and nucleotide biosynthesis, while pathways in one-carbon metabolism, particularly serine, glycine, and threonine interconversions, exhibited the strongest responses. Collectively, these results highlight the robustness and discriminative power of 13C-MFA and also demonstrate the importance of accurate biomass composition measurement in E. coli 13C-MFA, given the intrinsic sensitivity of the method to macromolecular stoichiometry.