13C flux ratio analysis with FRAPPPE reveals differences in metabolic fluxes between gut Bacteroidota and Escherichia coli

Gut bacteria shape the metabolism of their host and play an important role in human health. However, systems biology approaches to study their intracellular metabolic fluxes are largely underdeveloped. We present an experimental and computational workflow to quantify metabolic flux ratios in gut bacteria using 13C-labeled nutrient supplementation and a newly developed machine learning-based Flux Ratio Prediction Python PackagE (FRAPPPE). We apply FRAPPPE to investigate central carbon metabolism in two prevalent gut Bacteroidota, Bacteroides uniformis and Phocaeicola vulgatus, in comparison to Escherichia coli. FRAPPPE revealed altered tricarboxylic acid cycle bifurcation in Bacteroidota compared to E. coli under anaerobic conditions. Further, we used FRAPPPE to investigate co-metabolism of nucleosides and carbohydrates by B. uniformis and P. vulgatus. We found distinct species-specific patterns in how nucleosides affected growth and were utilized depending on the co-fed compound. We quantified co-metabolism and showed that the tested nucleosides were mainly contributing to anabolic metabolism closely related to the specific co-fed nucleoside. Together, these findings provide insights into central and nucleoside metabolism of two gut Bacteroidota, and showcase FRAPPPE as a generalizable workflow to investigate metabolic fluxes in gut bacteria.