Metabolic design considerations for recycling of respiratory CO₂ in leaves

Net carbon gain in plants is significantly reduced by the loss of assimilated carbon due to respiration and other metabolism in heterotrophic tissues. Avoiding this loss is difficult due to the essential nature of the metabolic processes involved but it could be possible to engineer metabolism to reuse the generated CO₂. Here, we employ constraint-based modelling to reveal that crassulacean acid metabolism (CAM) cycling is the most efficient native mechanism for nocturnal CO₂ reassimilation in a leaf. CAM cycling can, in principle, reassimilate 100% of the CO₂ generated at night leading to increased growth while requiring only a modest increase in energy consumption. Other native CO₂ fixation routes proved less efficient, running into system balance issues and energy limitations. We also tested the feasibility of the synthetic crotonyl-(CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle for nocturnal CO₂ reassimilation. This revealed an unexpected limitation in the supply of NADPH at night without generating additional metabolic CO₂ and working directly against the goal of CO₂ reassimilation. As a result, the nocturnal CETCH cycle was highly inefficient, increasing total flux through metabolism by 2.5-fold and using 100% of the available energy. This inefficiency could be partially addressed by switching the CETCH cycle to using NADH as the coenzyme or by distributing some of its enzymatic activity to the day phase of the model. We conclude that CAM cycling provides the most promising route for engineering enhanced carbon efficiency in C3 crops.