Substrate-informed metabolic engineering enables a genome-encoded seven-sugar chassis of Corynebacterium glutamicum for spent sulfite liquor valorization.
Efficient microbial conversion of heterogeneous carbohydrate mixtures remains a central bottleneck in industrial biotechnology, yet scalable chassis architectures capable of coordinated multi-sugar utilization are limited. Recently, substrate-informed metabolic alignment enabled balanced glucose-xylose co-utilization in Corynebacterium glutamicum. Here, we examine whether this alignment principle is scalable and transferable to complex industrial feedstocks. We progressively expanded the substrate spectrum of C. glutamicum through genome-encoded integration of mannose, xylose, arabinose, galactose, and rhamnose utilization modules, establishing a stable seven-sugar chassis. Rather than relying on adaptive evolution or high-copy expression, pathway capacities were quantitatively aligned with native uptake and central metabolism. Progressive expansion increased total volumetric carbon uptake and attenuated substrate hierarchy without compromising transcriptional stability. Complementary 13C tracer experiments showed that carbon from all seven sugars was incorporated into biomass in proportion to substrate utilization while revealing characteristic precursor-level incorporation patterns consistent with their distinct metabolic entry routes. Using authentic spent sulfite liquor (SSL) as a chemically heterogeneous validation substrate, the engineered strain achieved near-complete depletion of all fermentable sugars and substantially accelerated conversion kinetics compared to the wild type. Transfer of the aligned substrate module into a glucose-optimized glutarate production strain enabled sustained fed-batch conversion of SSL to 30 g L-1 glutarate without pathway erosion or performance decline. Together, these results demonstrate that substrate-informed metabolic engineering can provide a scalable and industrially transferable strategy for chassis development and position C. glutamicum as a robust multi-substrate platform for heterogeneous renewable feedstocks.