Biosynthesis of a Therapeutically Important Nicotinamide Mononucleotide through a Phosphoribosyl Pyrophosphate Synthetase 1 and 2 Engineered Strain of Escherichia coli

Abstract

Nicotinamide mononucleotide (NMN), a precursor of NAD+, can be synthesized by the conversion of nicotinamide with the help of nicotinamide phosphoribosyl transferase (NAMPT) via the salvage pathway. NMN has recently gained great attention as an excellent therapeutic option due to its long-term effective pharmacological activities. In this study, we constructed a recombinant strain of Escherichia coli by inserting NAMPT and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and PRPS2 (from Homo sapiens) genes to investigate the effect of PRPS1 and PRPS2 on NMN synthesis. The metabolically engineered strain of E. coli BL21 (DE3) exhibited 1.57 mM NMN production in the presence of Mg2+ and phosphates in batch fermentation studies. For further improvement in NMN production levels, effects of different variables were studied using a response surface methodology approach. A significant increment was achieved with a maximum of 2.31 mM NMN production when supplemented with 1% ribose, 1 mM Mg2+ and phosphate, and 0.5% nicotinamide in the presence of a lactose (1%) inducer. Additionally, insertion of the PRPS1 and PRPS2 genes in the phosphoribosyl pyrophosphate synthesis pathway and individual gene expression studies facilitated a higher NMN production at the intracellular level than the reported studies. The strain exhibited intracellular production of NMN from cheap substrates such as glucose, lactose, and nicotinamide. Hence, the overall optimized process can be further scaled up for the economical production of NMN using a recombinant strain of E. coli BL21 (DE3), which is the future perspective of the current study.

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

“…[12][13][14] Furthermore, researchers also conducted whole-cell biocatalytic systems using E. coli to produce NMN with excess addition of NAM in the mediums. [15][16][17][18] However, it is difficult to optimize the whole-cell systems due to their complicated intracellular environment. Therefore, we proposed to develop a biocatalytic cascade to mimic in vivo production, and further optimize its reaction condition for NMN biosynthesis.…”

“…Shoji et al [17] constructed artificial operons in E. coli to overexpress seven genes in the pentose phosphate pathway to enhance the pool size of PRPP for high NMN production. Kim et al [18] overexpressed Homo sapiens phosphoribosyl pyrophosphate synthetase (PRPS) 1 and 2 in E. coli for accomplishing the enhanced production of PRPP and ultimately NMN because PRPS catalyzes PRPP synthesis from ATP and D-ribose-5phosphate (R5P) in the biosynthesis of pyrimidine nucleotides. Both studies were conducted in the recombinant E. coli strains without optimization of individual gene expression.…”

“…In the whole-cell NMN production systems, glucose is commonly used for the synthesis of the ribose part. [15][16][17][18] Except for glucose, xylose is the most abundant sugar derived from biomass. However, microorganisms, especially E. coli, prefer glucose in a mixture of sugars as the primary source.…”

“…Although D ‐ribose is commercially available, its price needs to be considered for large‐scale production of NMN. In the whole‐cell NMN production systems, glucose is commonly used for the synthesis of the ribose part [15–18] . Except for glucose, xylose is the most abundant sugar derived from biomass.…”

A Combination of Nicotinamide and D-Ribose (RiaGev) Is Safe and Effective to Increase NAD+ Metabolome in Healthy Middle-Aged Adults: A Randomized, Triple-Blind, Placebo-Controlled, Cross-Over Pilot Clinical Trial

“…In individuals with thalassemia minor, their PRPP synthetase activities are below normal and the NAD + levels are significantly decreased in their red blood cells [ 33 ]. On the other hand, when human PRPP synthetases 1 and 2 were introduced into E. coli under the control of a strong promotor, the maximum amount of NMN was generated when D-ribose was supplied in the cell culture media [ 34 ]. Therefore, we predicted that formulating D-ribose with NAM will enhance NAD + production as well as possibly reduce side effects of NAM.…”