A Method to Constrain Genome-Scale Models with 13C Labeling Data

Current limitations in quantitatively predicting biological behavior hinder our efforts to engineer biological systems to produce biofuels and other desired chemicals. Here, we present a new method for calculating metabolic fluxes, key targets in metabolic engineering, that incorporates data from 13C labeling experiments and genome-scale models. The data from 13C labeling experiments provide strong flux constraints that eliminate the need to assume an evolutionary optimization principle such as the growth rate optimization assumption used in Flux Balance Analysis (FBA). This effective constraining is achieved by making the simple but biologically relevant assumption that flux flows from core to peripheral metabolism and does not flow back. The new method is significantly more robust than FBA with respect to errors in genome-scale model reconstruction. Furthermore, it can provide a comprehensive picture of metabolite balancing and predictions for unmeasured extracellular fluxes as constrained by 13C labeling data. A comparison shows that the results of this new method are similar to those found through 13C Metabolic Flux Analysis (13C MFA) for central carbon metabolism but, additionally, it provides flux estimates for peripheral metabolism. The extra validation gained by matching 48 relative labeling measurements is used to identify where and why several existing COnstraint Based Reconstruction and Analysis (COBRA) flux prediction algorithms fail. We demonstrate how to use this knowledge to refine these methods and improve their predictive capabilities. This method provides a reliable base upon which to improve the design of biological systems.Author Summary: While metabolic fluxes constitute the most direct window into a cell’s metabolism, their accurate measurement is non trivial. The gold standard for flux measurement involves providing a labeled feed where some of the carbon atoms have been substituted by isotopes with higher atomic mass (13C instead of 12C). The ensuing labeling found in intracellular metabolites is then used to computationally infer the metabolic fluxes that produced the observed pattern. However, this procedure is typically performed with small metabolic models encompassing only central carbon metabolism. The genomic revolution has afforded us easily available genomes and, with them, comprehensive genome-scale models of cellular metabolism. It would be desirable to use the 13C labeling experimental data to constrain genome-scale models: these data constrain fluxes very effectively and provide in the labeling data fit an obvious proof that the underlying model correctly explains measured quantities. Here, we introduce a rigorous, self-consistent method that uses the full amount of information contained in 13C labeling data to constrain fluxes for a genome-scale model where underlying assumptions are explicitly stated.