[1S04a] 14. Chemical Biotechnology - Part 2

3:15 PM - 3:30 PM JST(6:15 AM - 6:30 AM UTC)

[1Chemi-11] Coordinating Calvin Cycle and Glycolysis in Escherichia coli

Yu-Jen Lin¹, ○Hsien-Tse Chen¹, Pei-Yi Lin¹, Yi-Jyun Lai¹, Si-Yu Li¹ (1. National Chung Hsing University (Taiwan))

Keywords:

Calvin cycle,glycolysis,Escherichia coli,central metabolism rewiring,mixotroph,mass-action rebalancing

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.