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

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 <i>Escherichia coli</i> by inserting <i>NAMPT</i> and phosphoribosyl pyrophosphate synthetase 1 (<i>PRPS1</i>) and <i>PRPS2</i> (from <i>Homo sapiens</i>) genes to investigate the effect of <i>PRPS1</i> and <i>PRPS2</i> on NMN synthesis. The metabolically engineered strain of <i>E. coli</i> 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 <i>PRPS1</i> and <i>PRPS2</i> 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 <i>E. coli</i> BL21 (DE3), which is the future perspective of the current study.