Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in .

species thrive in different nutritional environments and can catabolize divergent carbon substrates. These capabilities have important implications for the role of these species in natural and engineered carbon processing. However, the metabolic phenotypes enabling to utilize mixed substrates remain poorly understood. Here, we employed a multi-omics approach involving stable isotope tracers, metabolomics, fluxomics, and proteomics in KT2440 to investigate the constitutive metabolic network that achieves co-utilization of glucose and benzoate, respectively a monomer of carbohydrate polymers and a derivative of lignin monomers. Despite nearly equal consumption of both substrates, metabolite isotopologues revealed nonuniform assimilation throughout the metabolic network. Gluconeogenic flux of benzoate-derived carbons from the tricarboxylic acid cycle did not reach the upper Embden-Meyerhof-Parnas pathway nor the pentose-phosphate pathway. These latter two pathways were populated exclusively by glucose-derived carbons through a cyclic connection with the Entner-Doudoroff pathway. We integrated the C-metabolomics data with physiological parameters for quantitative flux analysis, demonstrating that the metabolic segregation of the substrate carbons optimally sustained biosynthetic flux demands and redox balance. Changes in protein abundance partially predicted the metabolic flux changes in cells grown on the glucose:benzoate mixture on glucose alone. Notably, flux magnitude and directionality were also maintained by metabolite levels and regulation of phosphorylation of key metabolic enzymes. These findings provide new insights into the metabolic architecture that affords adaptability of to divergent carbon substrates and highlight regulatory points at different metabolic nodes that may underlie the high nutritional flexibility of species.