<jats:title>Abstract</jats:title><jats:p>Antimicrobial agents are urgently needed to tackle the growing threat of antibiotic-resistant pathogens. An important source of new antimicrobials is the large repertoire of cryptic gene clusters embedded in microbial genomes. Genome mining revealed a napsamycin/mureidomycin biosynthetic gene cluster in the chromosome of <jats:italic>Streptomyces roseosporus</jats:italic> NRRL 15998. The cryptic gene cluster was activated by constitutive expression of a foreign activator gene <jats:italic>ssaA</jats:italic> from sansanmycin biosynthetic gene cluster of <jats:italic>Streptomyces</jats:italic> sp. strain SS. Expression of the gene cluster was verified by RT-PCR analysis of key biosynthetic genes. The activated metabolites demonstrated potent inhibitory activity against the highly refractory pathogen <jats:italic>Pseudomonas aeruginosa</jats:italic> and characterization of the metabolites led to the discovery of eight acetylated mureidomycin analogues. To our surprise, constitutive expression of the native activator gene <jats:italic>SSGG_02995</jats:italic>, a <jats:italic>ssaA</jats:italic> homologue in <jats:italic>S. roseosporus</jats:italic> NRRL 15998, has no beneficial effect on mureidomycin stimulation. This study provides a new way to activate cryptic gene cluster for the acquisition of novel antibiotics and will accelerate the exploitation of prodigious natural products in <jats:italic>Streptomyces</jats:italic>.