Systematic Engineering of Escherichia coli for Efficient Production of Nicotinamide Mononucleotide From Nicotinamide

Abstract

Research studies on NAD + have proven its crucial role in aging and disease. Nicotinamide mononucleotide (NMN), as the key intermediate of NAD + , plays a significant role in supplying and maintaining NAD + levels. In the present study, a biocatalytic method for the efficient synthesis of NMN was established. First, Escherichia coli was systematically modified to make it more conducive to the biosynthesis and accumulation of NMN. Next, the performance of nicotinamide phosphoribosyltransferase from Vibrio bacteriophage KVP40 (VpNadV) was determined, which has the best catalytic activity to produce NMN from nicotinamide. The accumulation of extracellular NMN was further increased after the introduction of an NMN transporter. Fine-tuning of gene expression and copy number led to the synthesis of NMN at the yield of 2.6 g/L at the shake flask level. The introduction of a nicotinamide transporter, BcniaP, could not obviously increase the production of NMN at the shake flask level, but it decreased the production of NMN at the bioreactor level. Finally, the titer of NMN reached 16.2 g/L with a conversion ratio of 97.0% from nicotinamide, both of which are highest according to currently available reports. The fed-batch fermentation with direct supplementation of nicotinamide could facilitate the industrialscale production of NMN compared to that achieved by the whole-cell catalysis process. These results also represent the highest reported yield of NMN synthesized from nicotinamide in E. coli.

High level expression of nicotinamide nucleoside kinase from Saccharomyces cerevisiae and its purification and immobilization by one-step method

“…The most effective chemical synthesis method at present is the two-step method used in the 2017 Zhang’s group report: the β-isomer was eventually obtained in 85% yield after deprotection of the ester in methanolic ammonia ( Zhang and Sauve, 2017 ). Owing to the chemical production of NMN is time-consuming and laborious, the biosynthesis of NMN has become a hot spot ( Huang et al, 2022 ). Compared with traditional chemical catalysis, it has unique advantages such as mild conditions, low equipment requirements and strong stereoselectivity.…”

“…Zhou Jingwen of Jiangnan University and others transformed E. coli into nicotinamide and glucose, creating a record high yield of NMN. The NMN titer reached 16.2 g/L, and the nicotinamide conversion rate was 97.0% ( Huang et al, 2022 ). According to the current report, both were the highest.…”

“…According to the current report, both were the highest. Compared with whole cell catalysis, fed batch fermentation with direct addition of nicotinamide is more conducive to the industrial production of NMN ( Huang et al, 2022 ). Chemical synthesis using nicotinamide nucleoside (NR) as substrate depends on excessive phosphorus oxychloride and requires strict temperature operation which is not suitable for industrial ( Qian et al, 2022 ).…”

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell‐free protein synthesis with split GFP

“…Among them, an impressively high NMN titer of 93.5 g/L was achieved by enzymatic phosphorylation of nicotinamide riboside to NMN using a new nicotinamide riboside kinase. However, nicotinamide riboside is a more expensive substrate for NMN synthesis than NAM, the substrate used in our pathway (Huang et al, 2022). In addition, the NMN yield obtained under our optimized reaction conditions was substantially higher than those reported in these studies (Supporting Information: Table S5), suggesting that an improved pathway for NMN production was successfully established in this study.…”

Metabolic Engineering of Bacillus licheniformis for the Bioproduction of Nicotinamide Riboside from Nicotinamide and Glucose

“…coli for the production of NMN from NAM, leading to 1.89 g/L NMN titer in shake flasks . Subsequently, Huang et al optimized the expression of PRPP synthase (BaPRS), nicotinamide phosphoribosyltransferase (VpNadV), and NMN transporter (BmPnuC), which resulted in 2.6 g/L NMN in shake flasks . Although systematic engineering of E.…”