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A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis

Abstract

Steroids are essential triterpenoid molecules that are present in all eukaryotes and modulate the fluidity and flexibility of cell membranes. Steroids also serve as signalling molecules that are crucial for growth, development and differentiation of multicellular organisms1,2,3. The steroid biosynthetic pathway is highly conserved and is key in eukaryote evolution4,5,6,7. The flavoprotein squalene epoxidase (SQE) catalyses the first oxygenation reaction in this pathway and is rate limiting. However, despite its conservation in animals, plants and fungi, several phylogenetically widely distributed eukaryote genomes lack an SQE-encoding gene7,8. Here, we discovered and characterized an alternative SQE (AltSQE) belonging to the fatty acid hydroxylase superfamily. AltSQE was identified through screening of a gene library of the diatom Phaeodactylum tricornutum in a SQE-deficient yeast. In accordance with its divergent protein structure and need for cofactors, we found that AltSQE is insensitive to the conventional SQE inhibitor terbinafine. AltSQE is present in many eukaryotic lineages but is mutually exclusive with SQE and shows a patchy distribution within monophyletic clades. Our discovery provides an alternative element for the conserved steroid biosynthesis pathway, raises questions about eukaryote metabolic evolution and opens routes to develop selective SQE inhibitors to control hazardous organisms.

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Fig. 1: Identification of an AltSQE from P.tricornutum.
Fig. 2: Distribution of SQE across the tree of life.
Fig. 3: Characterization of AltSQEs from various species.

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Data availability

Gene sequences used in this study were deposited in GenBank under the accession numbers MH422131 to MH422144. All other data that support the findings of this study are available from the corresponding author upon request.

References

  1. Benveniste, P. Biosynthesis and accumulation of sterols. Annu. Rev. Plant Biol. 55, 429–457 (2004).

    Article  CAS  Google Scholar 

  2. Payne, A. H. & Hales, D. B. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr. Rev. 25, 947–970 (2004).

    Article  CAS  Google Scholar 

  3. Weete, J. D., Abril, M. & Blackwell, M. Phylogenetic distribution of fungal sterols. PLoS ONE 5, e10899 (2010).

    Article  Google Scholar 

  4. Gold, D. A., Caron, A., Fournier, G. P. & Summons, R. E. Paleoproterozoic sterol biosynthesis and the rise of oxygen. Nature 543, 420–423 (2017).

    Article  CAS  Google Scholar 

  5. Summons, R. E., Bradley, A. S., Jahnke, L. L. & Waldbauer, J. R. Steroids, triterpenoids and molecular oxygen. Phil. Trans. R. Soc. B 361, 951–968 (2006).

    Article  CAS  Google Scholar 

  6. Crowe, S. A. et al. Atmospheric oxygenation three billion years ago. Nature 501, 535–538 (2013).

    Article  CAS  Google Scholar 

  7. Desmond, E. & Gribaldo, S. Phylogenomics of sterol synthesis: insights into the origin, evolution, and diversity of a key eukaryotic feature. Genome Biol. Evol. 1, 364–381 (2009).

    Article  Google Scholar 

  8. Fabris, M. et al. Tracking the sterol biosynthesis pathway of the diatom Phaeodactylum tricornutum. New Phytol. 204, 521–535 (2014).

    Article  CAS  Google Scholar 

  9. Rampen, S. W., Abbas, B. A., Schouten, S. & Damsté, J. S. S. A comprehensive study of sterols in marine diatoms (Bacillariophyta): implications for their use as tracers for diatom productivity. Limnol. Oceanogr. 55, 91–105 (2010).

    Article  CAS  Google Scholar 

  10. Gallo, C., d’Ippolito, G., Nuzzo, G., Sardo, A. & Fontana, A. Autoinhibitory sterol sulfates mediate programmed cell death in a bloom-forming marine diatom. Nat. Commun. 8, 1292 (2017).

    Article  Google Scholar 

  11. Goldstein, J. L., DeBose-Boyd, R. A. & Brown, M. S. Protein sensors for membrane sterols. Cell 124, 35–46 (2006).

    Article  CAS  Google Scholar 

  12. Gill, S., Stevenson, J., Kristiana, I. & Brown, A. J. Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase. Cell Metab. 13, 260–273 (2011).

    Article  CAS  Google Scholar 

  13. Pollier, J. et al. The protein quality control system manages plant defence compound synthesis. Nature 504, 148–152 (2013).

    Article  CAS  Google Scholar 

  14. Dong, L. et al. Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast. Metab. Eng. 49, 1–12 (2018).

    Article  CAS  Google Scholar 

  15. Huysman, M. J. J. et al. AUREOCHROME1a-mediated induction of the diatom-specific cyclin dsCYC2 controls the onset of cell division in diatoms (Phaeodactylum tricornutum). Plant Cell 25, 215–228 (2013).

    Article  CAS  Google Scholar 

  16. Miettinen, K. et al. The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis. Nat. Commun. 8, 14153 (2017).

    Article  CAS  Google Scholar 

  17. Shanklin, J. & Cahoon, E. B. Desaturation and related modifications of fatty acids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 611–641 (1998).

    Article  CAS  Google Scholar 

  18. Zhu, G., Koszelak-Rosenblum, M., Connelly, S. M., Dumont, M. E. & Malkowski, M. G. The crystal structure of an integral membrane fatty acid α-hydroxylase. J. Biol. Chem. 290, 29820–29833 (2015).

    Article  CAS  Google Scholar 

  19. Liu, X. et al. Addressing various compartments of the diatom model organism Phaeodactylum tricornutum via sub-cellular marker proteins. Algal Res. 20, 249–257 (2016).

    Article  Google Scholar 

  20. Leber, R. et al. Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles. Mol. Biol. Cell 9, 375–386 (1998).

    Article  CAS  Google Scholar 

  21. Laranjeira, S. et al. Arabidopsis squalene epoxidase 3 (SQE3) complements SQE1 and is important for embryo development and bulk squalene epoxidase activity. Mol. Plant 8, 1090–1102 (2015).

    Article  CAS  Google Scholar 

  22. Bai, Y. et al. X-ray structure of a mammalian stearoyl-CoA desaturase. Nature 524, 252–256 (2015).

    Article  CAS  Google Scholar 

  23. Johnsson, N. & Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl Acad. Sci. USA 91, 10340–10344 (1994).

    Article  CAS  Google Scholar 

  24. Monier, A. et al. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus. Genome Res. 19, 1441–1449 (2009).

    Article  CAS  Google Scholar 

  25. Keeling, P. J. & Inagaki, Y. A class of eukaryotic GTPase with a punctate distribution suggesting multiple functional replacements of translation elongation factor 1α. Proc. Natl Acad. Sci. USA 101, 15380–15385 (2004).

    Article  CAS  Google Scholar 

  26. Szabová, J., Růžička, P., Verner, Z., Hampl, V. & Lukeš, J. Experimental examination of EFL and MATX eukaryotic horizontal gene transfers: coexistence of mutually exclusive transcripts predates functional rescue. Mol. Biol. Evol. 28, 2371–2378 (2011).

    Article  Google Scholar 

  27. Wang, D. Z. Neurotoxins from marine dinoflagellates: a brief review. Mar. Drugs 6, 349–371 (2008).

    Article  Google Scholar 

  28. Vasconcelos, V., Azevedo, J., Silva, M. & Ramos, V. Effects of marine toxins on the reproduction and early stages development of aquatic organisms. Mar. Drugs 8, 59–79 (2010).

    Article  CAS  Google Scholar 

  29. Trainic, M. et al. Infection dynamics of a bloom-forming alga and its virus determine airborne coccolith emission from seawater. iScience 6, 327–335 (2018).

    Article  Google Scholar 

  30. Moses, T. et al. Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum. Proc. Natl Acad. Sci. USA 111, 1634–1639 (2014).

    Article  CAS  Google Scholar 

  31. Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).

    Article  CAS  Google Scholar 

  32. Deslandes, L. et al. Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proc. Natl Acad. Sci. USA 100, 8024–8029 (2003).

    Article  CAS  Google Scholar 

  33. De Riso, V. et al. Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res. 37, e96 (2009).

    Article  Google Scholar 

  34. Berges, J. A., Franklin, D. J. & Harrison, P. J. Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades. J. Phycol. 37, 1138–1145 (2001).

    Article  Google Scholar 

  35. Lin, H.-Y. et al. Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum. Algal Res. 23, 58–65 (2017).

    Article  Google Scholar 

  36. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  37. Strand, T. A., Lale, R., Degnes, K. F., Lando, M. & Valla, S. A new and improved host-independent plasmid system for RK2-based conjugal transfer. PLoS ONE 9, e90372 (2014).

    Article  Google Scholar 

  38. Diner, R. E., Bielinski, V. A., Dupont, C. L., Allen, A. E. & Weyman, P. D. Refinement of the diatom episome maintenance sequence and improvement of conjugation-based DNA delivery methods. Front. Bioeng. Biotechnol. 4, 65 (2016).

    Article  Google Scholar 

  39. Siaut, M. et al. Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. Gene 406, 23–25 (2007).

    Article  CAS  Google Scholar 

  40. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  Google Scholar 

  41. Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).

    Article  CAS  Google Scholar 

  42. Johnson, L. K., Alexander, H. B. & Brown, C. T. Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes. Preprint at https://www.biorxiv.org/content/early/2018/09/18/323576 (2018).

  43. Burki, F. et al. Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista. Proc. R. Soc. B 283, 20152802 (2016).

    Article  Google Scholar 

  44. Yoshida, Y. et al. De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions. BMC Genomics 17, 182 (2016).

    Article  Google Scholar 

  45. Van Bel, M. et al. TRAPID: an efficient online tool for the functional and comparative analysis of de novo RNA-seq transcriptomes. Genome. Biol. 14, R134 (2013).

    Article  Google Scholar 

  46. Keller, O., Kollmar, M., Stanke, M. & Waack, S. A novel hybrid gene prediction method employing protein multiple sequence alignments. Bioinformatics 27, 757–763 (2011).

    Article  CAS  Google Scholar 

  47. Villar, E. et al. The Ocean Gene Atlas: exploring the biogeography of plankton genes online. Nucleic Acids Res. 46, W289–W295 (2018).

    Article  Google Scholar 

  48. Sunagawa, S. et al. Structure and function of the global ocean microbiome. Science 348, 1261359 (2015).

    Article  Google Scholar 

  49. Wilson, W. H. et al. Complete genome sequence and lytic phase transcription profile of a Coccolithovirus. Science 309, 1090–1092 (2005).

    Article  CAS  Google Scholar 

  50. Hurwitz, B. L. & Sullivan, M. B. The Pacific Ocean Virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology. PLoS ONE 8, e57355 (2013).

    Article  CAS  Google Scholar 

  51. Goodacre, N., Aljanahi, A., Nandakumar, S., Mikailov, M. & Khan, A. S. A Reference Viral Database (RVDB) to enhance bioinformatics analysis of high-throughput sequencing for novel virus detection. mSphere 3, e00069-18 (2018).

    Article  Google Scholar 

  52. Nishimura, Y. et al. Environmental viral genomes shed new light on virus–host interactions in the ocean. mSphere 2, e00359-16 (2017).

    Article  Google Scholar 

  53. Mihara, T. et al. Linking virus genomes with host taxonomy. Viruses 8, 66 (2016).

    Article  Google Scholar 

  54. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).

    Article  Google Scholar 

  55. Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    Article  CAS  Google Scholar 

  56. Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank R. De Clercq and T. Lapshina for technical assistance, Y. Bai, M. Johnson and R. Abbriano-Burke for support with confocal microscopy and M. Huysman and L. De Veylder for providing the P.tricornutum cDNA library. J.P. is a postdoctoral fellow of the Research Foundation-Flanders. E.V. is funded by the BOF project GOA01G01715. M.F. is supported by a CSIRO Synthetic Biology Future Science Fellowship, co-funded by CSIRO and the University of Technology Sydney.

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Contributions

J.P., E.V., U.K. and M.F. carried out the experiments. J.P., K.V., A.G. and M.F. designed the experiments. All authors contributed to writing of the manuscript.

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Correspondence to Alain Goossens.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Table 1.

Reporting Summary

Supplementary Dataset 1

Overview of all detected alternative and conventional SQE protein sequences in all queried organisms.

Supplementary Dataset 2

Maximum-likelihood phylogeny of eukaryotic and viral AltSQE proteins constructed from an alignment of homologues from 202 organisms and 403 informative aligned sites, rooted with ERG3. Proteins are coloured based on their phylogenetic affiliations and bootstrap values are mentioned at the right of every node.

Supplementary Dataset 3

Maximum-likelihood phylogeny of eukaryotic conventional SQE proteins constructed from an alignment of homologues from 235 organisms and 624 informative aligned sites, rooted with UbiH. Proteins are coloured based on their phylogenetic affiliations and bootstrap values are mentioned at the right of every node.

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Pollier, J., Vancaester, E., Kuzhiumparambil, U. et al. A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis. Nat Microbiol 4, 226–233 (2019). https://doi.org/10.1038/s41564-018-0305-5

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