The Sensitivity of 13C-Metabolic Flux Analysis to Biomass Composition Underscores the Need for Accurate Biomass Formulation in Escherichia coli
- 1University of Michigan
Abstract
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.
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BibTeX
@misc{cai2026sensitivity,
title = {{The Sensitivity of 13C-Metabolic Flux Analysis to Biomass Composition Underscores the Need for Accurate Biomass Formulation in Escherichia coli}},
author = {Y. Cai and M. R. Antoniewicz},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.21.760808},
url = {https://www.biorxiv.org/content/10.64898/2026.10.21.760808v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - The Sensitivity of 13C-Metabolic Flux Analysis to Biomass Composition Underscores the Need for Accurate Biomass Formulation in Escherichia coli
AU - Y. Cai
AU - M. R. Antoniewicz
PY - 2026
DA - 2026/10/21
PB - bioRxiv
DO - 10.64898/2026.10.21.760808
UR - https://www.biorxiv.org/content/10.64898/2026.10.21.760808v1
AB - 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.
ER -