Work

Coordinating Calvin Cycle and Glycolysis in Escherichia coli

Year: 2026

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide Engineering Escherichia coli to coassimilate glucose and CO 2 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 (Δ ldhA Δ frd ). Activation of the Rubisco-based engineered pathway not only enabled CO 2 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 CO 2 -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, Δ ldhA Δ frd, 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 G3 P /1,3-BPG/3PG node.

Source: ACS Synthetic Biology

DOI: 10.1021/acssynbio.5c00854

Authors: Yu-Jen Lin, Hsien-Tse Chen, Pei-Yi Lin, Yi-Jyun Lai, Si-Yu Li

Institution: National Chung Hsing University

Language: English

Topic: Microbial Metabolic Engineering and Bioproduction

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funder: National Science and Technology Council