Efficient One-Pot Synthesis of Cytidine 5'-Monophosphate Using an Extremophilic Enzyme Cascade System

A rapid in vitro enzymatic biosynthesis system has been developed as a biological manufacturing platform with potential industrial uses. Cytidine 5'-monophosphate (5'-CMP) is a key intermediate in the preparation of several nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, a highly efficient biosynthesis system was constructed for manufacturing 5'-CMP in vitro. Cytidine kinase (CK) was used for the biotransformation of cytidine to 5'-CMP, while polyphosphate kinase (PPK) was coupled for adenosine triphosphate regeneration. Both CK and PPK were selected from extremophiles, possessing great potential for biocatalytic synthesis. The effects of temperature, substrate concentration, and enzyme ratios were investigated to enhance the titer and yield of 5'-CMP. After optimization, 96 mM 5'-CMP was produced within 6 h, and the yield reached nearly 100%. This work highlights the ease of 5'-CMP production by an in vitro biomanufacturing platform and provides a green and efficient approach for the industrial synthesis of 5'-CMP.