Regulation–Metabolism Rewiring Enables Efficient Nicotinamide Mononucleotide Biosynthesis in Food-Grade Lactiplantibacillus plantarum

Abstract Nicotinamide mononucleotide (NMN) is a high-value functional ingredient, yet its efficient biosynthesis in food-grade microorganisms remains limited by an incomplete understanding of regulatory mechanisms. Here, we identified a GntR-family transcription factor (TFs), lp_0262, as a key regulator of NMN metabolism in Lactiplantibacillus plantarum. Characterization of lp_0262 revealed an unusual nonmonotonic regulatory behavior, where both its deletion and overexpression significantly promoted NMN accumulation. Comparative transcriptomic profiling of the Δlp_0262 unveiled a metabolic trade-off characterized by the robust upregulation of NMN biosynthetic genes alongside a paradoxical downregulation of the pentose phosphate pathway (PPP). To overcome this limitation, key oxidative PPP genes (zwf, pgl, and gnd) were reinforced in the Δlp_0262 chassis. The synergistic coupling of regulatory relief and metabolic flux optimization resulted in a maximal NMN titer of 300 μmol L–1, the highest reported for lactic acid bacteria (LAB) to date. These findings provide a promising strategy for engineering NMN-producing food-grade microbial cell factories.