Coordinating Calvin Cycle and Glycolysis in <i>Escherichia coli</i>

Engineering <i>Escherichia coli</i> to coassimilate glucose and CO₂ requires rewiring central metabolism so a Calvin-Benson-Bassham (CBB) module can compete with, rather than be overwhelmed by, glycolysis. We implemented a Rubisco-based engineered pathway, i.e., heterologous phosphoribulokinase (PrkA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and attenuated glycolysis by (i) CRISPRi repression of <i>gapA</i> (GAPDH) and (ii) deletion of major fermentative redox sinks (Δ<i>ldhA</i> Δ<i>frd</i>). Activation of the Rubisco-based engineered pathway not only enabled CO₂ fixation but also unexpectedly revived glycolytic throughput, yielding a coordinated "harmony" between the two pathways. The engineered strain (<i>E. coli</i> MZLF/pSLiP, pCCS01) sustained cometabolism, consuming glucose at 170 ± 1 mg L^-1 h^-1 by 84 h. Flux analysis indicated that 17.0 ± 0.1 mg L^-1 h^-1, 10% of total glucose uptake, was routed through the Rubisco-based engineered pathway, corresponding to a CO₂-fixation rate of 5.0 ± 0.1 mg L^-1 h^-1. Mechanistically, in the parental <i>E. coli</i> background (<i>ldhA</i>⁺, <i>frd</i>⁺) ATP is primarily supplied by glycolysis with redox balance via lactate/succinate formation. With the CBB module active in the engineered context (<i>gapA</i> repressed, Δ<i>ldhA</i> Δ<i>frd</i>, <i>prkA</i>⁺, <i>rbcLS</i>⁺), pyruvate allocation, ATP from acetate, and NADH reoxidation from ethanol production jointly determine flux partitioning, yielding a distinct fermentation profile. These findings show that successful central-metabolism rewiring must target not only core nodes (e.g., <i>gapA</i>) but also auxiliary redox circuits (<i>ldhA</i>, <i>frd</i>). Equally important is maintaining a moderate activity of the Rubisco-based engineered pathway, which allows restoration of the near-equilibrium state at the GAPDH-repressed G3<i>P</i>/1,3-BPG/3PG node.