Despite continual advances in antimicrobial therapies, infectious diseases pose a serious threat to global health owing to the ability of microbes to acquire resistance against antibiotics and transform themselves into superbugs. Infectious diseases are an even more serious threat to health during immunocompromised conditions such as HIV/AIDS, diabetes and old age. The recent increase in multidrug-resistant pathogens emphasizes the urgent need for the discovery of therapeutically active novel antimicrobial agents. Lysobacter species are widely distributed in nature and characterized by their ability to lyse other organisms by producing a broad range of proteases and antibiotics. Lysobacter sp. RH2180-5 was isolated from soil in Japan while screening for novel therapeutically active antibiotics using the silkworm infection model. Lysocin E, a therapeutically active novel cyclic peptide produced by Lysobacter sp. RH2180-5, has antimicrobial activity against clinical isolates of methicillin-susceptible and methicillin-resistant Staphylococcus aureus, including S. simulans, S. haemolyticus, S. pseudintermedius, Bacillus subtilis, B. cereus and Listeria monocytogenes with MICs ranging from 1–4 μg ml− 1. Lysocin E exhibits a novel mode of action that involves its binding to menaquinone in the cell membrane. In addition to lysocin E, Lysobacter sp. RH2180-5 produces at least eight more derivatives, most of which have antimicrobial activity. To further improve the production of lysocin E, analyze the derivatives and prepare novel derivatives with improved activity, the genetic analysis of the lysocin biosynthetic gene cluster is required. The critical feature of the lysocin E chemical structure involves 12 amino acids including one N-methylphenylalanine and one fatty acid side chain. On the basis of this structure, we speculated that lysocin E could be synthesized via non-ribosomal peptide synthetases (NRPSs). In this work, we sequenced the whole genome of RH2180-5 and analyzed the putative lysocin biosynthetic gene cluster. The 71 kb DNA contained two large multimodular NRPSs named lysocin E synthetase (Les) A and LesB with a 1.7 MDa core peptide. A total of 12 modules and 43 domains were detected where LesA and LesB contained a loading condensation domain and terminal thioesterase domain, respectively. The specificities of the adenylation domains suggested a linear mode of lysocin biosynthesis, and confirmed the association between the cluster and lysocin biosynthesis.