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Ancient plant-like terpene biosynthesis in corals

Abstract

Octocorals are major contributors of terpenoid chemical diversity in the ocean. Natural products from other sessile marine animals are primarily biosynthesized by symbiotic microbes rather than by the host. Here, we challenge this long-standing paradigm by describing a monophyletic lineage of animal-encoded terpene cyclases (TCs) ubiquitous in octocorals. We characterized 15 TC enzymes from nine genera, several of which produce precursors of iconic coral-specific terpenoids, such as pseudopterosin, lophotoxin and eleutherobin. X-ray crystallography revealed that coral TCs share conserved active site residues and structural features with bacterial TCs. The identification of coral TCs enabled the targeted identification of the enzyme that constructs the coral-exclusive capnellane scaffold. Several TC genes are colocalized with genes that encode enzymes known to modify terpenes. This work presents an example of biosynthetic capacity in the kingdom Animalia that rivals the chemical complexity generated by plants, unlocking the biotechnological potential of octocorals for biomedical applications.

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Fig. 1: Bioactive terpenoids extracted from different octocorals linked to their proposed biosynthetic precursor terpenes.
Fig. 2: Phylogeny of octocoral TCs and their terpene products.
Fig. 3: Structure of the coral cembrene A synthase ErTC-2 and other TCs.
Fig. 4: Possible timeline of TC evolution in octocorals.
Fig. 5: Directed approach to the discovery of capnellene synthase CiTC-1.

Data availability

Shotgun sequencing reads for C. imbricata are available at NCBI SRA under project accession PRJNA761189. The crystal structure of ErTC-2 has been deposited at PDB under the accession number 7S5L. NMR and electron ionization MS spectra of characterized compounds are available in the Supplementary Information. Structure elucidation data are shown in Supplementary Note 1. Sequences used in this study are listed in Supplementary Note 2. Accession numbers of proteins used for HMM building are listed in Supplementary Table 1. Accession data for the literature genomic and transcriptomic data used in this study are summarized in Supplementary Tables 2 and 3, respectively. Accession data for raw genomic data used for mapping are listed in Supplementary Table 4. Accession numbers for proteins used for the phylogenetic analysis are listed in Supplementary Table 10. The PDB accession numbers for reference protein structures in Fig. 3 are 4LZ0, 3P5R and 5JA0.

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Acknowledgements

We thank our University of California San Diego colleagues W. Fenical, L. Aluwihare and B. Duggan for critical feedback, access to GC–MS equipment and assistance with NMR measurements, respectively, C. Delbeek (California Academy of Sciences) for access to C. imbricata, J. Keasling and J. Blake-Hedges (University of California Berkeley) for assisting in genome sequencing, J. Bailey and the University of California San Diego Crystallography Facility for collecting the X-ray diffraction data used for phasing and J.P. Noel and G. Louie (Salk Institute for Biological Studies) for beamtime coordination. Use of the Stanford Synchrotron Radiation Lightsource is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-AC02-76SF00515, the Department of Energy Office of Biological and Environmental Research and the NIH and NIGMS (including P41GM103393). This work was supported by National Institutes of Health awards F32GM129960 to T.d.R. and R01GM085770 to B.S.M. and a Leopoldina postdoctoral fellowship (LPDS 2019-04) to I.B.

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Contributions

I.B., T.d.R. and B.S.M. conceived the project, designed the experiments, analyzed the data and wrote the paper with input from all authors. I.B. performed the protein expression and purification, enzymatic assays, product analyses and data analyses with help from T.d.R. who additionally performed the C. imbricata experiments. P.Y.-T.C. performed crystallography experiments and subsequent data analysis.

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Correspondence to Bradley S. Moore.

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Nature Chemical Biology thanks Thu-Thuy Dang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Radial depiction of the phylogenetic tree from Fig. 2.

The IDS clade is used as outgroup and bootstrap values for the branches are shown. The representative enzymes used for structure comparison in Fig. 3 are indicated by arrows.

Extended Data Fig. 2 Examples of octocoral derived terpenoid structures sharing a common terpene carbon backbone8.

Structures marked with an asterisk have been found as both enantiomers.

Extended Data Fig. 3 Genetic context of octocoral TCs.

Mapped transcripts (top), gene annotations (middle) and mapped long-reads (bottom) for the indicated regions are shown. a) Context of XsTC-1 from Xenia sp. b) Context of DgTC-1 from D. gigantea. c) Context of DgTC-2 from D. gigantea. All genes are coded on long eukaryotic contigs (see Supplementary Table 4) with continuous long read coverage between 60 and 130 (Oxford Nanopore for a, PacBio for b and c). While the expression of DgTC-1 and DgTC-2 is low, XsTC-1 is highly expressed. For gene IDs see Supplementary Tables 5-7. The co-occurrence of two different bases in the same position (marked by colors) results from the heterozygosity of D. gigantea and Xenia sp.

Extended Data Fig. 4 Octocoral gene clusters.

Genomic contigs from Xenia sp., Renilla muelleri and Trachythela sp. show the physical co-localization of TCs with genes coding for CYP and SHD enzymes. Genes on the Xenia scaffold that are marked in grey are either annotated as unknown proteins or show similarity to enzymes with non-biosynthetic functions. CYP = cytochrome P450 monooxygenase, IDS = isoprenyl diphosphate synthase, SDH = short-chain dehydrogenase, TC = terpene cyclase.

Extended Data Fig. 5 Putative biochemical pathway from 7 to oxidized Xenia diterpenoids.

A: Putative terpenoid cluster including Cytochrome P450 genes and short chain dehydrogenase genes additionally to the characterized terpene cyclase XsTC-1, which produces 7. B: Putative overview how different compounds isolated from Xenia spp. and closely related corals could be explained from 7 using CYP and SHD chemistry. The oxidative cleavage of the cyclobutene ring in 7 has been proposed in the literature28. The end points of these putative biochemical pathways all represent isolated molecules from different Xenia spp8. and could all result from CYP and SDH chemistry following the outlined scheme. Acylations and methylations are also observed in some compounds but could be the result of non-clustered specific or promiscuous enzymatic activity. CYP = cytochrome P450 monooxygenase, IDS = isoprenyl diphosphate synthase, SDH = short-chain dehydrogenase, TC = terpene cyclase, MT = methyl transferase, AT = acyl transferase.

Extended Data Fig. 6 Analysis of a dichloromethane extract of Capnella imbricata by GC-MS.

Signals for capnellene (11), capnellene-8β,10α-diol (12) and putative terpenoid compounds are labeled.

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Supplementary Figs. 1–21, Notes 1 and 2, Tables 1–11 and references 1–51.

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Burkhardt, I., de Rond, T., Chen, P.YT. et al. Ancient plant-like terpene biosynthesis in corals. Nat Chem Biol 18, 664–669 (2022). https://doi.org/10.1038/s41589-022-01026-2

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