A cell-free artificial anabolic pathway for direct conversion of CO2 to ethanol

Abstract

Biological CO₂ activation and conversion to high-value ethanol are a feasible and green strategy to close the carbon cycle. However, naturally evolved CO₂ utilization pathways involve carbon loss or ATP consumption. Herein, we report a complete anabolic pathway for direct conversion of CO₂ to ethanol by constructing and assembling three functional modules including CO₂ activation, formaldehyde → acetyl-CoA, and ethanol synthesis in a carbon-conserved and ATP-independent system. These artificial ethanol metabolic pathway fluxes were strengthened by screening efficient key enzymes for CO₂ activation, promoting formaldehyde assimilation, and employing a two-step reaction. ¹³C-labeled CO₂ demonstrated the feasibility of the pathway converting CO₂ into ethanol in vitro by detecting the carbon flow. With this anabolic pathway, we obtained an ethanol concentration of 0.37 mM at a conversion rate of 4.33 nmol CO₂ min⁻¹ mg⁻¹ enzyme and 0.5 mM R5P supply. This modularization strategy provides a new avenue in the construction of artificial metabolic pathways for ethanol synthesis from CO₂.