Industrial biosynthesis of β-nicotinamide mononucleotide (β-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.9-fold higher enzyme activity. Molecular dynamics simulations and structural analysis demonstrate the enhancement of its structural stability. High-density fermentation was achieved through a 5 L fermentation tank, with a titer reaching 208.3 U/mL, the highest in the current report. An ATP-cycling whole-cell catalytic system was employed and optimized by introducing a polyphosphate kinase 2 (PPK2) recombinant strain, and 15.16 g/L β-NMN was obtained through a series of batch transformation experiments. This study provides a new strategy for the efficient screening of highly active enzyme variants and offers a green and promising biotransformation system for NMN production.
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http://dx.doi.org/10.1021/acs.jafc.4c05520 | DOI Listing |
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