Chemical diversity and metabolic versatility in microbial natural product biosynthesis

In many marine macroorganisms (e.g. sponges, tunicates, soft corals), the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia we chose to investigate the ET-743 (Yondelis®) biosynthetic pathway. This molecule is an approved anti-cancer agent obtained in low abundance (10^-4-10^-5% w/w) from the tunicate Ecteinascidia turbinata, and is generated in suitable quantities for clinical use by a lengthy semi-synthetic process. Based on structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anti-cancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogs through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

In another effort, we are pursuing studies of diverse natural product cytochrome P450 enzymes as biocatalysts toward C-H bond activation against native and unnatural substrates. This work was initiated by characterization of the anchor-group directed pikromycin pathway PikC monooxygenase. More recently, we have identified and probed the function and specificity of an iterative P450 enzyme that catalyzes three sequential oxidation steps including 2^o hydroxylation, epoxidation and 1^o hydroxymethylation. Structural studies are providing deep insights into the basis for regio- and stereoselective C-H bond oxidation, and are facilitating efforts to apply these remarkable biocatalysts for creation of structurally diverse biologically active molecules.

This work was supported by NIH grants R01 GM078553, U01 U01 TW007404 and the H. W. Vahlteich Professorship.