Establishing a novel pathway for the biosynthesis of nicotinamide mononucleotide.

Nicotinamide mononucleotide (NMN) is a pivotal molecule within the realm of metabolic health, serving as a precursor to nicotinamide adenine dinucleotide (NAD⁺), a critical coenzyme in cellular energy metabolism. In recent years, the biological production of NMN has garnered significant interest. In this study, we developed the novel NRK-dependent synthesis routes for NMN production. Two strategies were designed to supply D-ribose-1-phosphate (R-1-P): (1) phosphorylation of exogenous D-ribose to ribose-5-phosphate (R-5-P) using engineered ribokinase (RK), followed by isomerization to R-1-P; (2) R-5-P synthesis from glucose through the pentose phosphate pathway. An optimized in vitro multi-enzyme cascade (XapA/PNP/NRK, PPM, NRK) identified NRK as the most efficient catalyst for NMN biosynthesis from D-ribose and niacinamide. In Escherichia coli, overexpression of this cascade, knockout of competing pathways, and secretion enhancement via a pelB signal peptide-fused PnuC transporter achieved an NMN titer of 62.0 mg L^-¹ .This work provides a viable alternative for the biosynthesis of NMN.