Exploring the <i>De Novo</i> NMN Biosynthesis as an Alternative Pathway to Enhance NMN Production

Abstract

Nicotinamide mononucleotide (NMN) serves as a precursor for NAD⁺ synthesis and has been shown to have positive effects on the human body. Previous research has predominantly focused on the nicotinamide phosphoribosyltransferase-mediated route (NadV-mediated route) for NMN biosynthesis. In this study, we have explored the <i>de novo</i> NMN biosynthesis route as an alternative pathway to enhance NMN production. Initially, we systematically engineered <i>Escherichia coli</i> to enhance its capacity for NMN synthesis and accumulation, resulting in a remarkable over 100-fold increase in NMN yield. Subsequently, we progressively enhanced the <i>de novo</i> NMN biosynthesis route to further augment NMN production. We screened and identified the crucial role of MazG in catalyzing the enzymatic cleavage of NAD⁺ to NMN. And the <i>de novo</i> NMN biosynthesis route was optimized and integrated with the NadV-mediated NMN biosynthetic pathways, leading to an intracellular concentration of 844.10 ± 17.40 μM NMN. Furthermore, the introduction of two transporters enhanced the uptake of NAM and the excretion of NMN, resulting in NMN production of 1293.73 ± 61.38 μM. Finally, by engineering an <i>E. coli</i> strain with optimized PRPP synthetase, we achieved the highest NMN production, reaching 3067.98 ± 27.25 μM after 24 h of fermentation at the shake flask level. In addition to constructing an efficient <i>E. coli</i> cell factory for NMN production, our findings provide new insights into understanding the NAD⁺ salvage pathway and its role in energy metabolism within <i>E. coli</i>.