Towards the conversion of CO2 into optically pure N-carbamoyl-l-aspartate and orotate by an in vitro multi-enzyme cascade

Abstract

A novel in vitro multi-enzymatic system was constructed for the conversion of CO₂ into optically pure N-carbamoyl-L-aspartate and orotate. Four cascade enzymes, carbamoyl phosphate synthase (CPS), aspartate carbamoyl-transferase (ATCase), dihydroorotase (DHOase), and dihydroorotate dehydrogenase (DHOD), were used for the biotransformation of feedstocks into products in one pot while polyphosphate kinase (PPK) was coupled for ATP regeneration. Under the optimal reaction conditions and substrate concentrations, 19.2 mM N-carbamoyl-L-aspartate was produced within 3.0 h, which enabled the synthesis of 15.5 mM orotate. The yields of N-carbamoyl-L-aspartate and orotate based on the added L-aspartic acid reached 96.0% and 93.3%, respectively. This work highlights the ease of producing optically pure N-carbamoyl-L-aspartate and orotate via an in vitro biomanufacturing platform and provides a novel pathway for the utilization of renewable feedstock for the synthesis of value-added commodity chemicals.