Metabolic Engineering of Pseudomonas putida KT2440 for β-Nicotinamide Mononucleotide Biosynthesis from Glucose and Aspartate
β-Nicotinamide mononucleotide (NMN) is an important intermediate in nicotinamide adenine dinucleotide (NAD+) metabolism and has attracted increasing interest as a bioactive compound and biomanufacturing product. In this study, Pseudomonas putida KT2440 was engineered for NMN production using a “block–enhance–transport” strategy. Deletion of nicB impaired nicotinic acid degradation and resulted in the accumulation of 66.2 μM nicotinic acid in cell extracts, whereas additional deletion of the putative NMN-consuming genes pncC and ushA did not lead to detectable NMN accumulation. Coexpression of endogenous pncB and engineered Francisella tularensis nadE* enabled low-level NMN formation through a Preiss–Handler pathway-based route. By contrast, overexpression of endogenous nadA, nadB, and nadC strengthened precursor supply through the NAD+ de novo biosynthetic pathway and resulted in approximately 0.17 mM NMN in cell extracts. Chromosomal integration of an engineered Salmonella enterica pnuC* transporter cassette was associated with pronounced extracellular NMN accumulation. Additional overexpression of genes involved in downstream NAD+ metabolism or phosphoribosyl pyrophosphate supply did not considerably improve production, possibly because of metabolic competition or expression burden. The best-performing strain, LW10, produced 1.28 mM extracellular NMN, corresponding to approximately 0.43 g/L, after 96 h of shake-flask cultivation in basal salt medium containing glucose and L-aspartate. These results suggest the feasibility of NMN biosynthesis in engineered P. putida KT2440 and highlight the importance of balancing precursor supply, competing reactions, and product transport. Thus, P. putida KT2440 represents an alternative chassis for further pathway balancing and process optimization toward fermentative NMN production.