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.