An Unexpected Split-Merge Pathway in the Assembly of the Symmetric Nonribosomal Peptide Antibiotic Closthioamide

Closthioamide (CTA) is a symmetric nonribosomal peptide (NRP) comprised of two diaminopropane-linked polythioamidated monomers. CTA is biosynthesized by Ruminiclostridium cellulolyticum via an atypical NRP synthetase (NRPS)-independent biosynthetic pathway. Although the logic for monomer assembly was recently elucidated, the strategy for the biosynthesis and incorporation of the diamine linker remained a mystery. By means of genome editing, synthesis, and in vitro biochemical assays, we demonstrate that the final steps in CTA maturation proceed through a surprising split-merge pathway involving the dual use of a thiotemplated intermediate. This pathway includes the first examples of an aldo-keto reductase catalyzing the reductive release of a thiotemplated product, and of a transthioamidating transglutaminase. In addition to clarifying the remaining steps in CTA assembly, our data shed light on largely unexplored pathways for NRPS-independent peptide biosynthesis.

Automated summary

The final steps of CTA maturation involve a surprising split-merge pathway utilizing a thiotemplated intermediate, with the involvement of an aldo-keto reductase and a transthioamidating transglutaminase. This sheds light on largely unexplored pathways for NRPS-independent peptide biosynthesis.