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

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 (<b>1</b>) and reductasporine (<b>2</b>), two bioactive TDs. As suggested by our phylogenetic analysis of CPAS genes, identified in our survey of crude eDNA extracts, reductasporine (<b>2</b>) 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

Discovery and Synthetic Refactoring of Tryptophan Dimer Gene Clusters from the Environment

Here we investigate bacterial tryptophan dimer (TD) biosynthesis by probing environmental DNA (eDNA) libraries for chromopyrrolic acid (CPA) synthase genes. Functional and bioinformatics analyses of TD clusters indicate that CPA synthase gene sequences diverge in concert with the functional output of their respective clusters, making this gene a powerful tool for guiding the discovery of novel TDs from the environment. Twelve unprecedented TD biosynthetic gene clusters that can be arranged into five groups (A–E) based on their ability to generate distinct TD core substructures were recovered from eDNA libraries. Four of these groups contain clusters from both cultured and culture independent studies, while the remaining group consists entirely of eDNA-derived clusters. The complete synthetic refactoring of a representative gene cluster from the latter eDNA specific group led to the characterization of the erdasporines, cytotoxins with a novel carboxy-indolocarbazole TD substructure. Analysis of CPA synthase genes in crude eDNA suggests the presence of additional TD gene clusters in soil environments