Substrate-informed metabolic engineering of Corynebacterium glutamicum enables balanced glucose-xylose co-utilization...

Efficient co-utilization of glucose and xylose-the predominant sugars in lignocellulosic and paper-derived hydrolysates-remains a major bottleneck in microbial bioprocessing due to substrate hierarchy and carbon catabolite repression. Here, we develop a substrate-informed metabolic engineering framework in Corynebacterium glutamicum that overcomes substrate hierarchy and carbon catabolite repression, enabling a balanced, largely transcription-independent glucose-xylose co-utilization regime at the level of central carbon metabolism. This regime is tailored to the sugar composition of cardboard hydrolysate (CBH), a waste-derived third-generation feedstock. A library of 34 engineered strains was constructed by systematically varying xylAB modules, transporter identity, promoter strength, and gene dosage. Integrated physiological, enzymatic, transcriptomic, and ¹³C-tracer analyses revealed strain XYL-6A as a representative of a distinct, kinetically balanced metabolic regime in which glucose- and xylose-derived fluxes merge early at the F6P/G3P node and maintain stable proportions independent of changing substrate levels or transcriptional adjustments. This regime arises from simple kinetic coordination of a compact, redox-neutral xylose-isomerase pathway with tuned transport capacity, eliminating the need for specialized feeding strategies, extensive regulatory rewiring, or attenuation of native glucose uptake. The optimized module translated directly into an industrial L-lysine producer, enabling high lysine yields from both defined mixtures and CBH (47.3 mmol C-mol^-1). These results demonstrate how substrate-informed pathway design can exploit intrinsic network connectivity to achieve robust mixed-sugar metabolism. More broadly, they illustrate a core synthetic-biology principle: simple, well-balanced modules can generate scalable and reliable metabolic behaviors, providing a practical foundation for valorizing heterogeneous carbon feedstocks.