Genome Editing Reveals Novel Thiotemplated Assembly of Polythioamide Antibiotics in Anaerobic Bacteria

Angew Chem Int Ed Engl. 2018 Oct 22;57(43):14080-14084. doi: 10.1002/anie.201807970. Epub 2018 Oct 3.


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.

Keywords: biosynthesis; clostridia; natural products; thioamides; thiotemplate systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / chemistry*
  • Anti-Bacterial Agents / pharmacology
  • Bacteria, Anaerobic / chemistry*
  • Bacteria, Anaerobic / genetics
  • CRISPR-Cas Systems
  • Carbon-13 Magnetic Resonance Spectroscopy
  • Chromatography, High Pressure Liquid
  • Clostridiales / chemistry*
  • Clostridiales / genetics
  • DNA Gyrase / drug effects
  • Gene Editing*
  • Genes, Bacterial
  • Introns
  • Mass Spectrometry
  • Multigene Family
  • Peptide Synthases / chemistry
  • Proton Magnetic Resonance Spectroscopy
  • Thioamides / chemistry*
  • Thioamides / pharmacology


  • Anti-Bacterial Agents
  • Thioamides
  • DNA Gyrase
  • Peptide Synthases
  • non-ribosomal peptide synthase