Streamlining Design, Engineering, and Applications of Nicotinamide Riboside Kinase for Sustainable Biosynthesis of Nicotinamide Mononucleotide in Flow

The market demand for β-nicotinamide mononucleotide (NMN), an antiaging health product, is rapidly expanding. Consequently, there is an urgent requirement for an environment-friendly and economical approach to synthesize NMN. In this study, we develop a biocatalytic synthesis method for the production of valuable NMN from chemically synthesized nicotinamide riboside (NR). To enhance the efficiency of the process, the biocatalyst human nicotinamide riboside kinase (HS-NRK) was engineered by introducing a beneficial substitution (G13S) near the ATP-binding site. As a result, a 100% conversion of 50 g L–1 NR was achieved by using the engineered variant NRKG13S. However, the NMN yield was limited to 78% (39 g L–1) due to biodegradation by endogenous enzymes present in Escherichia coli. To overcome this challenge, we identified and knocked out the deoD gene for NMN biodegradation. Through protein engineering and metabolic engineering, we achieved a high NMN yield of 93% within 2 h using an effective whole-cell biocatalysis approach. Finally, an immobilized biocatalyst in continuous operation was developed, which facilitated the enzymatic process and enabled the cost-effective and environmentally attractive production of NMN.