Reprogramming energy system for growth and bioproduction from CO₂ and methanol in Escherichia coli

Microbial CO₂ assimilation provides a strategy for sustainable biomanufacturing. However, CO₂ reduction requires substantial energy input, while native energy systems are tightly regulated and extensively consumed by endogenous metabolism, limiting assimilation efficiency. Here, we design and construct an orthogonal energy system in Escherichia coli, achieving an intracellular NUDH concentration exceeding 7.0 mM and an NUDH/NUD⁺ ratio above 55. Subsequently, we design a CO₂-formate-acetyl-CoA-pyruvate-malate pathway and reprogram key enzymes for NUDH-dependent operation. Integration of the OES-driven CAM* pathway with a rationally rewired native electron transport chain supports growth of the engineered E. coli on CO₂ and methanol, with a doubling time of 8.6 ± 0.3 h and a maximum OD₆₀₀ of 37.5 ± 5.8. Moreover, the platform extends CO₂ assimilation to biosynthesis of multiple chemicals from distinct CAM* pathway nodes, including mevalonate, lactate, 2,3-butanediol, and succinate. Together, these results establish a framework for reprogramming microbial energy metabolism and expanding one-carbon biotransformation.