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

Angew Chem Int Ed Engl. 2021 Feb 19;60(8):4104-4109. doi: 10.1002/anie.202011741. Epub 2020 Dec 23.


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.

Keywords: antibiotics; enzymes; natural products; nonribosomal peptides; thioamide.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldo-Keto Reductases / genetics
  • Aldo-Keto Reductases / metabolism
  • Anti-Bacterial Agents / analysis
  • Anti-Bacterial Agents / biosynthesis*
  • Anti-Bacterial Agents / chemistry
  • Biocatalysis
  • Chromatography, High Pressure Liquid
  • Clostridiales / genetics
  • Clostridiales / metabolism
  • Gene Editing
  • Multigene Family
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Thioamides / analysis
  • Thioamides / chemistry
  • Thioamides / metabolism*
  • Transaminases / genetics
  • Transaminases / metabolism
  • Transglutaminases / genetics
  • Transglutaminases / metabolism


  • Anti-Bacterial Agents
  • Thioamides
  • closthioamide
  • Aldo-Keto Reductases
  • Transglutaminases
  • Transaminases