Under standard laboratory conditions, Streptomyces formicae produces the polyketide antibiotics formicamycins and an uncharacterised antifungal molecule. However, genome sequencing shows S. formicae encodes as many as 45 secondary metabolite biosynthetic gene clusters (BGCs), most of which look novel. By inducing the cryptic pathways encoded in S. formicae, we can access the untapped biosynthetic potential for antimicrobial molecules. Here, we show that deleting the entire formicamycin BGC induces production of the potent antimicrobial peptide, LL-A0341. LL-A0341 is a cyclic depsipeptide first isolated from S. canus in the 1960s, but no BGC has been identified which has limited its further development. Using a combination of proteomics, transcriptomics and metabolomics, we identified the BGC responsible for LL-A0341 production in S. formicae and confirmed this by CRISPR/Cas9 deletion of core genes. We then used gene editing to rewire the LL-A0341 BGC to increase compound yields and isolated enough for both structural and bioactivity studies. LL-A0341 is a potent inhibitor of Gram-positive microorganisms, including Staphylococcus aureus and Mycobacterium smegmatis. Using DiSC-35 assays, qPCR and surface plasmon resonance (SPR), we show that LL-A0341 targets the bacterial cell membrane by binding to the phospholipid, cardiolipin. This work shows how mining the genomes of talented Actinomycetes opens the door for antibiotic discovery and the further development of clinically relevant molecules.