Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743 - PubMed (original) (raw)

. 2011 Nov 18;6(11):1244-56.

doi: 10.1021/cb200244t. Epub 2011 Sep 20.

Benjamin Janto, Josh Earl, Azad Ahmed, Fen Z Hu, Luisa Hiller, Meg Dahlgren, Rachael Kreft, Fengan Yu, Jeremy J Wolff, Hye Kyong Kweon, Michael A Christiansen, Kristina Håkansson, Robert M Williams, Garth D Ehrlich, David H Sherman

Affiliations

Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743

Christopher M Rath et al. ACS Chem Biol. 2011.

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 anticancer 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 semisynthetic process. On the basis of 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 anticancer 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 analogues 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.

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Figures

Figure 1

Figure 1

Tetrahydroisoquinoline natural products, biosynthetic pathways, and a novel workflow. a) ET-743 (1) and: saframycin A (2), saframycin Mx1 (3), and safracin (4). b) ET-743 core modular NRPS proteins (EtuA1-3) and previously characterized Sfm, Saf, and Sac NRPS biosynthetic systems. NRPS domains are: AL-acyl ligase, T-thiolation, C-condensation, A-adenylation, RE-reductive. c) The experimental workflow: Ecteinascidia turbinata in its natural environment (Cory Walter, Mote Marine Laboratory) is collected (Erich Bartels, Mote Marine Laboratory), and subjected to meta-omic analysis. ET-743 related secondary metabolites were evaluated by Liquid chromatography FTICR mass spectrometry (LC-FTICR-MS/MS), total metagenomic and 16S DNA by 454 metagenomic sequencing with contig assembly, and total metaproteome by nano-LC-FTICR-MS/MS (nLC) and nLC-Orbitrap-MS/MS.

Figure 2

Figure 2

ET-743 related metabolites identified from the tunicate/microbial sample. a) LC-FTICR-MS total ion chromatogram and extracted ion chromatograms for M + H+ (b, c, f, h), and (M – H2O) + H+ (c, e, g, i) for ET-743 (1), ET-597 (19), ET-594 (21), and ET-583 (18). Y axis is in arbitrary units. All identified compounds were verified by collision induced dissociation MS/MS (Table S1).

Figure 3

Figure 3

Multiple sequence alignment tree. 16S rRNA gene sequences reported in previous E. turbinata analyses (26, 27) were aligned with 16S rRNA gene sequences representing the most abundant bacterial populations in our tunicate samples. A 16S rRNA gene-containing contig (00422) clusters with previously identified E. frumentensis.

Figure 4

Figure 4

ET-743 biosynthesis. a) Gene organization and names on the contiguous 35 kb gene cluster. Names relate to proposed function for each protein: formula image-NRPS with domains illustrated, formula image-DNA processing, formula image-fatty-acid enzymes, formula imagehydroxylase, formula imagemethyltransferases, formula imageamidotransferases, formula imagemonoxygenase, formula image-pyruvate cassette, formula image-regulatory enzymes, formula image-drug transporter, **EtuU-**unknown function. b) Proposed biosynthetic pathway for ET-743. Named intermediates (characterized), enzymes (if assigned) and enzyme intermediates (thioester-bound) are shown.

Figure 5

Figure 5

EtuA2 RE and SfmC reactions with (26). a) The proposed biochemical activity of EtuA2 RE-domain in transforming activated didepsipeptide acyl-thioester (10) to the aldehyde (11). b) The analogous reaction for SfmC is the transformation of (26) to (27) as reported by Koketsu (25). The reaction of (26) to (27) was investigated with no enzyme control (c), EtuA2 RE-domain (d), SfmC (e), and an authentic standard of (27) (f). The aldehyde-dipeptide product (27) was monitored as the Na+ adduct in positive ion mode by LC-FTICR MS with an EIC at +/−20 ppm. Inset spectra are shown over the peak elution window (g–i).

Figure 6

Figure 6

Synthetic peptides as authentic standards to verify metaproteomics peptide assignments. a) Total ion chromatogram for the standard peptide mixture on the LTQ-orbitrap. b–g) extracted ion chromatograms generated at +/− 0.1 m/z for each of the synthetic peptides in the mixture. Chromatograms are presented as time versus normalized intensity. Maximum intensity in each normalized total or extracted ion chromatogram is noted. *Denotes that the experimental retention time for doubly protonated tryptic LLDVGGGTAINAIALAK was obtained on a different LC system with a different gradient and column, as compared to the authentic standard. In the case of all other synthetic standard versus experimental identifications the LC system and gradient were identical, although a different column was used. ◊ denotes the elution time of the experimental MS2 spectra assigned to each of the peptides. Peptide MS2 sequence coverage for metaproteomics versus authentic standard synthetic peptides (h–m). Only b and y ion assignments are shown although other ions (e.g., a, b - H2O, b - NH3, y - H2O, and y - NH3) could also be assigned. Multiple bars indicate that a given fragment can be assigned to multiple charge states.

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