Design and analysis of synthetic carbon fixation pathways based on novel enzymatic reactions

Biological carbon fixation is currently limited to seven naturally occurring pathways, each with its own limitations and constraints. In recent years, computational analyses of known biochemical reaction networks have identified dozens of theoretical carbon fixation pathways, some of which may have the potential to outperform their natural counterparts. This mix and match approach, however, cannot account for those reactions that have not been reported to occur in nature, which heavily limits the possible solution space. Here, we use a bioretrosynthetic approach coupled with expert biochemical knowledge to identify eight novel pathways that leverage enzyme promiscuity and the latent biochemical reaction space. We analyze the thermodynamic, stoichiometric, and kinetic parameters of these pathways and compare them to the ubiquitous Calvin-Benson-Bassham cycle and previously proposed synthetic CO ₂ fixation cycles, highlighting advantages and disadvantages. This work highlights the need for enzyme engineering and design in the quest for efficient biological carbon fixation.