Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors

Significance Here we use an informatics-based approach to natural product discovery that is broadly applicable to the isolation of medicinally relevant metabolites from environmental microbiomes. Combining metagenome sequencing and bioinformatics approaches with a defined set of metagenomic tools provides a template for the targeted discovery of compounds from the global metagenome. The power of this approach is demonstrated by surveying ketosynthase domain amplicon sequencing data from 185 soil microbiomes for biosynthetic gene clusters encoding epoxyketone proteasome inhibitors, leading to the isolation and characterization of seven epoxyketone natural products, including compounds with unique warhead structures. We believe this approach is applicable to any conserved biosynthetic gene and provides a higher-throughput cost-effective alternative to whole genome sequencing discovery methods. In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A–E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.