The NAD salvage pathway enzyme NMNAT-C sustains dark-phase NAD⁺ homeostasis in cyanobacteria

Nicotinamide adenine dinucleotide (NAD⁺) is a crucial cofactor in cyanobacteria, which serve as model organisms for studying photosynthesis. Maintaining NAD⁺ homeostasis in cyanobacteria is critically important, and it is currently believed that multiple pathways contribute to NAD⁺ biosynthesis in these organisms. However, the specific contribution of each pathway to NAD⁺ supplementation under both light and dark conditions, which determines NAD⁺ homeostasis, has not yet been studied. In this study, we identified NMNAT-C, a cyanobacterial nicotinamide nucleotide adenylyltransferase (NMNAT), as a key player in NAD⁺ homeostasis, particularly during dark phases. NMNAT-C showed opposite-phase oscillations in expression, aligned with NAD⁺ fluctuations during light–dark cycles. Genetic and biochemical tests revealed that deleting NMNAT-C in one cyanobacterium (Synechococcus elongatus PCC 7942) accelerated NAD⁺ depletion during dark periods, increased sensitivity to dark stress, and impacted growth rate. Conversely, induced overexpression of NMNAT-C temporarily raised NAD⁺ levels but also caused adverse effects over time. Metabolomic analysis indicated that NMNAT-C plays a role in mediating the metabolic crosstalk between the NAD⁺ salvage pathway and the de novo pathway. Our results identify NMNAT-C as a key regulator of NAD⁺ dynamics that aligns with daily cycles and suggest that this enzyme plays a crucial role in maintaining NAD⁺ homeostasis through the NAD⁺ salvage pathway.