Arixanthomycins A–C: Phylogeny-Guided Discovery of Biologically Active eDNA-Derived Pentangular Polyphenols

Soil microbiomes are a rich source of uncharacterized natural product biosynthetic gene clusters. Here we use short conserved biosynthetic gene sequences (natural product sequence tags) amplified from soil microbiomes as phylogenetic markers to correlate genotype to chemotype and target the discovery of novel bioactive pentangular polyphenols from the environment. The heterologous expression of an environmental DNA-derived gene cluster (the ARX cluster), whose ketosynthase beta (KS_β) sequence tag was phylogenetically distinct from any known KS_β sequence, led to the discovery of the arixanthomycins. Arixanthomycin A (<b>1</b>) exhibits potent antiproliferative activity against human cancer cell lines

Mining Soil Metagenomes to Better Understand the Evolution of Natural Product Structural Diversity: Pentangular Polyphenols as a Case Study

Sequence-guided mining of metagenomic libraries provides a means of recovering specific natural product gene clusters of interest from the environment. In this study, we use ketosynthase gene (KS) PCR amplicon sequences (sequence tags) to explore the structural and biosynthetic diversities of pentangular polyphenols (PP). In phylogenetic analyses, eDNA-derived sequence tags often fall between closely related clades that are associated with gene clusters known to encode distinct chemotypes. We show that these common “intermediate” sequence tags are useful for guiding the discovery of not only novel bioactive metabolites but also collections of closely related gene clusters that can provide new insights into the evolution of natural product structural diversity. Gene clusters corresponding to two eDNA-derived KS_β sequence tags that reside between well-defined KS_β clades associated with the biosynthesis of (C24)-pradimicin and (C26)-xantholipin type metabolites were recovered from archived soil eDNA libraries. Heterologous expression of these gene clusters in <i>Streptomyces albus</i> led to the isolation of three new PPs (compounds <b>1</b>–<b>3</b>). Calixanthomycin A (<b>1</b>) shows potent antiproliferative activity against HCT-116 cells, whereas arenimycins C (<b>2</b>) and D (<b>3</b>) display potent antibacterial activity. By comparing genotypes and chemotypes across all known PP gene clusters, we define four PP subfamilies, and also observe that the horizontal transfer of PP tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures, suggesting that only a fraction of the “natural product-like” chemical space that can theoretically be encoded by these secondary metabolite tailoring genes has likely been sampled naturally

Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast

The use of DNA sequencing to guide the discovery of natural products has emerged as a new paradigm for revealing chemistries encoded in bacterial genomes. A major obstacle to implementing this approach to natural product discovery is the transcriptional silence of biosynthetic gene clusters under laboratory growth conditions. Here we describe an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and TAR to enable single-marker multiplexed promoter engineering of large gene clusters. mCRISTAR highlights the first application of the CRISPR/Cas9 system to multiplexed promoter engineering of natural product biosynthetic gene clusters. In this method, CRISPR/Cas9 is used to induce DNA double-strand breaks in promoter regions of biosynthetic gene clusters, and the resulting operon fragments are reassembled by TAR using synthetic gene-cluster-specific promoter cassettes. mCRISTAR uses a CRISPR array to simplify the construction of a CRISPR plasmid for multiplex CRISPR and a single auxotrophic selection to improve the inefficiency of using a CRISPR array for multiplex gene cluster refactoring. mCRISTAR is a simple and generic method for multiplexed replacement of promoters in biosynthetic gene clusters that will facilitate the discovery of natural products from the rapidly growing collection of gene clusters found in microbial genome and metagenome sequencing projects

Malleilactone, a Polyketide Synthase-Derived Virulence Factor Encoded by the Cryptic Secondary Metabolome of Burkholderia pseudomallei Group Pathogens

Sequenced bacterial genomes are routinely found to contain gene clusters that are predicted to encode metabolites not seen in fermentation-based studies. Pseudomallei group <i>Burkholderia</i> are emerging pathogens whose genomes are particularly rich in cryptic natural product biosynthetic gene clusters. We systematically probed the influence of the cryptic secondary metabolome on the virulence of these bacteria and found that disruption of the MAL gene cluster, which is natively silent in laboratory fermentation experiments and conserved across this group of pathogens, attenuates virulence in animal models. Using a promoter exchange strategy to activate the MAL cluster, we identified malleilactone, a polyketide synthase-derived cytotoxic siderophore encoded by this gene cluster. Small molecules targeting malleilactone biosynthesis either alone or in conjunction with antibiotics could prove useful as therapeutics to combat melioidosis and glanders

Mutations in the Proteolipid Subunits of the Vacuolar H⁺‑ATPase Provide Resistance to Indolotryptoline Natural Products

Indolotryptoline natural products represent a small family of structurally unique chromopyrrolic acid-derived antiproliferative agents. Like many prospective anticancer agents before them, the exploration of their potential clinical utility has been hindered by the limited information known about their mechanism of action. To study the mode of action of two closely related indolotryptolines (BE-54017, cladoniamide A), we selected for drug resistant mutants using a multidrug resistance-suppressed (MDR-sup) <i>Schizosaccharomyces pombe</i> strain. As fission yeast maintains many of the basic cancer-relevant cellular processes present in human cells, it represents an appealing model to use in determining the potential molecular target of antiproliferative natural products through resistant mutant screening. Full genome sequencing of resistant mutants identified mutations in the c and c′ subunits of the proteolipid substructure of the vacuolar H⁺-ATPase complex (V-ATPase). This collection of resistance-conferring mutations maps to a site that is distant from the nucleotide-binding sites of V-ATPase and distinct from sites found to confer resistance to known V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies, and direct c/c′ subunit mutagenesis all suggest that indolotryptolines are likely a structurally novel class of V-ATPase inhibitors. This work demonstrates the general utility of resistant mutant selection using MDR-sup <i>S. pombe</i> as a rapid and potentially systematic approach for studying the modes of action of cytotoxic natural products

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

Natural Product Discovery through Improved Functional Metagenomics in <i>Streptomyces</i>

Because the majority of environmental bacteria are not easily culturable, access to many bacterially encoded secondary metabolites will be dependent on the development of improved functional metagenomic screening methods. In this study, we examined a collection of diverse <i>Streptomyces</i> species for the best innate ability to heterologously express biosynthetic gene clusters. We then optimized methods for constructing high quality metagenomic cosmid libraries in the best <i>Streptomyces</i> host. An initial screen of a 1.5 million-membered metagenomic library constructed in <i>Streptomyces albus</i>, the species that exhibited the highest propensity for heterologous expression of gene clusters, led to the identification of the novel natural product metatricycloene (<b>1</b>). Metatricycloene is a tricyclic polyene encoded by a reductive, iterative polyketide-like gene cluster. Related gene clusters found in sequenced genomes appear to encode a largely unexplored collection of structurally diverse, polyene-based metabolites

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

The Chemical Arsenal of <i>Burkholderia pseudomallei</i> Is Essential for Pathogenicity

Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe–host response in infection and pathogenicity. The bacterial disease melioid­osis is conferred by the highly virulent, antibiotic-resistant pathogen <i>Burkholderia pseudomallei</i> (<i>BP</i>). Whereas some macromolecular structures have been shown to influence <i>BP</i> virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that <i>BP</i>-encoded small-molecule biosynthesis is indispensible for <i>in vivo BP</i> pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for <i>in vivo</i> virulence. NMR structural characterization of these metabolites identified a new class of lipo­peptide biosurfactants/biofilm modulators (the mallei­peptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for <i>BP</i>. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioid­osis-specific therapeutics

Environmental DNA-Encoded Antibiotics Fasamycins A and B Inhibit FabF in Type II Fatty Acid Biosynthesis

In a recent study of polyketide biosynthetic gene clusters cloned directly from soil, we isolated two antibiotics, fasamycins A and B, which showed activity against methicillin-resistant <i>Staphylococcus aureus</i> and vancomycin-resistant <i>Enterococcus faecalis</i>. To identify the target of the fasamycins, mutants with elevated fasamycin A minimum inhibitory concentrations were selected from a wild-type culture of <i>E. faecalis</i> OG1RF. Next-generation sequencing of these mutants, in conjunction with <i>in vitro</i> biochemical assays, showed that the fasamycins inhibit FabF of type II fatty acid biosynthesis (FASII). Candidate gene overexpression studies also showed that fasamycin resistance is conferred by <i>fabF</i> overexpression. On the basis of comparisons with known FASII inhibitors and <i>in silico</i> docking studies, the chloro-<i>gem</i>-dimethyl-anthracenone substructure seen in the fasamycins is predicted to represent a naturally occurring FabF-specific antibiotic pharmacophore. Optimization of this pharmacophore should yield FabF-specific antibiotics with increased potencies and differing spectra of activity. This study demonstrates that culture-independent antibiotic discovery methods have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use