Multivariate Modular Metabolic Engineering for High Titer Uridine Triphosphate Production in Escherichia coli

Uridine triphosphate (UTP) holds great potential for applications in the food and medicine fields. Compared with the chemical synthesis route, microbial biosynthesis of UTP through metabolic engineering approaches offers a sustainable and environmentally friendly alternative. However, the tightly regulated UTP metabolic pathway has presented challenges in building efficient microbial cell factories. To address this issue, we applied a modular metabolic engineering strategy to overcome four distinct bottlenecks in UTP production in Escherichia coli. First, by blocking the UTP catabolic module, we achieved a substantial accumulation of UTP up to 304.67 μM. Second, in the UTP synthetic module, a uridine 5′-monophosphate (UMP) kinase variant (MtUMPKR141A/K148A) was obtained based on crystal structure analysis, resulting in a 10.47-fold increase in catalytic capacity. Furthermore, by fusion expression of the UMP kinase variant and orotidine 5′-phosphate decarboxylase, the conversion of UMP to UTP was improved, leading to UTP concentrations reaching 11.36 mM. Third, in the UTP precursor module, several key metabolism-related genes were engineered to enhance the supply of precursors, resulting in an 87.9% increase in UTP production. Finally, by implementing electron transport chain regulation with an ATP-sensing switch, the optimal strain E. coli K16 achieved an intracellular UTP concentration of 28.40 mM (13.75 g/L), which was 544.37-fold higher than that of the base strain. This study represents the design of a de novo UTP-producing strain through metabolic engineering, opening new avenues for UTP and its derivatives production.