Tunable dynamic engineering of cellular NAD boosts the production of antibiotics in actinomycetes

Nicotinamide adenine dinucleotide (NAD) homeostasis is crucial for secondary metabolism in antibiotic-producing actinomycetes. However, NAD-based dynamic control strategies for boosting antibiotic titers have not been reported. We identified SACE_1905, an alcohol dehydrogenase in Saccharopolyspora erythraea , which converts NADH to NAD + . Overexpressing SACE_1905 lowered the cellular NADH/NAD + ratio, facilitating carbon source utilization and erythromycin biosynthetic precursor supply, concurrently improving cell growth and erythromycin yield. To balance primary and secondary metabolism, we developed a strategy that fine-tunes SACE_1905 expression with inducible clustered regularly interspaced short palindromic repeat interference (CRISPRi) to dynamically modulate the NADH/NAD + ratio, which we named diNAD. Optimized diNAD redirected carbon flux, maximizing erythromycin biosynthesis at a moderate NADH/NAD + ratio during the stationary phase. Based on its utility in actinorhodin and avermectin overproduction in Streptomyces coelicolor and Streptomyces avermitilis , we show that diNAD is effective in augmenting antibiotic titers in actinomycetes.