Abstract: In order to improve the production of β-NMN by multi-enzyme cascade reaction of E. coli with nicotinamide and D-ribose. Based on the engineering bacterium B20 constructed in the laboratory, the recombinant bacterium B26 was constructed by replacing the key enzyme nicotinamide phosphoribosyl transferase（NAMPT). Then, the pH of the reaction system, the addition mode of niacinamide, the concentration of Mg²⁺, the starting substrate of polyphosphokinase(PPK）and the addition strategy of inorganic polyphosphate were optimized by single factor experiments. The results showed that there was no significant difference in yield between the two engineering strains B20 and B26, but the biomass of the engineering strain B26 expressing the key enzyme CpNAMPT^Y15S was 1.78 times that of B20, The total production of NMN can increase by 71.6%. The results of the single factor experiment determined that in a 20 mL catalytic system containing a final concentration of 15 mmol·L⁻¹ Mg²⁺, 5 mmol·L⁻¹ AMP, and 25 mmol·L⁻¹ inorganic phosphate, nicotinamide with a final concentration of 25 mmol·L⁻¹ was added in batches at 0 h and 6 h, while maintaining a pH of 7.0 as the optimal conditions for the NMN catalyzed reaction with ribose and nicotinamide as direct substrates. Under these conditions, the multi-enzyme cascade reaction was carried out for 12 h. The final NMN production reached 9.09 g·L⁻¹. By screening the dominant strains and optimizing the reaction conditions, the production of NMN biosynthesized by Escherichia coli was effectively improved, which provided a cost-effective reference method for the large-scale production of NMN.