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

Abstract Enzymatic synthesis of β‐nicotinamide mononucleotide (NMN) from D‐ribose has garnered widespread attention due to its cheap material, 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 this method is impeded by the low activity of rate‐limiting enzymes. The ribose‐phosphate diphosphokinase (PRS) and nicotinamide phosphoribosyltransferase (NAMPT), that control the rate of the reaction, were engineered to improve the reaction efficacy. The actives of mutants PRS‐H150Q and NAMPT‐Y15S were 334% and 57% higher than that of their corresponding wild‐type enzymes, respectively. Furthermore, by adding pyrophosphatase, the byproduct pyrophosphate which can inhibit the activity of NAMPT was degraded, leading to a 6.72% increase in NMN yield. Following with reaction‐process reinforcement, a high yield of 8.10 g L −1 NMN was obtained after 3 h of reaction, which was 56.86‐fold higher than that of the stepwise reaction synthesis (0.14 g L −1 ), indicating that the in vitro enzymatic synthesis of NMN from D‐ribose and niacinamide is an economical and feasible route.