Fig. 1: ATP-driven CO₂-reduction as a novel entry-point for carbon fixation.

A Single-step reduction of CO₂ by formate dehydrogenases (FDH) is thermodynamically and kinetically constrained under ambient CO₂-conditions (see main text). B Design principles for the CO₂ reduction pathways proposed here. Aerobic conversion of CO₂ to formate at ambient CO₂ concentrations is pursued by energizing the process with ATP-hydrolysis in short, cyclic pathways. The order and number of the indicated steps varies among the proposed routes, while multiple reactions may also be catalyzed within a single enzyme (e.g. carboxylation and ATP-hydrolysis in the case of biotin-dependent carboxylases). C Proposed synthetic autotrophy based on extending the CO₂ reduction pathway with a subsequent module to assimilate formate into multi-carbon products, shown for an example route consisting of: ACK (promiscuous) acetate kinase⁸⁴, FPR formyl-phosphate reductase⁸⁴, FLS formolase⁸⁵, DHAK dihydroxyacetone kinase. D Proposed synthetic photorespiration based on a CO₂ reduction pathway, circumventing the inefficient carbon-releasing reactions of natural photorespiration (glycine decarboxylation). A more detailed depiction is shown in Supplementary Fig. 26. CBB cycle Calvin-Benson-Bassham cycle, GCS glycine-cleavage-system, RuBP ribulose bisphosphate, SHMT serine hydroxymethyltransferase, 3PG 3-phosphoglycerate, THF tetrahydrofolate. E The CORE cycle incorporates CO₂ in the form of bicarbonate by acetyl-CoA carboxylation (#1), producing malonyl-CoA that is subsequently reduced (#2) to malonate semialdehyde (MSA). The proposed BKACE reaction (#3) then condenses MSA with acetyl-CoA to produce formyl-CoA and acetoacetate. Formyl-CoA can be used as a CoA donor to activate acetoacetate to acetoacetyl-CoA (#4), releasing formate. Finally, 2 molecules of acetyl-CoA are regenerated from acetoacetyl-CoA by a β-ketothiolase reaction (#5). As an alternative to the CoA-transferase variant, formyl-CoA can be hydrolyzed instead (#6), while acetoacetate is activated at the expense of additional ATP (via a kinase/ligase/synthetase, Supplementary Fig. 1). F Generic scheme of the BKACE reaction. The Claisen-like condensation reaction accepts various β-keto acids that react with acetyl-CoA to form an acyl-CoA ester and acetoacetate^28,29,129. In the reaction proposed here for the CORE cycle, MSA (the smallest β-keto acid) is used to produce formyl-CoA (the smallest CoA-ester).