Biotechnological production of reduced and oxidized NAD⁺ precursors

Dysregulation of nicotinamide adenine dinucleotide (NAD⁺) homeostasis by increased activity of NAD⁺ consumers or reduced NAD⁺ biosynthesis plays an important role in the onset of prevalent, often age-related, diseases, such as diabetes, neuropathies or nephropathies. To counteract such dysregulation, NAD⁺ replenishment strategies can be used. Among these, administration of vitamin B₃ derivatives (NAD⁺ precursors) has garnered attention in recent years. However, the high market price of these compounds and their limited availability, pose important limitations to their use in nutritional or biomedical applications. To overcome these limitations, we have designed an enzymatic method for the synthesis and purification of (1) the oxidized NAD⁺ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), (2) their reduced forms NMNH and NRH, and (3) their deaminated forms nicotinic acid mononucleotide (NaMN) and nicotinic acid riboside (NaR). Starting from NAD⁺ or NADH as substrates, we use a combination of three highly overexpressed soluble recombinant enzymes; (a) a NAD+ pyrophosphatase, (b) an NMN deamidase, and (c) a 5'-nucleotidase, to produce these six precursors. Finally, we validate the activity of the enzymatically produced molecules as NAD⁺ enhancers in cell culture.