Thimmappa R et al. (2022), Biosynthesis of saponin defensive compounds in ...

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Nat Chem Biol 2022 Jul 01;187:774-781. doi: 10.1038/s41589-022-01054-y.

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Biosynthesis of saponin defensive compounds in sea cucumbers.

Thimmappa R , Wang S , Zheng M , Misra RC , Huang AC , Saalbach G , Chang Y , Zhou Z , Hinman V , Bao Z , Osbourn A .

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Soft-bodied slow-moving sea creatures such as sea stars and sea cucumbers lack an adaptive immune system and have instead evolved the ability to make specialized protective chemicals (glycosylated steroids and triterpenes) as part of their innate immune system. This raises the intriguing question of how these biosynthetic pathways have evolved. Sea star saponins are steroidal, while those of the sea cucumber are triterpenoid. Sterol biosynthesis in animals involves cyclization of 2,3-oxidosqualene to lanosterol by the oxidosqualene cyclase (OSC) enzyme lanosterol synthase (LSS). Here we show that sea cucumbers lack LSS and instead have two divergent OSCs that produce triterpene saponins and that are likely to have evolved from an ancestral LSS by gene duplication and neofunctionalization. We further show that sea cucumbers make alternate sterols that confer protection against self-poisoning by their own saponins. Collectively, these events have enabled sea cucumbers to evolve the ability to produce saponins and saponin-resistant sterols concomitantly.

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	Fig. 1. Evolution of divergent OSCs in sea cucumbers.a, Presence or absence of saponin chemical defenses in slow-moving, soft-bodied echinoderms. The tree is drawn as in ref. 17. b, Biosynthetic origin of steroidal and triterpene saponins and usual and unusual sterols in sea stars and sea cucumbers. Usual sterol, characterized by a common C5 unsaturation as well as the absence of methyl groups at carbon positions 4 and 14. *Unusual sterols with C7 and C9(11) unsaturation. Solid and dashed arrows represent single and multiple steps, respectively. Glc, glucose. Fuc, fucose; MeGlc, methyl glucose; Xyl, xylose; Qui, quinovose. c, Complementation of the LSS-deficient yeast strain Gil77 with cloned OSC genes. pYES2, empty vector control. Yeast was spotted from stock cultures undiluted (−) and diluted tenfold and 100-fold. Ergo, ergosterol; Gal, galactose; SD-URA, synthetic defined medium without uracil. d–f, GC–MS profile of yeast extracts expressing clade I OSC candidates (d), clade II OSC1 (e) and OSC2 candidates (f). In the LDS experiments, ketoconazole (50 µg ml−1) was included in the medium to limit in vivo modifications of OSC1 products by the endogenous yeast CYP51 enzyme (Extended Data Fig. 2c and Methods). GC–MS peaks in d–f were extracted ion chromatograms for the ion m/z 426. The corresponding total ion chromatograms and mass spectra are shown in Extended Data Fig. 2b–h. The lower chromatograms in d–f show GC traces for an equimolar mixture of the standards lanosterol, lanostadienol and parkeol. Pm, sea star P. miniata; Sp, sea urchin S. purpuratus; Aj, sea cucumber A. japonicus; Pp, sea cucumber P. parvimensis. Superscripts ‘a’ and ‘b’ for A. japonicus LDS and A. japonicus PS denote different accessions of A. japonicus. Rt; retention time.
	Fig. 2. Biosynthesis of defense saponins in sea cucumbers.a, Antifungal activity, saponin profiles and OSC transcript levels for different sea cucumber tissues. Top, yeast growth (mean ± s.d., P. parvimensis, n = 2; A. japonicus, n = 3). Across all tissues, a 100 µg ml−1 crude extract was used with methanol as a control. Middle, presence (+) or absence (−) of saponins in P. parvimensis and A. japonicus based on LC–MS profiles (Extended Data Fig. 4a). Bottom, heatmap showing green (low) to red (high) OSC gene expression generated from reads per kb of exon model per million mapped reads (RPKM) values (A. japonicus, n = 3). RPKM mean ± s.d. values are given in the source data. OD600, optical density at 600 nm. b, Comparison of sea cucumber (Sc) sterol-biosynthetic genes with those of humans (Hu), sea urchins (Su) and sea stars (Ss). Presence or absence of sterol genes was scored based on pairwise ortholog identity as shown in Supplementary Table 8.Source data
	Fig. 3. Sea cucumbers synthesize diverse triterpene saponins and unusual sterols.Roles of PS, LDS and C7D in the biosynthesis of unusual sterols (Δ7(8) and Δ9(11)) and triterpene saponins (Δ7(8) and Δ9(11)). The different colored arrows represent taxa-specific sterol- or saponin-biosynthetic routes. Solid and dashed arrows represent single and multi-step reactions, respectively.
	Fig. 4. A single active residue underlies functional divergence of LDS and PS from LSS in echinoderms.a, Superpositioned homology models of sea star (P. miniata LSS), sea urchin (S. purpuratus LSS) and divergent sea cucumber OSCs (P. parvimensis LDS and P. parvimensis PS) showing variation at position 444 position near the B and/or C rings of lanosterol. Colored circles next to amino acid residues in the models represent different cyclization roles. Dashed lines represent a hydrogen bond between Y503 and H232, and numbering on B and/or C rings of lanosterol represents cationic regions. b, Structure-based sequence alignment of position 444 and its associated positions across echinoderm and holozoan OSCs. Clades are colored according to OSC product specificity. OSC sequence numbering is according to that of human LSS. Further information about the sequences used is provided in Supplementary Table 2. GoPS, G. obscuriglobus PS, Dre, Danio rerio; Branchi, Branchiostoma floridae; Amphi, Amphimedon queenslandica; Sacco, Saccoglossus kowalevskii; Helob, Helobdella robusta; Af, Asterias forbesi; Ar, Asterias rubens; Lsp, Leptasterias sp.; Hsp, Henricia sp.; Es, Echinaster spinulosus; Ap, Acanthaster plancii (COTS); Ep, Echinarachnius parma; Sg, Sphaerechinus granularis; Sc, Stichopus chloronotus. c, Complementation of an LSS-deficient yeast strain with A. japonicus PSa wild type (WT) and mutants thereof. Yeast was spotted from stock cultures undiluted (−) and diluted tenfold and 100-fold. d,e, GC–MS profiles of yeast extracts from strains expressing A. japonicus PSa wild type and active site mutants (d) and the bacterial OSC G. obscuriglobus PS (e). The corresponding total ion chromatograms for d,e are shown in Extended Data Figs. 8d and 9c, respectively. Standards were lanosterol, lanostadienol and parkeol.
	Extended Data Fig. 1. Phylogeny of predicted amino acid sequences of echinoderm OSCs.Major clades are colored. Candidates with red stars were functionally characterized in yeast in this study. Scale bar 0.1 amino acid substitutions per site. OSCs sequence details are provided in Supplementary Table 2.
	Extended Data Fig. 2. Functional analysis of sea urchin, sea star and sea cucumber OSCs in yeast.a, Complementation of the LSS-deficient yeast strain (Gil77) with echinoderm OSCs. Yeast transformants spotted undiluted (−), 10- and 100-folds dilution and growth recorded 7 days after spotting. GC-MS total ion chromatograms (TICs) of yeast extracts expressing b, sea star PmLSS and sea urchin SpLSS, c, sea cucumber LDS, d, sea cucumber PS. Superscripts ‘a’ and ‘b’ indicate different accessions of A. japonicus. Peaks marked with asterisks in c and d indicate background modifications of OSC products in yeast. e, GC-MS TICs of yeast extract expressing AjLDSb treated with different concentrations of ketoconazole (CYP51 inhibitor) in the medium (1-50 µg/ml). f-h, GC mass spectra of lanosterol (2), lanostadienol (4) and parkeol (3). pYES2, yeast containing the empty vector (ev). Standards, 2, lanosterol; 4, lanostadienol; 3, parkeol. Ergo = ergosterol, glu = glucose and gal = galactose.
	Extended Data Fig. 3. Characterization of sea cucumber saponins.a, Thin layer chromatography (TLC) of P. parvimenis sea cucumber crude extract and its semi-pure fractions (Fr1-Fr7). b, 96 well yeast growth inhibition (YGI) assay. c, Effect of sea cucumber crude extract and its semi-pure fractions on yeast growth (1-100 µg/ml). Optical density (OD600) was shown as mean ± SD (error bar) with n = 2 replicates. Fr, fraction (see Source Extended Data Fig. 3). d, LC-MS (−) total (TIC) and extracted ion chromatograms (EIC) of fractions Fr5 and Fr6. Saponin peaks of Fr5 and Fr6 are marked with their masses. Fr5; (M-H)- m/z 1391 and m/z 1393, Fr6; (M-H)- m/z 1377 and m/z 1379. e, Q Exactive high resolution LC-MS2 mass fragmentation spectra of saponins observed in fr5 (1391 and 1393) and fr6 (1377 and 1379), their deduced saponin structures showing nature of fragmentation (blue lines and red numbers) and high-resolution masses of parent and product ions etc. Glc; glucose, MeGlc; methyl glucose, Qui; quinovose, Xyl; xylose, Ag; aglycone. Aglycone mass is without the δ lactone moiety because it is lost as CO2 (M-H-CO2). Structural details on saponin sugar chains are given in Supplementary Fig. 4. Source data
	Extended Data Fig. 4. LC-MS (−) analysis of sea cucumber tissue extracts.a, LC-MS (−) TICs of sea cucumber adult tissue extracts and b, early stages. All tissue wise replicate profiles are shown. Aj, A. japonicus n = 3; Pp, P. parvimensis n = 2. Rt; retention time. Saponin peaks with same retention times and masses are connected by a dotted line and annotated with compound names or their masses. c-d, Q Exactive high resolution LC-MS analysis showing detection of saponin ions in A. japonicus (Aj) and P. parvimensis (Pp) early-stage extracts. Saponin ions highlighted in bold were subjected for LC-MS2 and its corresponding data shown in Extended Data Fig. 5b.
	Extended Data Fig. 5. Antifungal activity, saponin content and OSC gene expression in sea cucumber juvenile stages.a, Top, inhibition of yeast growth by crude extracts of early growth stages of A. japonicus and P. parvimensis. Methanol (MeOH) used as a control (mean ± SD, n = 2) (see Source Extended Data Fig. 5). Middle, presence (+) or absence (−) of saponins in P. parvimensis (Pp) and A. japonicus (Aj) based on LC-MS profiles shown in Extended Data Fig. 4b–d. Bottom, heat map showing low (green) to high (red) OSC gene expression generated from RPKM values (Aj, n = 3). b, Q Exactive high resolution LC-MS2 mass fragmentation spectra of saponins observed in early-stage extracts, their deduced saponin structures and high-resolution masses of parent and product ions observed. Glc; glucose, MeGlc; methyl glucose, Qui; quinovose, Xyl; xylose, Ag; aglycone. Structural details on saponin sugar chains are given in Supplementary Fig. 4. Source data
	Extended Data Fig. 6. Sea stars and sea cucumbers make unusual sterols.a, Representative GC-MS profiles (TICs) of sea urchin, sea star and sea cucumber adult tissues. The peaks at 10.1, 10.4 and 11.52 min are lathosterol (7), episterol (22), and avenasterol (21), respectively. Peak eluting at 9.98 min unique to sea cucumbers and was characterized to be 14α-methylcholest-9(11)-en-3β-ol (11) (Supplementary Tables 5–7). b, Levels of cholesterol (5), lathosterol (7) and 14α-methylcholest-9(11)-en-3β-ol (11) in adult tissues of A. japonicus (mean ± SD, n = 2). Dw, dry weight (see Source Extended Data Fig. 6). c, Unusual sterols renders sea star and sea cucumber membranes saponin resistant. 3D conformations of cholesterol (5), lathosterol (7) and 14α-methylcholest-9(11)-en-3β-ol (11) showing flat conformation of side chain in 5 and bent conformation in 7 and 11. 3D conformations were optimized using Frog2 server with default parameters60. d, GC mass spectrum of the sterols observed. Avenasterol (21) and episterol (22) and were identified based on known spectra in the literature and others based on authentic standards. All sterols are TMS derivatives. Source data
	Extended Data Fig. 7. Sea stars and sea cucumbers evolved unusual sterol pathways.a, Canonical cholesterol biosynthetic pathway of animals including sea urchins and sea stars. b, Sea star and sea cucumber specific biosynthetic pathway of Δ7 sterols, lathosterol (7), avenasterol (21) and episterol (22). Sea stars make lathosterol (7) from cholesterol (5) that is made de novo whereas sea cucumbers make lathosterol (7) through diet derived cholesterol (5) or other Δ5 sterols. c, Neighbor-joining tree of C7Ds (cholesterol 7-desaturse) hits from echinoderms and other functionally characterized sequences. Sequence names are uniprot sequence identifiers. d, Sequence alignment of C7D/DAF36 active site motifs of echinoderm as well as other functionally characterized sequences. Residue positions marked with red arrows are part of the Rieske motif which co-ordinates iron and sulfur required in cholesterol 7-desaturation.
	Extended Data Fig. 8. A single active site residue (position 444) determines cyclization mechanism and product specificity of LSS, LDS and PS OSCs.a, The cyclization mechanism for lanosterol (2), lanostadienol (4), parkeol (3) and cycloartenol. Protonation, cyclization, and rearrangement of 2, 3 oxidosqualene (1) to a central protosteryl cation (C20). Rearrangement of C20 cation lead to either C8 or C9 cations depending on type of OSC involved. Deprotonation at C7 of C8 cation lead to lanostadienol (4) and deprotonation at C11 of C9 cation lead to parkeol (3). Lanosterol (2) and cycloartenol are derived from C8 and C9 cations, respectively. b, OSC sequence alignment of 24 active site residue positions of echinoderms and others (sequences given in Supplementary Notes 2). c, OSC active site region is shown in yellow and region encompassing 5Å around lanosterol is in blue. d, GC-MS TICs of yeast extracts expressing AjPSa-WT and its mutants AjPSa-L436F, AjPSa-L436Q. Peaks marked with asterisks are undesired modifications of the PS product in yeast. e-f, TICs and EICs (m/z 426) of yeast extracts expressing SpLSS-WT and its mutants SpLSS-F440L, SpLSS-F440Q. Standards: 2, lanosterol; 4, lanostadienol; 3, parkeol.
	Extended Data Fig. 9. Discovery of Gemmata obscuriglobus parkeol synthase (GoPS).a, OSCs sequence alignment of residue position 444 from diverse taxa. Bacterial group-I OSCs with variation at residue position 444 are shown in green. Sequences used in phylogeny and sequence alignment are given in Supplementary Notes 2. b, Neighbor-joining tree of representative OSCs from diverse taxa. Holozoan clade (blue arc) includes all animal representatives. Bacterial squalene hopane cyclases (SHC) used as outgroups. Bacterial group-I OSCs with ‘L’ natural mutation (yellow). Bootstrap percentages higher than 50 are shown (500 replicates). c, GC-MS TICs of yeast extract expressing GoPS. Peaks marked with asterisks are undesired modifications of the PS product in yeast. Standards, 2, lanosterol; 4, lanostadienol; 3, parkeol.
	Extended Data Fig. 10. Model for evolutionary origin of saponins and unusual sterols in sea stars and sea cucumbers.a, Phylogeny of sea stars, sea urchins and sea cucumbers based on ref. 16. b, Genomic neighborhood of OSC genes. OSC genes are shown in different shades of green, and CYP51 genes in orange. Red cross in CYP51 denote gene loss. Colored stars denote OSC mutations at amino acid residue position 444. Scaffolds and gene modules are not to scale. c, Presence, or absence of saponins in sea urchins, sea stars and sea cucumbers. d, Major sterols of sea urchins, sea stars and sea cucumbers.

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