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

Abstract

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.

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

“…end-capped β-alanine (βAla) polymers attached to the peptidyl carrier protein (PCP) CtaE (5,6). However, as all of the biosynthetic intermediates identified to date from both wild-type and knockout strains contain thioamides (5)(6)(7)14), the origin of this PHBA-βAla conjugate was a mystery.…”

“…end-capped β-alanine (βAla) polymers attached to the peptidyl carrier protein (PCP) CtaE (5,6). However, as all of the biosynthetic intermediates identified to date from both wild-type and knockout strains contain thioamides (5)(6)(7)14), the origin of this PHBA-βAla conjugate was a mystery. Further complicating matters, the biosynthetic enzymes expected to be responsible for forming this CtaE-linked intermediate-CtaD, CtaG, and CtaI-belong to protein families more commonly associated with primary metabolism than with NRP assembly, making the a priori prediction of their substrates difficult.…”

“…The CTA biosynthetic locus also encodes a second PCP (Fig. 1B), CtaH, with no proven role in CTA assembly (5,6). Given the conservation of this second PCP in similar biosynthetic gene clusters, we predicted that CtaH would be critical for CTA maturation.…”

“…However, a notable exception came with the recent discovery of a thiotemplated yet NRPS-independent pathway for the biosynthesis of closthioamide (CTA; Fig. 1 A and B) (5,6).…”

“…Knockout studies and in vitro biochemical assays led to the identification of the enzymes responsible for sulfur insertion and late-stage intermediate dimerization (Fig. 1C) (5,6). The biosynthetic strategy for assembly of the amide backbone of CTA has remained a mystery, however.…”