Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment

Abstract

Natural product discovery from environmental genomes (metagenomics) has largely been limited to the screening of existing environmental DNA (eDNA) libraries. Here, we have coupled a chemical-biogeographic survey of chromopyrrolic acid synthase (CPAS) gene diversity with targeted eDNA library production to more efficiently access rare tryptophan dimer (TD) biosynthetic gene clusters. A combination of traditional and synthetic biology-based heterologous expression efforts using eDNA-derived gene clusters led to the production of hydroxysporine (1) and reductasporine (2), two bioactive TDs. As suggested by our phylogenetic analysis of CPAS genes, identified in our survey of crude eDNA extracts, reductasporine (2) contains an unprecedented TD core structure: a pyrrolinium indolocarbazole core that is likely key to its unusual bioactivity profile. This work demonstrates the potential for the discovery of structurally rare and biologically interesting natural products using targeted metagenomics, where environmental samples are prescreened to identify the most phylogenetically unique gene sequences and molecules associated with these genes are accessed through targeted metagenomic library construction and heterologous expression.

Bacterial natural product biosynthetic domain composition in soil correlates with changes in latitude on a continent-wide scale

“…One of the ultimate goals of studying NP biosynthetic diversity is to guide the identification of new bioactive metabolites. One way NPST data can assist with this is by identifying environments that are rich in gene cluster families that are known to encode clinically important metabolites (29)(30)(31)(32)(33)(34)(35). Biosynthetic gene clusters that encode families of structurally related metabolites generally share a common evolutionary ancestor and therefore exhibit high sequence identity.…”

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.…”

Using natural products for drug discovery: the impact of the genomics era

“…Screening is especially challenging for large complex metagenomes such as soil, which can contain up to 10 5 unique species and require multimillion-membered mega-libraries for sufficient coverage [87, 88]. Conventional function- and sequence-based methods involve screening libraries for easily observable phenotypes or the presence of target DNA sequences, respectively (Figure 2a).…”

“…New chemical functionalities can also be discovered using this method. A new subclass of natural tryptophan dimers possessing a pyrrolinium indolocarbazole core was uncovered by focusing on eDNA-derived gene clusters with sequence tags that are not closely associated with characterized tryptophan dimer BGCs [88]. In general, while targeted metagenome mining may not be suitable for the discovery of fundamentally distinct chemical entities, it can be used to survey the biosynthetic potential of environmental samples as well as identify and recover novel variants of pharmaceutically relevant BGCs for downstream analyses (Figure 2b).…”