NEXT-FBA: A hybrid stoichiometric/data-driven approach to improve intracellular flux predictions

Genome-scale metabolic models (GEMs) have been widely utilized to understand cellular metabolism. The application of GEMs has been advanced by computational methods that enable the prediction and analysis of intracellular metabolic states. However, the accuracy and biological relevance of these predictions often suffer from the many degrees of freedom and scarcity of available data to constrain the models adequately. Here, we introduce Neural-net EXtracellular Trained Flux Balance Analysis, (NEXT-FBA), a novel computational methodology that addresses these limitations by utilizing exometabolomic data to derive biologically relevant constraints for intracellular fluxes in GEMs. We achieve this by training artificial neural networks (ANNs) with exometabolomic data from Chinese hamster ovary (CHO) cells and correlating it with ¹³C-labeled intracellular fluxomic data. By capturing the underlying relationships between exometabolomics and cell metabolism, NEXT-FBA predicts upper and lower bounds for intracellular reaction fluxes to constrain GEMs. We demonstrate the efficacy of NEXT-FBA across several validation experiments, where it outperforms existing methods in predicting intracellular flux distributions that align closely with experimental observations. Furthermore, a case study demonstrates how NEXT-FBA can guide bioprocess optimization by identifying key metabolic shifts and refining flux predictions to yield actionable process and metabolic engineering targets. Overall, NEXT-FBA aims to improve the accuracy and biological relevance of intracellular flux predictions in metabolic modelling, with minimal input data requirements for pre-trained models.