Protein Engineering and Dual-Module Optimization for Efficient NMN Production in E. coli.

Area of Science:

• Biotechnology
• Enzymatic Synthesis
• Metabolic Engineering

Background:

• Nicotinamide mononucleotide (NMN) is a crucial NAD+ precursor with significant interest as a supplement.
• Existing NMN synthesis methods face challenges in efficiency and scalability.

Purpose of the Study:

• To engineer a novel, efficient dual-module enzymatic system for NMN biosynthesis.
• To enhance the stability and activity of key enzymes involved in NMN production.
• To optimize the system for high conversion rates using readily available substrates.

Main Methods:

• Construction of a two-module enzymatic reaction system for NMN synthesis from uridine and nicotinamide.
• Engineering of a more stable Nicotinamide Riboside Kinase (NRK) mutant (KlmNRK_M4) through structural and evolutionary analysis.
• Knockout of endogenous degradative enzyme genes in *Escherichia coli* to improve substrate and intermediate conversion.
• Optimization of reaction conditions for the dual-module system.

Main Results:

• Achieved efficient synthesis of NMN from Nicotinamide Riboside (NR) in module 1 using an NRK and ATP regeneration system.
• Successfully synthesized NMN from uridine and nicotinamide in module 2 via synergistic catalysis of NRK and pyrimidine nucleoside phosphorylase (PyNP).
• Obtained a highly stable KlmNRK_M4 mutant with improved enzymatic properties.
• Attained a high NMN conversion rate of 81.1% using 300 mM uridine and nicotinamide under optimized conditions.

Conclusions:

• The developed dual-module recombination system presents a novel and efficient pathway for NMN biosynthesis.
• Enzyme engineering and metabolic pathway optimization significantly enhance NMN production efficiency.
• This study provides a scalable and cost-effective approach for the industrial production of NMN.