Nicotinamide Mononucleotide Production in Metabolically Engineered Saccharomyces cerevisiae

Nicotinamide mononucleotide (NMN) is an essential precursor in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺), a critical cofactor in cellular metabolism and energy regulation. With the growing interest in NMN for its antiaging and therapeutic benefits, microbial production systems, particularly Saccharomyces cerevisiae, offer a promising alternative to traditional chemical synthesis. This study explored the optimization of NMN production in S. cerevisiae BY4742 using both constitutive and inducible promoters. Yeast strains were engineered to express human nicotinamide phosphoribosyl transferase (h-NAMPT) and yeast phosphoribosyl pyrophosphate synthetase (PRS5 and PRS2) to enable the direct conversion of nicotinamide (NAM) to NMN. The genes were expressed under the control of GAL1 (inducible) and TEF1 (constitutive) promoters in the plasmids. The results demonstrated that strains with the TEF1 constitutive promoter produced higher levels of intracellular NMN and NAD⁺ compared with those using the GAL1 inducible promoter. Additionally, fermentation in a rich R-SD medium further enhanced NMN production, with the scTEF2g strain (overexpressing plasmid-based h-NAMPT and PRS5 genes under the TEF1 promoter) achieving 151.71 mg/L NMN, a 3-fold increase in NMN yield compared to the control strain. This is the highest intracellular NMN produced in recombinant yeast from NAM in a flask. This work highlights the importance of gene regulation through promoter selection and culture optimization in maximizing NMN yields, presenting yeast-based systems as a promising platform for NMN production from NAM.