Targeted Genome Mining Discovery of the Ramoplanin Congener Chersinamycin from the Dynemicin-Producer Micromonospora chersina DSM 44154.

Abstract

The availability of genome sequence data combined with bioinformatic genome mining has accelerated the identification of biosynthetic gene clusters (BGCs). Ramoplanins and enduracidins are lipodepsipeptides produced by Actinoplanes ramoplaninifer ATCC 33076 and Streptomyces fungicidicus B-5477, respectively, that exhibit excellent in vitro activity against a broad spectrum of Gram-positive pathogens. To explore if ramoplanin/enduracidin-like BGCs exist within genomes of organisms sequenced to date, we devised a targeted genome mining strategy that employed structure-activity relationships to identify conserved, essential biosynthesis genes from the ramoplanin and enduracidin BGCs. Five microorganisms were found to contain ramoplanin-like BGCs: the enediyne antibiotic producer Micromonospora chersina strain DSM 44151(dynemycin); the glycopeptide antibiotic producers Amycolatopsis orientalis strain B-37 (norvancomycin), Amycolatopsis orientalis strain DSM 40040 (vancomycin), and Amycolatopsis balhimycina FH1894 strain DSM 44591 (balhimycin); and Streptomyces sp. TLI_053. A single compound from fermentation of M. chersina was purified to homogeneity and found to possess good antibiotic activity against several Gram-positive bacterial test strains (1-2 g/mL), comparing favorably to ramoplanin family members. We named this compound chersinamycin and elucidated its covalent structure, which differs distinctly from ramoplanins and enduracidins. Further, the chersinamycin BGC was validated through insertional gene inactivation using CRISPR-Cas9 gene editing. In addition to the information gained by comparing and contrasting the sequence and organization of these five new BGCs, the amenability of M. chersina to genetic manipulation provides a much-needed tool to investigate the fundamental aspects of lipodepsipeptide biosynthesis and to facilitate metabolic engineering efforts for the production of novel antibiotics capable of combating antibiotic-resistant infections.