Decoupling Central Metabolism from Catabolite Repression Enables Robust Cellulosic Sugar Co-consumption in E. coli

Lignocellulosic biomass is the most abundant and sustainable carbon source for bioproduction, but its efficient utilization is hampered by the heterogeneous mixture of sugars released upon hydrolysis. Most industrial strains consume these mixed sugars sequentially due to strong regulation and cross-inhibition, leading to complex processes and reduced carbon efficiency. To address this, we leverage a novel central metabolism that decouples central carbon flux from native regulatory feedback by employing a Gluconate-Bypass of glycolysis. We demonstrate that the Gluconate-Bypass effectively alleviates feedback regulation in E. coli, enabling co-consumption of glucose and xylose. Further strain engineering leads to the first robust co-utilization of four major lignocellulosic sugars: glucose, xylose, arabinose, and galactose. By decoupling central carbon flux from native regulatory feedback, this architecture provides a feedstock-agnostic platform that maintains high and robust consumption regardless of extreme fluctuations in sugar composition.