Identification of the Biosynthetic Gene Cluster for the anti-MRSA Lysocins through Gene Cluster Activation Using Strong Promoters of Housekeeping Genes and Production of New Analogs in Lysobacter sp. 3655

The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natural products. In an effort to discover new antibiotics, we found a cryptic biosynthetic gene cluster (BGC) in Lysobacter sp. 3655 that shared a high similarity with the putative lysocin BGC identified in silico previously from Lysobacter sp. RH2180-5. Lysocins are cyclic lipodepsipeptides with potent activity against MRSA (methicillin-resistant Staphylococcus aureus) using a novel mode of action, but the lysocin BGC had not been experimentally verified so far. Using an activity-guided screening, we isolated the main antibiotic compound and confirmed it to be lysocin E. However, the putative lysocin BGC was barely transcribed in the wild type, in which lysocins were produced only in specific conditions and in a negligible amount. To activate the putative lysocin BGC, we screened for strongly transcribed housekeeping genes in strain 3655 and found several powerful promoters. Upon engineering the promoters into the BGC, the lysocin gene transcription was significantly enhanced and the lysocin yield was markedly increased. With readily detectable lysocins production in the engineered strains, we showed that lysocin production was abolished in the gene deletion mutant and then restored in the complementary strain, even when grown in conditions that did not support the wild type for lysocin production. Moreover, the engineered strain produced multiple new lysocin congeners. The determination of the lysocin BGC and the Lysobacter promoters will facilitate the ongoing efforts for yield improvement and new antibiotic biosynthesis using synthetic biology strategies.