Balancing nitrogen metabolism to efficiently drive anti-tuberculosis ilamycins biosynthesis in Streptomyces atratus

The deep-sea-derived Streptomyces atratus SCSIO ZH16 is a promising host for producing nanomole-level anti-tuberculosis ilamycins. However, limited research on regulating the ilamycins biosynthetic gene cluster (BGC) has hindered industrial production. Our previous study found that nitrogen metabolism-related genes were upregulated in strains with enhanced ilamycins production. Since amino acids from nitrogen metabolism are key precursors, we aimed to optimize ilamycins production by balancing BGC expression and nitrogen metabolism. Using RNA-seq and hierarchical clustering, we identified the native promoter P₂₀₆₀₅ and its modified version P_20605-400, which regulate the positive regulator IlaB in ilamycins BGC. To synchronously boost ilamycins synthesis and precursor supply, we analyzed P₂₀₆₀₅'s function via bioinformatics and validated it using an indigoidine biosynthetic model. The engineered strain ΔilaR::P_20605-400-ilaB::P_ermE*-phoP achieved over a dozen-fold increase in ilamycins yield. Fermentation was successfully scaled up in 5-L and 500-L bioreactors, reaching titers of 2,546.4 mg/L and 1,993.9 mg/L, respectively, significantly surpassing previously reported yields. This study highlights the industrial potential of ilamycins and provides insights into enhancing peptide compound production in Streptomyces.