Unlocking the potential of CMP synthesis: Merging enzyme design, host genome editing, and fermentation optimization strategy

Cytidine-5′-monophosphate (CMP) and its derivatives play an important role in the fields of food, medicine and cosmetics. In this study, we used crude enzyme preparations of nucleoside kinase (NK) and polyphosphate kinase (PPK) to synthesize CMP in a synergistic reaction. A variant NK enzyme A220L/A236V/V237A/H252A was developed through a rational channel engineering approach, and its activity was increased by 9.17 times compared with the wild type. However, when cytidine was used as a substrate, the accumulation of a large amount of byproducts hindered the reaction efficiency, resulting in CMP yields that were always less than 80 %. By knocking out the cdd, rihA and rihC genes related to cytidine metabolism in the Escherichia coli host genome, the CMP yield was significantly increased to more than 95 %. Subsequently, we optimized the fermentation process in a 5-L bioreactor to further improve the crude enzyme activity. The optimized crude enzyme coupling reaction successfully generated 291 mM CMP within 20 h with a yield of up to 97 %. This study significantly improved the production efficiency of CMP by integrating key enzyme engineering, host bacteria modification and fermentation process optimization, laying a solid foundation for its industrial-scale production.