Improvement of an enzymatic cascade synthesis of nicotinamide mononucleotide via protein engineering and reaction-process reinforcement

Enzymatic synthesis of β-Nicotinamide mononucleotide (NMN) has garnered widespread attention due to its advantages of minimal environmental impact, the use of mild reaction conditions, and the ability to produce highly pure products with the desired optical properties. However, the overall NMN yield of enzymatic synthesis, which begins with ribose and niacinamide, is impeded by the low activity of rate-limiting enzymes. In this study, the enzymes that control the rate of the reaction, ribose-phosphate diphosphokinase (PRS) and nicotinamide phosphoribosyltransferase (NAMPT), were engineered to improve the reaction efficacy. The actives of single mutants PRS-H150Q and NAMPT-Y15S were 4.34 times and 1.57 times that of their corresponding wild type enzymes, respectively. Furthermore, the byproduct pyrophosphate, which can inhibit NAMPT, was degraded by pyrophosphatase (PPase), leading to a 6.72% increase in NMN yield. Following with reaction-process reinforcement, a yield of 8.10 g/L NMN was obtained from 3.64 g/L NAM after 3 hours of reaction, which was 56.86-fold higher than that of the stepwise reaction synthesis (0.14 g/L), indicating that the in vitro enzymatic synthesis of NMN from ribose and niacinamide is an economical and feasible route.