Closthioamide (CTA) is a unique symmetric nonribosomal peptide with six thioamide moieties that is produced by the Gram-positive obligate anaerobe Ruminiclostridium cellulolyticum. CTA displays potent inhibitory activity against important clinical pathogens, making it a promising drug candidate. Yet, the biosynthesis of this DNA gyrase-targeting antibiotic has remained enigmatic. Using a combination of genome mining, genome editing (targeted group II intron, CRISPR/Cas9), and heterologous expression, we show that CTA biosynthesis involves specialized enzymes for starter unit biosynthesis, amide bond formation, thionation, and dimerization. Surprisingly, CTA biosynthesis involves a novel thiotemplated peptide assembly line that markedly differs from known nonribosomal peptide synthetases. These findings provide the first insights into the biosynthesis of thioamide-containing nonribosomal peptides and offer a starting point for the discovery of related natural products.
Closthioamide (CTA) is au nique symmetric nonribosomal peptide with six thioamide moieties that is produced by the Gram-positive obligate anaerobe Ruminiclostridium cellulolyticum. CTAdisplays potent inhibitory activity against important clinical pathogens,m aking it ap romising drug candidate.Y et, the biosynthesis of this DNAg yrase-targeting antibiotic has remained enigmatic. Using ac ombination of genome mining,g enome editing (targeted group II intron, CRISPR/Cas9), and heterologous expression, we show that CTAbiosynthesis involves specialized enzymes for starter unit biosynthesis,a mide bond formation, thionation, and dimerization. Surprisingly,C TA biosynthesis involves an ovel thiotemplated peptide assembly line that markedly differs from knownn onribosomal peptide synthetases.T hese findings providet he first insights into the biosynthesis of thioamidecontaining nonribosomal peptides and offer astarting point for the discovery of related natural products.
Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.
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