Pseudomonas species are widely acknowledged to degrade diverse carbon substrates, including lignocellulose derivatives. In the first research chapter, we conducted a multi-omics investigation of P. putida mt-2, a soil bacterium, to elucidate the metabolic network directing lignocellulose derivatives to carbon dioxide (CO2)-producing and biomass-synthesizing reactions. Carbon use efficiency (CUE) describes the relative investment of processed organic matter to biomass versus CO2. Microbial metabolic strategies were proposed to dictate differences in substrate-specific CUEs. Using 13C-metabolomics with P. putida mt-2, we observed co-assimilation of cellulose-derived sugar with lignin aromatics. However, metabolic flux analysis determined a two-fold lower investment of the sugar towards CO2 due to metabolic allocation of sugar-derived carbons in glycolytic pathways and aromatic substrate-derived carbons in the tricarboxylic acid cycle. This non-uniform metabolic routing resulted in nearly two-fold higher sugar investment to biomass production than the aromatic compound. Therefore, inherent segregation of metabolic fluxes may underlie disproportionate CUE during microbial conversion of divergent carbon substrates. Due to their versatile metabolic potentials, Pseudomonads are employed as cellular factories for substrate conversion to valuable products. Leveraging these capabilities requires understanding what controls nutritional adaptation. Conversion of lignocellulose derivatives relies on glycolytic and gluconeogenic fluxes for the metabolism of sugars and phenolic acids, respectively. Each pathway configuration was hypothesized to be driven thermodynamically by substrate carbon influx, presumably facilitated by substrate-dependent expression of catabolic enzymes. In the second research chapter, we tested this hypothesis using a multi-omics analysis of P. putida KT2440, a biotechnologically relevant strain, subjected to switches between glucose and ferulate. Indeed, proteins for ferulate uptake and the aromatic cleavage pathway were detected only during growth on ferulate, while several proteins for initial glucose catabolism were only found or elevated during growth on glucose. However, kinetic 13C-metabolomics data revealed rapid uptake of the introduced carbon source after the substrate switch. Metabolome and proteome profiling highlighted a metabolite-level regulation for the flux directionality of the assimilated substrate, but a protein-level gatekeeping regulation hinders rapid adaptation to instantaneous shifts in metabolic regimes. Collectively, the findings from my thesis advance new metabolic considerations for interpreting dynamics in microbial carbon cycling and strategies for rational pathway engineering efforts.