Posttranslational β-methylation and macrolactamidination in the biosynthesis of the bottromycin complex of ribosomal peptide antibiotics

Bottromycins are unique peptide antibiotics that contain a macrolactamidine, several non-proteinogenic amino acids and a thiazole. We hypothesised that the bottromycins originate from a ribosomally biosynthesised precursor that is elaborated to the mature antibiotics via a series of unusual. posttranslational modifications, including beta-methylation of Phe, Val and Pro residues, and proteolytic macrolactamidine formation. To investigate this hypothesis, we generated a draft genome sequence of the bottromycin producer Streptomyces bottropensis DSM 40262 in which we identified a gene encoding a polypeptide containing a sequence corresponding to the putative precursor of the bottromycins. Deletion of the gene encoding this putative precursor peptide abolished production of bottromycins in S. bottropensis. Bottromycin production was restored in the resulting mutant by in trans expression of the entire operon containing the precursor peptide gene. In contrast to other posttranslationally modified ribosomal peptide antibiotics, the bottromycin precursor peptide lacks an N-terminal "leader" sequence. Instead, it contains a C-terminal "follower" sequence that is removed during posttranslational processing and that is presumably required for correct maturation of the antibiotic. Comparative sequence analyses of the proteins encoded by the genes flanking the precursor peptide gene identified ten enzymes that are likely to catalyse the posttranslational modifications required for bottromycin assembly. A gene cluster that is essentially identical to the S. bottropensis bottromycin biosynthetic gene cluster was identified in the complete genome sequence of the plant pathogen Streptomyces scabies 87.22 and LC-MS analyses confirmed that S. scabies is a novel producer of bottromycins. Our findings set the stage for engineered biosynthesis of novel bottromycin analogues and delineate a plausible pathway for bottromycin biosynthesis.