Meta-omic Characterization of the Marine Invertebrate Microbial Consortium That Produces the Chemotherapeutic Natural Product ET-743

Abstract

In many macroorganisms, 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.

Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance

“…Interestingly, within this non-chelating subset, four compounds show an outstandingly high toxicity. Even though they contain the substructure motive by which they were identified, their structure is very different from the other compounds: They are derivatives of the marine natural product Trabectedin (ecteinascidin-743, Figure S12 D), which is currently undergoing clinical trials (phase III) in the USA, while it is approved for cancer treatment in Europe [105][106][107].…”

Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses

“…In a number of bacterial-eukaryote symbioses, bacterial partners have been implicated in the production of complex molecules derived from polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways (3,8,9). Examples include pederin, made by bacteria that live in rove beetles of the genus Paederus, and structurally related metabolites, the onnamides and psymberin, produced by bacteria that live in marine sponges (Fig.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…Nonetheless, the natural products whose biosynthetic loci have been identified/cloned using metagenomic approaches might be suitable candidates for the preclinical drug discovery pipeline and are listed in Table S1. Recently, a biosynthetic gene cluster (NRPS pathway) for Yondelis (Et-743), an approved anticancer drug, was cloned and analyzed using metagenomic sequencing of the total DNA from a tunicate-microbial consortium (Rath, Janto et al 2011). This molecule is obtained in low quantity from the tunicate Ecteinascidia turbinate and generated for clinical use by a cumbersome semisynthetic method.…”

“…This molecule is obtained in low quantity from the tunicate Ecteinascidia turbinate and generated for clinical use by a cumbersome semisynthetic method. The work by Rath et al (2011) laid the foundation for direct production of the drug and its analogues using metagenomic approaches. The scope of metagenomics, together with metabolic engineering and other contemporary technologies, in the drug discovery pipeline is broad and can be envisaged by the proportion of small molecule compounds and biologicals in the total number of approved drugs by the US FDA.…”

“…The development and use of broad-range host vectors and alternate hosts (see Table S2) have addressed this issue considerably. An increasing number of biosynthetic gene loci are being cloned from metagenomic samples both from symbiotic microorganisms as well as from environmental samples, which will provide new lead molecules for the pre-clinical phases in the drug discovery pipeline (Rath, Janto et al 2011). …”