Coordinating Calvin Cycle and Glycolysis in Escherichia coli

Engineering Escherichia coli 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 gapA (GAPDH) and (ii) deletion of major fermentative redox sinks (ΔldhA Δfrd). 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 (E. coli 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 E. coli background (ldhA⁺, frd⁺) ATP is primarily supplied by glycolysis with redox balance via lactate/succinate formation. With the CBB module active in the engineered context (gapA repressed, ΔldhA Δfrd, prkA⁺, rbcLS⁺), 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., gapA) but also auxiliary redox circuits (ldhA, frd). 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 G3P/1,3-BPG/3PG node.