Custom carbon cycling

Science 354, 900–904 (2016)

In addition to the widespread and well-known Calvin–Benson–Bassham (CBB) cycle, five other carbon-fixation pathways exist in nature. Schwander et al. now add a designer in vitro option to that portfolio. Beginning with a collection of enoyl–CoA carboxylases/reductases (ECRs), the authors composed several theoretical CO₂ fixation cycles, then calculated their Gibbs free energy profiles and estimated their consumption of ATP and NADPH per molecule of CO₂ converted. Guided by this analysis, they chose a crotonyl–CoA/ethylmalonyl–CoA/hydroxybutyryl–CoA (CETCH) cycle and selected enzymes to experimentally catalyze each step in the pathway. Further optimization of the CETCH cycle involved rational structure-guided engineering of three enzymes to exhibit desirable activities, the addition of enzymes for ATP and NADPH regeneration, protection against oxidative damage from H₂O₂ production, and incorporation of metabolic proofreading enzymes to correct for unwanted side reactions. In total, the optimized CETCH cycle comprised 17 enzymes originating from nine different organisms across all three domains of life (including bacteria, archaea, plants, and humans) and had CO₂-fixation efficiency comparable with the best estimates for the CBB cycle. A reliance on ECR activity for autotrophic CO₂ fixation is unprecedented in nature, and realization of the CETCH cycle demonstrates the potential not only for improving carbon fixation but also for the rational design of custom metabolic pathways.