Actinomycin analogs from soil-derived Streptomyces sp. PSU-S4-23 with activity against MRSA

Genome-based discovery provides a powerful approach for identifying bioactive natural products. Using this approach, we identified actinomycin production in Streptomyces sp. PSU-S4-23. The genome contains a nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster closely matching the actinomycin D reference (BGC0000296), including two NRPS genes encoding the canonical five-module assembly line and a neighboring cytochrome P450 consistent with oxidative tailoring. Culture extracts were tested and showed antibacterial activity against Gram-positive species Staphylococcus aureus (both MSSA and MRSA), Bacillus subtilis, Bacillus cereus, Enterococcus faecalis , and Staphylococcus epidermidis with additional inhibition of the Gram-negative pathogens Pseudomonas aeruginosa and Acinetobacter baumannii in agar diffusion assays. LC–MS/MS profiling of active fractions revealed a tight chromatographic window with ions diagnostic for actinomycins, featuring [M + H] ⁺ at m/z 1,269.6, 1,255.6, and 1,271.6 corresponding to actinomycin X ₂ , actinomycin D, and actinomycin I, respectively. Comparative genomics placed PSU-S4-23 within Streptomyces and closest to S. caeni CGMCC 4.7426 ^T , while ANIb (94.69%) and dDDH (68.2%) values were below species thresholds, indicating a distinct lineage. Overall, the agreement between the actinomycin-like BGC and LC–MS/MS evidence establishes Streptomyces sp. PSU-S4-23 as a producer of actinomycin analogs, predominantly actinomycin X ₂ and underscores genome mining as an efficient strategy to prioritize strains for antibiotic discovery.