Systematic design and in vitro validation of novel one-carbon assimilation pathways

The utilization of one-carbon (C₁) assimilation pathways to produce chemicals and fuels from low-cost C₁ compounds could greatly reduce the substrate-related production costs, and would also alleviate the pressure of the resource supply for bio-manufacturing. However, the natural C₁ assimilation pathways normally involve ATP consumption or the loss of carbon resources as CO₂, resulting in low product yields, making the design of novel pathways highly pertinent. Here we present several new ATP-independent and carbon-conserving C₁ assimilation cycles with 100% theoretical carbon yield, which were discovered by computational analysis of metabolic reaction set with 6578 natural reactions from MetaCyc database and 73 computationally predicted aldolase reactions from ATLAS database. Then, kinetic evaluation of these cycles was conducted and the cycles without kinetic traps were chosen for further experimental verification. Finally, we used the two engineered enzymes Gals and TalB^F178Y for the artificial reactions to construct a novel C₁ assimilation pathway in vitro and optimized the pathway to achieve 88% carbon yield. These results demonstrate the usefulness of computational design in finding novel metabolic pathways for the efficient utilization of C₁ compounds and shedding light on other promising pathways.