Engineering Orthogonal Carbon Dissimilation: A Gluconate Bypass Platform for Robust Stationary-Phase Biomanufacturing

Abstract

Two-stage bioprocesses which decouple cell growth from product synthesis are an attractive approach to biomanufacturing. However high levels of production in stationary phase cultures often suffer from a progressive decline in metabolism. We demonstrate that in E. coli pyruvate accumulation, an inevitable consequence of high-flux metabolism, acts as a major inhibitor of stationary-phase glucose uptake. We present a novel central metabolism to optimize stationary phase production, a gluconate-bypass, which circumvents this challenge by rerouting carbon flux around glycolysis. This redesign achieves two critical outcomes: first, it decouples glucose uptake from pyruvate inhibition; second, glucose oxidation intrinsically co-generates the NADPH cofactor. Validated using NADPH-dependent L-alanine as a representative model, the GBP creates a self-regulating host that achieved a record titer of 197 g/L with a 1.6-fold extension of production longevity. This work establishes the GBP as a generalizable platform for robust stationary phase biosynthesis.