Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic.

Major advances over the past decade in the field of ancient DNA are providing access to past paleogenomic diversity, but the diverse functions and biosynthetic capabilities of this growing paleome remain largely elusive. We investigated the dental calculus of 12 Neanderthals and 52 anatomically modern humans ranging from 100,000 years ago to the present and reconstructed 459 bacterial metagenome-assembled genomes. We identified a biosynthetic gene cluster shared by seven Middle and Upper Paleolithic individuals that allows for the heterologous production of a class of previously unknown metabolites that we name "paleofurans." This paleobiotechnological approach demonstrates that viable biosynthetic machinery can be produced from the preserved genetic material of ancient organisms, allowing access to natural products from the Pleistocene and providing a promising area for natural product exploration.

A Specialized Polythioamide-Binding Protein Confers Antibiotic Self-Resistance in Anaerobic Bacteria.

Understanding antibiotic resistance mechanisms is central to the development of anti-infective therapies and genomics-based drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from less-explored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers self-resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum. Bioinformatics, biochemical analyses, and X-ray crystallography revealed CtaZ as a founding member of a new group of GyrI-like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand-optimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrI-like domain in self-resistance and unprecedented thioamide binding, this work aids in uncovering related drug-resistance mechanisms.

Alternative Benzoxazole Assembly Discovered in Anaerobic Bacteria Provides Access to Privileged Heterocyclic Scaffold.

Benzoxazole scaffolds feature prominently in diverse synthetic and natural product-derived pharmaceuticals. Our understanding of their bacterial biosynthesis is, however, limited to ortho-substituted heterocycles from actinomycetes. We report an overlooked biosynthetic pathway in anaerobic bacteria (typified in Clostridium cavendishii) that expands the benzoxazole chemical space to meta-substituted heterocycles and heralds a distribution beyond Actinobacteria. The first benzoxazoles from the anaerobic realm (closoxazole A and B) were elucidated by NMR and chemical synthesis. By genome editing in the native producer, heterologous expression in Escherichia coli, and systematic pathway dissection we show that closoxazole biosynthesis invokes an unprecedented precursor usage (3-amino-4-hydroxybenzoate) and manner of assembly. Synthetic utility was demonstrated by the precursor-directed biosynthesis of a tafamidis analogue. A bioinformatic survey reveals the pervasiveness of related gene clusters in diverse bacterial phyla.

Enzyme-Primed Native Chemical Ligation Produces Autoinducing Cyclopeptides in Clostridia.

Clostridia coordinate many important processes such as toxin production, infection, and survival by density-dependent communication (quorum sensing) using autoinducing peptides (AIPs). Although clostridial AIPs have been proposed to be (thio)lactone-containing peptides, their true structures remain elusive. Here, we report the genome-guided discovery of an AIP that controls endospore formation in Ruminiclostridium cellulolyticum. Through a combination of chemical synthesis and chemical complementation assays with a mutant strain, we reveal that the genuine chemical mediator is a homodetic cyclopeptide (cAIP). Kinetic analyses indicate that the mature cAIP is produced via a cryptic thiolactone intermediate that undergoes a rapid S→N acyl shift, in a manner similar to intramolecular native chemical ligation (NCL). Finally, by implementing a chemical probe in a targeted screen, we show that this novel enzyme-primed, intramolecular NCL is a widespread feature of clostridial AIP biosynthesis.