Semi-Rational Design of CpNampt Mutants for Enhanced Nicotinamide Mononucleotide (NMN) Synthesis in Saccharomyces cerevisiae

β-Nicotinamide mononucleotide (NMN) serves as a direct precursor of the essential cofactor NAD⁺, playing a crucial role in regulating mitochondrial function and delaying cellular aging. Synthetic biology, with its environmental and economic advantages, has become an important approach for the production of natural medicines, with the optimized design of metabolic pathways at its core. In this study, we focused on the NMN‑consuming pathway mediated by the nma1 and pof1 genes in Saccharomyces cerevisiae and consequently generated their double‑knockout mutant, DHSX02. Futhermore, a highly efficient NMN phosphoribosyltransferase (CpNampt) derived from Chitinophaga pinensis was mutated via AI-assisted design and introduced into the DHSX02 strain to enhance NMN synthesis. The results showed that the NMN yield in the DHSX02 strain increased by 20.7% compared to the wild-type strain. After multiple rounds of optimization, the strains carrying the mutated CpNampt exhibited significantly enhanced NMN production, with the I229A mutant strain achieving a total yield of 60.64 mg/L in 12h, a 4.4-fold increase compared to the wild-type strain. This work demonstrates an effective synthetic biology strategy, combining metabolic engineering and AI-assisted enzyme design to construct an efficient microbial cell factory for green NMN production.