Engineering Bacillus subtilis for the biosynthesis of nicotinamide mononucleotide

Nicotinamide mononucleotide (NMN) has emerged as a high-value metabolite with promising applications in food, pharmaceutical, and healthcare industries. Herein, we adopted a multilayered metabolic engineering strategy to systematically optimize the NMN biosynthetic pathway in Bacillus subtilis . Integrated expression of ywlF - prs was chosen over plasmid-based overexpression in terms of cell growth and NMN titer. Furthermore, we combined gntP - gntK - gntZ overexpression with gntR deletion to channel carbon flux toward the PPP, thus boosting PRPP availability. The engineered strain exhibited a 526.39% higher NMN titer in sodium gluconate-supplemented medium than the parental strain cultivated in glucose medium. Replacement of LB medium with phosphate-rich ZYM medium further improved NMN production by 53.60%. Finally, native prs knockout and overexpression of the feedback-insensitive PRS (L135I) mutant elevated the final NMN titer to 4607.33 mg/L, representing a 2299.65% improvement over the parental strain under initial culture conditions. This work reveals the coordinated effects of precursor supply, pathway regulation, and enzyme engineering for efficient NMN biosynthesis.