Modular in vivo engineering of the reductive methylaspartate cycles for synthetic CO₂ fixation

Biological carbon fixation is currently limited to seven naturally occurring pathways. Synthetic carbon fixation pathways have the potential to surpass natural aerobic pathways in efficiency, but none have been realized in living cells. Here, we present the reductive methylaspartate cycles, a family of four energy-efficient aerobic CO₂ fixation pathway variants. These cycles have the potential to outperform the yields and/or rates of the Calvin cycle and previously proposed synthetic CO₂ fixation cycles. To realize these designs, we adopted a modular engineering approach in Escherichia coli. Several pathway modules, up to a cascade of 11 enzymes, were realized via engineering and evolution. We demonstrate crotonyl- coenzyme A carboxylase-dependent growth under both elevated and ambient CO₂ conditions, and we show in vitro activity of the other CO₂-fixing 2-oxoglutarate carboxylase, which requires further activity optimization for mesophilic temperatures. This article demonstrates important steps toward realizing efficient synthetic carbon fixation pathways in living organisms.