Reconstruction of a 30,000-Year-Old Biosynthetic Gene Cluster Reveals Lasso Peptides Formed by a Highly Tolerant Cyclase

Abstract Microbial metagenomes preserved in prehistoric dental calculus samples provide access to past bacterial biodiversity and evolutionary dynamics across archeological time scales. Consequently, this sequence space represents a largely unexplored resource for natural product discovery. Phylogeny-guided genome mining of human dental calculus metagenomes spanning recent times to the Middle and Upper Paleolithic revealed a distinct lasso cyclase lineage associated with oral Actinomyces species. Heterologous expression of a biosynthetic gene cluster reconstructed from an approximately 30,000-year-old sample yielded two lasso peptides, palassoptides A1 and A2. Structure analyses established a canonical class II lasso topology with an eight-residue macrolactam ring. Notably, both palassoptides feature an unprecedented amino acid composition for lasso peptides. Mutational analysis of the PalA2 precursor further showed that PalC accepts all possible substitutions at core position 1 except for Pro, revealing the broadest respective tolerance reported for a lasso cyclase to date. Homologous gene clusters with highly conserved yet variable precursor sequences are widespread among modern oral Actinomyces, indicating long-term preservation of this biosynthetic lineage within the human oral microbiome. The two palassoptides exhibited combination-dependent antibacterial activity against selected Gram-positive bacteria. These findings demonstrate that ancient metagenomes can reveal chemically uncharacterized natural products and expand the known structural diversity of natural products.