Substrate-informed metabolic engineering enables a genome-encoded seven-sugar chassis of Corynebacterium glutamicum...

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.