Design and tailoring of an artificial DNA scaffolding system for efficient production of nicotinamide mononucleotide

•ATP regeneration system was constructed by coupling PPK with KLUNRK.•An artificial DNA scaffold was developed to enhance ATP regeneration.•Length and enzyme ratio of DNA scaffold was adjusted to enhance NMN production.•A 2.37-fold increase in NMN yield was obtained in a long-stranded scaffold L1. Nicotinamide mononucleotide (NMN), a molecule with special anti-aging activity, is extensively used in the development of health products. Recently, the phosphorylation of nicotinamide ribose (NR) based on nicotinamide ribose kinase (NRK) represents an important way to produce NMN, but it needs consume a large amount of ATP. Herein, polyphosphate kinase (PPK) from E. coli was coupled with NRK derived from Kluyveromyces marxianus to achieve ATP regeneration, thereby improving the biosynthesis of NMN by 1.22-fold. Then, an artificial DNA scaffold based on zinc-finger proteins was developed to assemble the NRK and PPK in vitro, effectively reducing the loss of intermediate metabolites. Furthermore, optimizing the length of the DNA scaffold increased the NMN conversion rate by 2.37-fold compared to using NRK alone. Meanwhile, this system also worked much better at a low initial ATP concentration, with NMN synthesis being 1.13-fold higher at 28 mM ATP compared to 42 mM ATP. This DNA scaffold system can be used as a platform for the construction and production of many biochemicals synthesized via multi-enzyme cascade reactions. [Display omitted]