Nicotinamide mononucleotide (NMN) is an endogenous substance in humans with high safety and thermal stability, and its application in cosmetics, medical health, and functional foods has received widespread attention. However, the synthesis process of NMN has problems such as high cost, time-consuming and low yield, which limits the large-scale industrial application of NMN to a certain extent. Nicotinamide phosphate ribose transferase (Nampt) is a critical enzyme in a technical route in the biological synthesis of NMN, which can catalyze the synthesis of NMN by Nicotinamide (NAM) and Phosphoribosyl pyrophosphate (PRPP). Screening and expression of a Nampt with excellent enzymatic properties and stability is the key to the synthesis of NMN by this method. At present, the main problems in the technical route of NMN production by Nampt are that the catalytic activity of Nampt is low. And the source of Nampt is small and limited, so we isolated the microorganism with high NMN production, Enterobacter chengduensis 2021T4.7, and optimized its fermentation conditions. The yield of NMN is up to 67.66 μM. In addition, we synthesized Nampt and constructed related recombinant high-yield engineering bacteria. We semi-rationally designed the Nampt structure derived from mice and obtained mutant mNampt-V365L with NMN yields as high as 135.99 μM increased by 62% compared with wild type. Here, we screened high-yield NMN natural strains, and obtained high-yield strains through the semi-rational design optimization of Nampt enzyme, which provided new chassis microorganisms and new ideas for the conversion rate of NMN.