Modular in vivo engineering of the reductive methylaspartate cycles for synthetic CO ₂ fixation

Biological carbon fixation is currently limited to seven naturally occurring pathways. Synthetic carbon fixation pathways have the potential to surpass aerobic natural pathways in efficiency, but none have been realized in living cells. Here, we present the reductive methylaspartate cycles (rMASP), a novel family of 4 energy-efficient aerobic CO ₂ fixation pathway variants. These cycles have the potential to outperform the yields and/or rates of the Calvin cycle and previously proposed synthetic CO ₂ 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-CoA carboxylase dependent growth for both elevated and ambient CO ₂ conditions, and show in vitro activity of the other CO ₂ -fixing 2-oxoglutarate carboxylase, which requires further activity optimization for mesophilic temperatures. This work demonstrates important steps toward realizing efficient synthetic carbon fixation pathways in living organisms.