Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont

Journal name:
Nature Chemical Biology
Volume:
10,
Pages:
648–655
Year published:
DOI:
doi:10.1038/nchembio.1573
Received
Accepted
Published online

Abstract

The Japanese marine sponge Discodermia calyx contains a major cytotoxic compound, ​calyculin A, which exhibits selective inhibition of protein phosphatases 1 and 2A. It has long been used as a chemical tool to evaluate intracellular signal transduction regulated by reversible protein phosphorylation. We describe the identification of the biosynthetic gene cluster of ​calyculin A by a metagenome mining approach. Single-cell analysis revealed that the gene cluster originates in the symbiont bacterium 'Candidatus Entotheonella' sp. A phosphotransferase encoded in the gene cluster deactivated ​calyculin A to produce a newly discovered diphosphate, which was actually the biosynthetic end product. The diphosphate had been previously overlooked because of the enzymatic dephosphorylation that occurred in response to sponge tissue disruption. Our work presents what is to our knowledge the first evidence for the biosynthetic process of ​calyculin A along with a notable phosphorylation-dephosphorylation mechanism to regulate toxicity, suggesting activated chemical defense in the most primitive of all multicellular animals.

At a glance

Figures

  1. Calyculin A from the marine sponge Discodermia calyx.
    Figure 1: Calyculin A from the marine sponge Discodermia calyx.

    (a) The marine sponge Discodermia calyx. Scale bar, 5 cm. (b) Structure of ​calyculin A. The β-branches at the C3 and C7 positions are highlighted.

  2. The biosynthetic gene cluster and proposed pathway of calyculin A.
    Figure 2: The biosynthetic gene cluster and proposed pathway of ​calyculin A.

    (a) ORFs encoded in the putative biosynthetic gene cluster. The ORFs related to NRPS and PKS are highlighted in red, and the putative phosphotransferases are in blue. The other ORFs include tailoring enzymes. (b) The domain organization and biosynthetic model for calyculin assembly. The KS clade and the putative substrate of the A domain are indicated above each domain. Domains: C, condensation; ER, enoylreductase; HC, heterocyclization; Ox, oxidation; PCP, peptidyl-carrier protein. KS clades: KS0, nonelongating ketosynthase; DB, double bond; SB, single bond; Oxz, oxazole; AA, amino acid.

  3. Monitoring of the conversion of phosphocalyculin A to calyculin A.
    Figure 3: Monitoring of the conversion of phosphocalyculin A to ​calyculin A.

    ODS HPLC profiles for (i) the product of CalQ-catalyzed reaction with ​calyculin A as the substrate, (ii) the ​methanol extract of the frozen sponges, (iii) the fresh sponges soaked in ​ethanol, (iv) the ​methanol extract of the sponges lyophilized after being flash-frozen in liquid nitrogen, (v) the authentic reference of phosphocalyculin A and (vi) the authentic reference of ​calyculin A. All of the chromatograms were detected by UV at 340 nm. The 2JP,P value was measured on the proton-decoupled 31P NMR spectrum.

  4. Symbiont bacteria bearing calyculin NRPS-PKS genes.
    Figure 4: Symbiont bacteria bearing calyculin NRPS-PKS genes.

    (a) Phase contrast image of D. calyx homogenate. A filamentous bacterium is designated as 'F'. A small filamentous bacterium appearing in bright color is designated as 'S'. (b) Differential interference contrast image of the filamentous bacteria. (c) CARD-FISH imaging with the calyculin NRPS-PKS gene as a probe. (d) PCR products with the calyculin NRPS-PKS–specific primer pair using dissected cells (F or S) as templates. F1–4, single filament (F); F5–7, four filaments (F); S1-4, four filaments (S); S4–7, eight filaments (S); P, D. calyx metagenomic DNA; N, negative control. In a, scale bars are 20 μm; in b and c, scale bars are 10 μm.

  5. Conversion of phosphocalyculin A to calyculin A after wounding the sponge tissue.
    Figure 5: Conversion of phosphocalyculin A to ​calyculin A after wounding the sponge tissue.

    The ratio of each compound in the analyzed samples was calculated as the percentage of the peak area of that component divided by the sum of the peak area of phosphocalyculin A and ​calyculin A (n = 3; error bars represent s.d.). No conversion was observed in unwounded sponge tissue (control).

  6. Phylogenetic tree of phosphotransferases involved in antibiotic resistance systems.
    Figure 6: Phylogenetic tree of phosphotransferases involved in antibiotic resistance systems.

    The neighbor-joining method (CLC sequence viewer and Geneious Tree Builder) was used to generate the phylogenetic tree with a bootstrap test of 100 replicates. Bootstrap values given in percentages are shown at the nodes. Clade names are provided on the basis of the phosphorylation site of aminoglycoside antibiotics.

Compounds

1 compounds View all compounds
  1. Phosphocalyculin A
    Compound 1 Phosphocalyculin A

Accession codes

Primary accessions

NCBI Reference Sequence

Referenced accessions

NCBI Reference Sequence

References

  1. Döderlein, L. Studien an japanischen Lithistiden. Zeitschr. f. wiss. Zool. 40, 62104 (1883).
  2. Kato, Y. et al. Calyculin A, a novel antitumor metabolite from the marine sponge Discodermia calyx. J. Am. Chem. Soc. 108, 27802781 (1986).
  3. Ishihara, H. et al. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem. Biophys. Res. Commun. 159, 871877 (1989).
  4. Wakimoto, T., Matsunaga, S., Takai, A. & Fusetani, N. Insight into binding of calyculin A to protein phosphatase 1: isolation of hemicalyculin A and chemical transformation of calyculin A. Chem. Biol. 9, 309319 (2002).
  5. Kita, A. et al. Crystal structure of the complex between calyculin A and the catalytic subunit of protein phosphatase 1. Structure 10, 715724 (2002).
  6. Fagerholm, A.E., Habrant, D. & Koskinen, A.M.P. Calyculins and related marine natural products as serine-threonine protein phosphatase PP1 and PP2A inhibitors and total syntheses of calyculin A, B, and C. Mar. Drugs 8, 122172 (2010).
  7. Bewley, C.A. & Faulkner, D.J. Lithistid sponges: star performers or hosts to the stars. Angew. Chem. Int. Ed. 37, 21622178 (1998).
  8. Dumdei, E.J., Blunt, J.W., Munro, M.H.G. & Pannell, L.K. Isolation of calyculins, calyculinamides, and swinholide H from the New Zealand deep-water marine sponge Lamellomorpha strongylata. J. Org. Chem. 62, 26362639 (1997).
  9. Fu, X., Schmitz, F.J., Kelly-Borges, M., McCready, T.L. & Holmes, C.F.B. Clavosines A–C from the marine sponge Myriastra clavosa: potent cytotoxins and inhibitors of protein phosphatases 1 and 2A. J. Org. Chem. 63, 79577963 (1998).
  10. Kehraus, S., König, G.M. & Wright, A.D. A new cytotoxic calyculinamide derivative, geometricin A, from the Australian sponge Luffariella geometrica. J. Nat. Prod. 65, 10561058 (2002).
  11. Edrada, R.A. et al. Swinhoeiamide A, a new highly active calyculin derivative from the marine sponge Theonella swinhoei. J. Nat. Prod. 65, 11681172 (2002).
  12. Schirmer, A. et al. Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissolute. Appl. Environ. Microbiol. 71, 48404849 (2005).
  13. Paul, V.J., Arthur, K.E., Ritson-Williams, R., Ross, C. & Sharp, K. Chemical defenses: from compounds to communities. Biol. Bull. 213, 226251 (2007).
  14. Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 27, 9961047 (2010).
  15. Hrvatin, S. & Piel, J. Rapid isolation of rare clones from highly complex DNA libraries by PCR analysis of liquid gel pools. J. Microbiol. Methods 68, 434436 (2007).
  16. Calderone, C.T., Kowtoniuk, W.E., Kelleher, N.L., Walsh, C.T. & Dorrestein, P.C. Convergence of isoprene and polyketide biosynthetic machinery: Isoprenyl-S-carrier proteins in the pksX pathway of Bacillus subtilis. Proc. Natl. Acad. Sci. USA 103, 89778982 (2006).
  17. Stachelhaus, T., Mootz, H.D. & Marahiel, M.A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6, 493505 (1999).
  18. Kato, Y., Fusetani, N., Matsunaga, S., Hashimoto, K. & Koseki, K. Isolation and structure elucidation of calyculins B, C, and D, novel antitumor metabolites, from the marine sponge Discodermia calyx. J. Org. Chem. 53, 39303932 (1988).
  19. Kwan, D.H. & Leadlay, P.F. Mutagenesis of a modular polyketide synthase enoylreductase domain reveals insights into catalysis and stereospecificity. ACS Chem. Biol. 5, 829838 (2010).
  20. Piel, J. et al. Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc. Natl. Acad. Sci. USA 101, 1622216227 (2004).
  21. Tang, M.-C., He, H.-Y., Zhang, F. & Tang, G.-L. Baeyer-Villiger oxidation of acyl carrier protein–tethered thioester to acyl carrier protein-linked thiocarbonate catalyzed by a monooxygenase domain in FR901464 biosynthesis. ACS Catal. 3, 444447 (2013).
  22. Julien, B., Tian, Z.-Q., Reid, R. & Reeves, C.D. Analysis of the ambruticin and jerangolid gene clusters of Sorangium cellulosum reveals unusual mechanisms of polyketide biosynthesis. Chem. Biol. 13, 12771286 (2006).
  23. Teufel, R. et al. Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement. Nature 503, 552556 (2013).
  24. Reid, R. et al. A model of structure and catalysis for ketoreductase domains in modular polyketide synthases. Biochemistry 42, 7279 (2003).
  25. Haines, A.S. et al. A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis. Nat. Chem. Biol. 9, 685692 (2013).
  26. Nguyen, T. et al. Exploiting the mosaic structure of trans-acyltransferase polyketide synthases for natural product discovery and pathway dissection. Nat. Biotechnol. 26, 225233 (2008).
  27. Butcher, R.A. et al. The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 104, 15061509 (2007).
  28. Kusebauch, B., Busch, B., Scherlach, K., Roth, M. & Hertweck, C. Functionally distinct modules operate two consecutive α,β→β,γ double-bond shifts in the rhizoxin polyketide assembly line. Angew. Chem. Int. Ed. 49, 14601464 (2010).
  29. Moldenhauer, J. et al. The final steps of bacillaene biosynthesis in Bacillus amyloliquefaciens FZB42: direct evidence for β,γ dehydration by a trans-acyltransferase polyketide synthase. Angew. Chem. Int. Ed. 49, 14651467 (2010).
  30. Müller, I. et al. A unique mechanism for methyl ester formation via an amide intermediate found in myxobacteria. ChemBioChem 7, 11971205 (2006).
  31. Matsunaga, S., Wakimoto, T. & Fusetani, N. Isolation of four new calyculins from the marine sponge Discodermia calyx. J. Org. Chem. 62, 26402642 (1997).
  32. Olano, C. et al. Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation. Mol. Microbiol. 52, 17451756 (2004).
  33. Matsunaga, S., Wakimoto, T., Fusetani, N. & Suganuma, M. Isolation of dephosphonocalyculin A from the marine sponge, Discodermia calyx. Tetrahedr. Lett. 38, 37633764 (1997).
  34. Schmidt, E.W., Obraztsova, A.Y., Davidson, S.K., Faulkner, D.J. & Haygood, M.G. Identification of the antifungal peptide-containing symbiont of the marine sponge Theonella swinhoei as a novel δ-proteobacterium, “Candidatus Entotheonella palauensis”. Mar. Biol. 136, 969977 (2000).
  35. Pernthaler, A., Pernthaler, J. & Amann, R. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68, 30943101 (2002).
  36. Kimura, M. et al. Calyxamides A and B, cytotoxic cyclic peptides from the marine sponge Discodermia calyx. J. Nat. Prod. 75, 290294 (2012).
  37. Wilson, M.C. et al. Discovery of an environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506, 5862 (2014).
  38. Paul, V.J. & Van Alstyne, K.L. Activation of chemical defenses in the tropical green algae Halimeda spp. J. Exp. Mar. Biol. Ecol. 160, 191203 (1992).
  39. Freeman, M.F. et al. Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides. Science 338, 387390 (2012).
  40. Kaasalainen, U. et al. Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis. Proc. Natl. Acad. Sci. USA 109, 58865891 (2012).
  41. Wiens, M. et al. Okadaic acid: a potential defense molecule for the sponge Suberites domuncula. Mar. Biol. 142, 213223 (2003).
  42. Konoki, K. et al. Binding of diarrheic shellfish poisoning toxins to okadaic acid binding proteins purified from the sponge Halichondria okadai. Bioorg. Med. Chem. 18, 76077610 (2010).
  43. Shakya, T. et al. A small molecule discrimination map of the antibiotic resistance kinome. Chem. Biol. 18, 15911601 (2011).
  44. Wittstock, U. & Gershenzon, J. Constitutive plant toxins and their role in defense against herbivores and pathogens. Curr. Opin. Plant Biol. 5, 300307 (2002).
  45. Wolfe, G.V., Steinke, M. & Kirst, G.O. Grazing-activated chemical defence in a unicellular marine alga. Nature 387, 894897 (1997).
  46. Teeyapant, R. & Proksch, R. Biotransformation of brominated compounds in the marine sponge Verongia aerophoba—evidence for an induced chemical defense? Naturwissenschaften 80, 369370 (1993).
  47. Thoms, C. & Schupp, P.J. Activated chemical defense in marine sponges—a case study on Aplysinella rhax. J. Chem. Ecol. 34, 12421252 (2008).
  48. Fieseler, L. et al. Widespread occurrence and genomic context of unusually small polyketide synthase genes in microbial consortia associated with marine sponges. Environ. Microbiol. 8, 921927 (2004).
  49. Beyer, S., Kunze, B., Silakowski, B. & Müller, R. Metabolic diversity in myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-(poly)peptide gene cluster from the epothilone producing strain Sorangium cellulosum So ce90. Biochim. Biophys. Acta 1445, 185195 (1999).
  50. Ginolhac, A. et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl. Environ. Microbiol. 70, 55225527 (2004).
  51. Moffitt, M.C. & Neilan, B.A. Evolutionary affiliations within the superfamily of ketosynthases reflect complex pathway associations. J. Mol. Evol. 56, 446457 (2003).
  52. He, R. et al. Porphyrins from a metagenomic library of the marine sponge Discodermia calyx. Mol. Biosyst. 8, 23342338 (2012).
  53. Ayuso-Sacido, A. & Genilloud, O. New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes: detection and distribution of these biosynthetic gene sequences in major taxonomic groups. Microb. Ecol. 49, 1024 (2005).
  54. Schmieder, R., Lim, Y.W., Rohwer, F. & Edwards, R. TagCleaner: identification and removal of tag sequences from genomic and metagenomic datasets. BMC Bioinformatics 11, 341354 (2010).
  55. Simpson, J.T. et al. ABySS: a parallel assembler for short read sequence data. Genome Res. 19, 11171123 (2009).
  56. Huang, X., Wang, J., Aluru, S., Yang, S.-P. & Hiller, L. PCAP: a whole-genome assembly program. Genome Res. 13, 21642170 (2003).
  57. Ishikawa, J. & Hotta, K. FramePlot: a new implementation of the Frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content. FEMS Microbiol. Lett. 174, 251253 (1999).
  58. Delcher, A.L., Harmon, D., Kasif, S., White, O. & Salzberg, S.L. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27, 46364641 (1999).
  59. Bachmann, B.O. & Ravel, J. Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. Methods Enzymol. 458, 181217 (2009).
  60. Weisburg, W.G., Barns, S.M., Pelletier, D.A. & Lane, D.J. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697703 (1991).

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Author information

  1. These authors contributed equally to this work.

    • Toshiyuki Wakimoto &
    • Yoko Egami

Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

    • Toshiyuki Wakimoto,
    • Yoko Egami,
    • Yu Nakashima,
    • Yukihiko Wakimoto,
    • Takahiro Mori,
    • Takayoshi Awakawa &
    • Ikuro Abe
  2. Japan Science and Technology Agency, CREST, Tokyo, Japan.

    • Toshiyuki Wakimoto,
    • Yoko Egami,
    • Takahiro Mori,
    • Takayoshi Awakawa &
    • Ikuro Abe
  3. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan.

    • Takuya Ito,
    • Hiromichi Kenmoku &
    • Yoshinori Asakawa
  4. Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

    • Jörn Piel

Contributions

T.W. and I.A. designed the research. T.W. collected sponge specimens. Y.E. constructed libraries and isolated cal genes. T.W., Y.E., T.I., H.K., Y.A., J.P. and I.A. sequenced and analyzed cal genes. T.W. and Y.E. performed the cell separation and elucidation of chemical structures as well as bioconversion experiments. Y.E. and Y.N. conducted the single-cell studies. Y.E., Y.N., Y.W., T.M. and T.A. characterized the enzymes. T.W., Y.E. and I.A. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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