Novel Biosynthetic Pathway for Nicotinamide Mononucleotide Production from Cytidine in Escherichia coli

Nicotinamide mononucleotide, known as NMN, is an important nicotinamide adenine dinucleotide (NAD+) precursor. It is integral in cellular metabolism, energy generation, and processes associated with aging. Since NMN provides healthy value, it becomes a major focus for the biotechnological industry. This study presents a new biosynthetic pathway for producing NMN without limits on intracellular PRPP (5′-phosphoribosyl pyrophosphate) metabolic flux. The route started by converting cytidine into 1-phosphoribose via pyrimidine-nucleoside phosphorylase (PyNP), after transforming into nicotinamide riboside (NR) through either purine-nucleoside phosphorylase (XapA) or nicotinate riboside kinase (NRK). NR was phosphorylated by NRK in the presence of nicotinamide (NAM) to produce NMN. We established an in vitro enzyme activity verification system for the feasibility check. The optimization of multienzyme cascade reactions was figured out for the NMN biosynthesis. Finally, the enzymes of PyNP and NRK were expressed in the cytidine-producing strain; we established a de novo biosynthesis pathway from glucose to NMN, achieving a production titer of 33.71 mg/L at a shake-flask scale.