Improving Biosynthesis Efficiency of Nicotinamide Mononucleotide by ATP Recycling Engineering and Condition Optimization

Nicotinamide mononucleotide (NMN) is a very important bioactive nucleotide that is of great help to human health. However, its widespread application has been limited by its high production costs, especially the cost of the core substrates, coenzyme, and enzymes. In this study, the ADP/GDP-polyphosphate phosphotransferase <i>Rh</i>PPK2 originating from <i>Rhodobacter sphaeroides</i> was successfully expressed in <i>Escherichia coli</i> with high-level solubility, and the enzyme activity in the lysate supernatant reached 21.9 ± 0.65 U/mL. And then, the temperature profiles, pH profiles, and kinetic parameters of purified re<i>Rh</i>PPK2 were systematically characterized, which demonstrate its potential for application in enzymatic ATP regeneration systems. Furthermore, the introduction of re<i>Rh</i>PPK2 for ATP regeneration significantly enhanced NMN production efficiency, achieving a 2.3-fold increase compared to the conventional ATP supplementation method. Finally, the production efficiency of NMN was further improved by a single-factor experiment and L9(3⁴) orthogonal design, and the yield was up to 14.6 ± 0.51 g/L, about 5.4 times of the initial yield. This research substantially reduced NMN production costs and established a robust foundation for industrial-scale NMN production.