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

Abstract

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.T he advent of cost-effective high-throughput sequencing and an increasingly sophisticated understanding of bacterial secondary metabolite biosynthesis have led to two important revelations with respect to the search for new natural products: first, that the biosynthetic potential of most cultured bacteria, as judged by the number of biosynthetic gene clusters (BGCs) observed in sequenced genomes, is far greater than previously estimated (1, 2); second, that the number of bacterial species in most environments is at least 100× greater than the number of species that is readily cultured (3, 4). These observations suggest that conventional "phenotype-first" natural products isolation approaches have only examined a small fraction of earth's bacterial biosynthetic potential.There are now a number of genomic search engines available that allow researchers to rapidly scan microbial whole genome sequences for BGCs encoding new natural products (5-7). Unfortunately, the large DNA contigs that these search strategies require as input are not readily available from complex metagenomes. In response to the need for a more robust metagenomic search strategy, our group recently developed an informatics platform called eSNaPD (8, 9) (environmental Surveyor of Natural Product Diversity) with the specific aim of facilitating sequence-guided discovery of new bacterial natural products from complex metagenomes (Fig. 1).The eSNaPD software is designed to bioinformatically assess short DNA sequences that have been amplified from environmental metagenomes by degenerate PCR targeting c...

Urban park soil microbiomes are a rich reservoir of natural product biosynthetic diversity

“…We have shown eSNaPD classification to be a robust indicator of a functional relationship between a microbiome-derived gene cluster yielding an AD or KS domain sequence tag and a curated known gene cluster (20)(21)(22). Based on empirical data from previous metagenomic analyses (20)(21)(22)(23), the homology cutoffs used in this study (e value < 10 −100 ) will identify gene clusters with a high likelihood of encoding either the same metabolite or a novel derivative (congener) of the metabolite encoded by the matching curated gene cluster.…”

“…AD and KS amplicons were assigned to known biosynthetic gene clusters using the eSNaPD program (18,19). Empirical exploration with the eSNaPD algorithm has shown that e values as high as 10 −40 to −60 return reliable gene cluster predictions (20)(21)(22). We used an expectation value to 10 −100 for this analysis.…”

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…Five studies also chose to use shotgun metagenomic methods, which do not require the use of DNA primers, to identify prokaryote DNA from metagenomes (not shown in Fig. 6; Delmont et al 2011;Nakai et al 2011;Inskeep et al 2013;Costa et al 2015;Owen et al 2015).…”

Mass spectrometry tools and workflows for revealing microbial chemistry

“…Genome mining is a process in which molecules are discovered by predicting what compound will be biosynthesized based on the sequences of gene clusters. Interpretation of NP-encoding genes and their organization can predict new pathways for discovery of silent or ‘cryptic’ biosynthetic gene clusters 128, 132, 186–188 . Combining the developments in tandem MS methods enable one to correlate the production of NPs with the genomic capacity of the microorganism in query or in an interactive complex sample.…”