Date of Award
Doctor of Philosophy (PhD)
Most microbial natural product discovery programs rely on the growth of bacteria in the laboratory, yet it is now well established that the vast majority of bacteria in the environment have not been cultured, particularly from the diverse soil microbiota. By extracting DNA directly from soil samples to construct large archived environmental DNA (eDNA) libraries, thousands of genomes from both cultured and as yet uncultured bacteria can be simultaneously screened for gene clusters encoding natural products of interest. Several natural products with pharmaceutically relevant biological activity arise from the dimerization of tryptophans, such as staurosporine, rebeccamycin, and violacein. To discover novel tryptophan dimers (TDs), we have designed a metagenomics-based TD natural product discovery and development pipeline that consists of seven steps: 1) soil eDNA extraction; 2) eDNA library construction; 3) homology-based screening; 4) bioinformatics analysis; 5) heterologous expression; 6) characterization of compounds and their biosynthesis; 7) target identification. Using a degenerate primer set that targets the CPA synthase gene, one of the conserved genes of tryptophan dimer biosynthesis, we screened the equivalent of ~1 million (over 1 tera base pairs) bacterial genomes from the eDNA libraries, resulting in the discovery of 14 unprecedented TD gene clusters, almost tripling the number of TD gene clusters that have previously been characterized. Using heterologous expression strategies that involve 1) shuttling of pathways into diverse bacterial hosts, 2) overexpression of positive transcriptional regulator, 3) synthetic refactoring of complete pathways, and 4) co-expression of deficient biosynthetic genes, we successfully expressed nine of the 14 gene clusters. This led to the functional characterization of three novel TD families (i.e. indolotryptoline, carboxy-indolocarbazole, and bisindolylmaleimide), consisting of 15 novel natural products (e.g. BE 54017s, borregomycins, erdasporines) with therapeutically relevant bioactivities (e.g. antitumor, antibacterial). Linking biologically active natural products to their cellular targets remains a challenging and critical process in the development of therapeutic agents and small-molecule probes, especially for cytotoxic agents that might serve as anticancer agents. Using multidrug resistance-suppressed (MDR-sup) fission yeast resistant mutant screening, the molecular target of the indolotryptoline family of TD was identified and validated to be the proteolipid subunits of vacuolar H+-ATPase (V-ATPase) at a putative binding site that is distinct from the previously described V-ATPase inhibitors. Together, we demonstrate the utility of this pipeline in the isolation, characterization, and development of novel natural products from the soil bacterial metagenome.
Chang, Fang-Yuan, "Metagenomics-Based Tryptophan Dimer Natural Product Discovery and Development Pipeline" (2015). Student Theses and Dissertations. 276.