Enzymatic Synthesis of NAD

Nicotinamide adenine dinucleotide (NAD⁺) is an essential metabolite involved in numerous biological processes, functioning both as a redox cofactor in cellular energy metabolism and as a co-substrate for NAD⁺-utilizing enzymes, including sirtuins and poly(ADP-ribose) polymerases. Given its central role in maintaining physiological homeostasis and its involvement in a wide range of disease processes, there is growing interest in developing NAD⁺ and its analogs as chemical probes and potential therapeutic agents. However, the chemical synthesis of NAD⁺ analogs is often hampered by low yields, poor stereoselectivity, and the formation of undesired byproducts. In contrast, enzymatic synthesis offers a more efficient and selective strategy using readily available starting materials under mild conditions. Herein, we report a two-step enzymatic strategy for the synthesis of NAD⁺. In the first step, a ten-enzyme coupled reaction cascade converts glucose into nicotinic acid adenine dinucleotide (NaAD). In the second step, the isolated NaAD is subsequently converted to NAD⁺ by NAD⁺ synthetase. This modular enzymatic platform provides an efficient, scalable approach to NAD⁺ production and establishes a versatile foundation for the biosynthesis of NAD⁺ analogs for biochemical and biomedical applications. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Transformation of plasmid DNA into Rosetta(DE3) cells Basic Protocol 2: Recombinant expression, purification, and activity assessment of NMA1 Basic Protocol 3: Recombinant expression, purification, and activity assessment of Ta1145 Basic Protocol 4: Recombinant expression, purification, and activity assessment of PrsA Basic Protocol 5: Two-step enzymatic synthesis of NAD.