Design of NAMPTs with Superior Activity by Dual-Channel Protein Engineering Strategy

The nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed substitution reaction plays a pivotal role in the biosynthesis of nucleotide compounds. However, industrial applications are hindered by the low activity of NAMPTs. In this study, a novel dual-channel protein engineering strategy was developed to increase NAMPT activity by enhancing substrate accessibility. The best mutant (CpNAMPT^{Y13G+Y15S+F76P}) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing CpNAMPT^{Y13G+Y15S+F76P} as a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L^-1 within 3 h with an impressive substrate conversion rate of 99.8%. Further analysis revealed that the newly generated substrate channel, formed through crack propagation, facilitated substrate binding and enhanced byproduct tolerance. In addition, three NAMPTs from different sources were designed based on the dual-channel protein engineering strategy, and the corresponding dual-channel mutants with improved enzyme activity were obtained, which proved the effectiveness and practicability of the approach.