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Ancient defensive terpene biosynthetic gene clusters in the soft corals

Abstract

Diterpenes are major defensive small molecules that enable soft corals to survive without a tough exterior skeleton, and, until now, their biosynthetic origin has remained intractable. Furthermore, biomedical application of these molecules has been hampered by lack of supply. Here, we identify and characterize coral-encoded terpene cyclase genes that produce the eunicellane precursor of eleutherobin and cembrene, representative precursors for the >2,500 terpenes found in octocorals. Related genes are found in all sequenced octocorals and form their own clade, indicating a potential ancient origin concomitant with the split between the hard and soft corals. Eleutherobin biosynthetic genes are colocalized in a single chromosomal region. This demonstrates that, like plants and microbes, animals also harbor defensive biosynthetic gene clusters, supporting a recombinational model to explain why specialized or defensive metabolites are adjacently encoded in the genome.

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Fig. 1: Terpene biosynthesis in the eleutherobin producer E. caribaeorum.
Fig. 2: Terpene biosynthetic pathways in octocorals are often found in animal-encoded gene clusters.
Fig. 3: Synthesis of coral terpenes from synthetic GGPP analogs in vitro.
Fig. 4: A distinct TPS clade is found in all sequenced octocoral genomes and transcriptomes.

Data availability

All supporting data are described in the publication, and associated raw data files are available upon request. All sequencing data (raw reads and assembled TPS genes and contigs) were deposited in GenBank (accession numbers in Supplementary Table 3). The following are sequencing data reported in this study: SRR15783032, SRR15817518, SRR15817517, OK081311, OK081312, OK081313, OK081314, OK081315, OK081316, OK081317, OK081318, OK081319 and OK081320; octocoral SRA data obtained from NCBI: DRR253190, SRR8506632, SRR12021959, SRR6782832, SRR6820379, SRR5123105, ERR2192493, SRR14295591, ERR2190350, ERR2190370, ERR2191368, SRR4449115, SRR14295593, SRR9330360, SRR14295588, SRR10873896, SRR12904788, SRR14295605, SRR14295600, SRR8297742, SRR7585363, SRR7174588, SRR7174589, SRR7174590, SRR7174591, SRR13925246, SRR8293935, SRR8486075, SRR8486076, SRR8486077, SRR8486078, SRR8486079, SRR8486080, SRR8486081, SRR8486082, SRR8486083, SRR6039601, SRR6039602, SRR6039603, SRR6039604, SRR6039605, SRR6039606, SRR12876609, SRR12876610, SRR12876613, SRR12876614, SRR12876620, SRR12876621, SRR12876622, SRR12876624, SRR12876625, SRR12876628, SRR12876629, SRR12876631, SRR12876632, SRR12876633, SRR12876635, SRR12876636, SRR12876637, SRR12876638, SRR12876639, SRR12876640, SRR12876644, SRR12876647, SRR12876648, SRR12876649, SRR12876650, SRR12876653, SRR12876654, SRR12876657, SRR12876658, SRR12876659, SRR12876660, SRR12876661, SRR12876663, SRR12876664, SRR935078, SRR935079, SRR935080, SRR935081, SRR935082, SRR935083, SRR935084, SRR935085, SRR935086, SRR935087, SRR935088, SRR935089, ERR3040053, ERR3040054, SRR12587798, SRR12587799, SRR12587800, SRR12587801, SRR12587803, SRR12587805, SRR12587806, SRR12587807, SRR12587808, SRR5949848, SRR7521178, SRR7521179, SRR7521180, SRR7521181, ERR3664727, ERR3664728, ERR3664729, ERR3664730, SRR13925244, SRR12573942, SRR12573944, SRR12573945, SRR12573946, ERR3026434, ERR3026435, SRR9278440, SRR9278441, SRR9278446, SRR9278447, SRR8113906, SRR8113907, SRR8113908 and SRR8113909; proteins used for HMM: BAM78698.1, BAM78697.1, WP_030430753.1, ADI87447.1, ADI87448.1 and AXN72980.1; PDB accession numbers for EcTPS1 structural homologs: 4OKM, 6TBD, 3KB9, 3V1V, 6VKZ, 5A0I, 4ZQ8, 1HM7, 6Q4S and 5UV0 and PDB accession numbers for EcAT1 structural homologs: 7KVW, 3FP0, 6N8E, 6AD3, 5U89, 6MFZ, 4ZXH, 5T3E, 5ISW and 4JN3.

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Acknowledgements

The coral photos in the graphical abstract and Fig. 1 were provided by B. Miller and J. Simpson, respectively, and are used with permission. We thank J.M. Winter for helpful discussions, J. Skalicky for assistance with NMR data acquisition and J.A. Maschek for assistance with GC–MS data acquisition. NMR, MS and sequencing data were acquired at University of Utah core facilities. This work was funded by National Institutes of Health grant GM122521 and the ALSAM Foundation.

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P.D.S. and E.W.S. designed the research. P.D.S. performed all experiments. Z.L. and P.D.S. performed bioinformatics analysis. All authors contributed to writing the manuscript.

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Correspondence to Eric W. Schmidt.

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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 Predicted structure of EcTPS1.

3D model of EcTPS1 protein structure (blue ribbon) superimposed on selinadiene synthase (green ribbon, PDB# 4OKZ) in complex with dihydrofarnesyl pyrophosphate (carbon = grey, phosphorus=orange, oxygen=red). The structural comparison and alignment were performed in i-TASSER. This figure reveals the predicted close structural similarity between EcTPS1 and other type I TPS proteins despite a lack of sequence similarity.

Extended Data Fig. 2 Metagenomic analysis of E. caribaeorum.

Binning plot of E. caribaeorum metagenome. Each points represent a unique assembled contig from the metagenome. They are plotted on the two dimensions that result from dimension-reduction by BH-tSNE and GC content of the contig. The points were grouped by DBSCAN and supervised by taxonomic identification of each contig. The elu BGC containing contig is part of the E. caribaeorum genome. Only a small percentage of the metagenome originates in bacteria and dinoflagellates. A small amount of sponge DNA is present because the coral forms a tight association with a sponge.

Extended Data Fig. 3 Phylogenetic analysis of octocoral terpene cyclases.

TPS gene distributions in marine invertebrate transcriptomes. The TPS genes were grouped by a threshold of 65% protein sequence identity. A single TPS group (group 1) is found in soft corals, hard corals, sponges, and dinoflagellates, and represents a dinoflagellate-encoded TPS group. The remaining groups (TPS groups 2–36) are only detected in octocorals genomes and transcriptomes and thus represent octocoral terpene cyclases such as EcTPS1 and EcTPS6.

Extended Data Fig. 4 GCMS analysis of enzymatic terpene cyclase reactions.

Raw data GC-MS total ion current chromatograms of control and sample enzyme assays. The x-axis is time, while the y-axis is the ion counts. Note that because of the robust synthesis of 4 and 5 by EcTPS1 and EcTPS6, respectively, the scale is zoomed in by 10-fold in the control experiments.

Extended Data Fig. 5 Characterization and kinetic analysis of terpene cyclases.

SDS-PAGE analysis of EcTPS1 elution fraction. EcTPS1 was expressed and purified over five times, providing a similar result. B) SDS-PAGE analysis of EcTPS6 elution fraction. EcTPS6 was expressed and purified three times, providing a similar result. C) Relationship between production of 4 and buffer pH D) GCMS calibration curve for quantification of 4. E) Michaelis-Menten analysis of EcTPS1 upon incubation with GGPP. Independent reactions were run in triplicate (n = 3 biological replicates) and analyzed once by GCMS (n = 1 technical replicate) and normalized to an internal standard. Data are presented as mean values + /- SD.

Extended Data Fig. 6 Proposed cyclization cascade for EcTPS1.

A) Proposed cyclization cascade starting from GGPP to form [2H2]-klysimplexin R (4). B) Relative free energies of intermediates and transition state structures in kcal mol−1, calculated with mPW1PW91/6-311 + G(d,p) in a water CPCM model.

Extended Data Fig. 7 Phylogenetic analysis of marine terpene cyclases.

Maximum likelihood phylogenetic tree of TPS protein sequences. The dinoflagellate series includes transcripts from dinoflagellates, as well as from corals and sponges but attributed to dinoflagellates. The tree shows that octocoral TPS sequences form a clade that is distinct from terpene cyclases of other origin. All orders of octocorals are included, and thus the results reveal that these genes do not result from horizontal transfer after the split of the octocoral orders.

Extended Data Fig. 8 TPS BGCs in octocorals.

TPS-containing contigs, presumably harboring biosynthetic gene clusters (BGCs), identified in previously published octocoral genomes.

Extended Data Fig. 9 Phylogenetic analysis of octocoral cytochrome P450 genes.

Maximum likelihood phylogenetic analysis of octocoral cytochrome P450 genes. Many TPS-linked P450s cluster together, indicating a potential common origin in octocoral specialized metabolism.

Extended Data Fig. 10 Predicted protein structure of EcAT1.

3D model of EcAT1 protein structure (green ribbon) showing sesquiterpene alcohol substrate, 15-decalonectrin, bound to the active site. This structure and ligand binding model were obtained using the i-TASSER web server on the basis of similarity to trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides (TRI3, PDB# 3fp0).

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Supplementary Figs. 1–3, Tables 1–7 and Notes 1 and 2.

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Scesa, P.D., Lin, Z. & Schmidt, E.W. Ancient defensive terpene biosynthetic gene clusters in the soft corals. Nat Chem Biol 18, 659–663 (2022). https://doi.org/10.1038/s41589-022-01027-1

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