Protein Engineering and Dual-Module Optimization for Efficient NMN Production in <i>E. coli</i>

Nicotinamide mononucleotide (NMN) has received widespread attention as a supplement of NAD⁺ in cells. In this study, a dual-module reaction system was constructed to synthesize NR using uridine and nicotinamide, and further to efficiently synthesize NMN. First, module 1 was constructed, which catalyzed the synthesis of NMN from NR using an efficient NRK and ATP regeneration system. Then module 2 was constructed by introducing pyrimidine nucleoside phosphorylase (PyNP) to synthesize NMN from uridine and NAM under the synergistic catalysis of NRK. Based on the fact that NRK has both phosphorylation and group transfer functions in the dual-module system, the mutant <i>Klm</i>NRK_M4 with nearly 4-fold increased stability was obtained through predicted structure and evolutionary conservation analysis. At the same time, the <i>pncC</i>, <i>deoD</i>, <i>ushA</i>, <i>nadR</i> and <i>deoB</i> genes encoding endogenous degradative enzymes in <i>Escherichia coli</i> affect substrate and intermediate conversion were knocked out. Finally, by optimizing the reaction conditions of the dual-module recombination system, a high NMN conversion rate of 81.1% was achieved using 300 mM uridine and nicotinamide as substrates. This study provides a novel and efficient pathway for the biosynthesis of NMN.