CO₂ fixation for malate synthesis energized by starch via in vitro metabolic engineering

Carbon dioxide (CO₂) is an appealing carbon feedstock for the sustainable production of biocommodities. Here we designed three in vitro artificial enzymatic pathways featuring the ATP-excess, ATP-deficit, and ATP-balanced pathways for the biotransformation of starch and CO₂ to malate. This ATP-balanced pathway without exogenous ATP donors can auto-regulate its carbon fluxes from glyceraldehyde 3-phosphate to 3-phosphoglycerate via either the ATP-generating pathway (a part of glycolysis) or no-ATP-generating pathway from a hyperthermophilic archaeon Thermococcus kodakarensis. The ATP-balanced pathway enabled to produce up to 52.4 mM malate with 95.3% of the theoretical yield, that is, 2 mol of malate synthesized from 1 mol of glucose of starch and 2 mol of CO₂. This pathway also enabled to produce high-yield malate regardless of ATP/ADP ratios. Anaerobic reaction conditions and/or the addition of a reducing agent dithiothreitol were of importance for creating an anoxic environment for biocatalysis of enzyme cocktails and for mitigating the deactivation of enzymes and degradation of intermediates. This new pathway could provide a green route for direct conversion of CO2 to many building blocks, a promising alternative of petrochemical-based production of biocommodities.