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A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid A

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A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid A A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid a-Onocerin Aldo Almeida, Lemeng Dong, Bekzod Khakimov, Jean-Etienne Bassard, Tessa Moses, Frederic Lota, Alain Goossens, Giovanni Appendino, Soren Bak To cite this version: Aldo Almeida, Lemeng Dong, Bekzod Khakimov, Jean-Etienne Bassard, Tessa Moses, et al.. A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid a-Onocerin. Plant Physiology, American Society of Plant Biologists, 2018. hal-02885734 HAL Id: hal-02885734 https://hal.archives-ouvertes.fr/hal-02885734 Submitted on 30 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid a-Onocerin1[OPEN] Aldo Almeida,a Lemeng Dong,a Bekzod Khakimov,b Jean-Etienne Bassard,a Tessa Moses,c,d Frederic Lota,e Alain Goossens,c,d Giovanni Appendino,f and Søren Baka,2 aDepartment of Plant and Environmental Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark bDepartment of Food Science, University of Copenhagen, DK-1958 Frederiksberg C, Denmark cGhent University, Department of Plant Biotechnology and Bioinformatics, 9052 Ghent, Belgium dVIB Center for Plant Systems Biology, 9052 Ghent, Belgium eAlkion Biopharma SAS, 91000 Evry, France fDipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy ORCID IDs: 0000-0001-5284-8992 (A.A.); 0000-0002-1435-1907 (L.D.); 0000-0001-9366-4727 (T.M.); 0000-0002-9330-5289 (F.L.); 0000-0002-1599-551X (A.G.); 0000-0003-4100-115X (S.B.). 8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid a-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, a-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase,OsONS1,wasidentified that produces a-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana.Incontrast,inLycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce a-onocerinintheN. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the a-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum a-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the a-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, a-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism. Triterpenoids are a class of isoprenoids characterized 2,3-oxide (SQO), which is synthesized from squalene by by an amazing structural diversity. Triterpenoids are squalene epoxidases (SQEs). The next step in triterpe- derived mainly from the 30-carbon precursor squalene- noid biosynthesis is the cyclization of SQO mediated by oxidosqualene cyclases (OSCs; Augustin et al., 2011). 1 Cyclization initiates with the acid-catalyzed epoxide The research leading to these results has received funding to ring opening of SQO and continues through a series of TriForC from the European Community’s Seventh Framework Pro- gramme [FP7/2007-2013] under grant agreement 613692. T.M. was methyl and hydride shifts (Abe, 2014) that give a series supported by the Special Research Funds from Ghent University. of conformationally discrete carbocation intermediates 2 Address correspondence to [email protected]. (Van Tamelen et al., 1982; Boar et al., 1984). Carbocation The author responsible for distribution of materials integral to the shifting in the catalytic cavity of the OSC is a dynamic findings presented in this article in accordance with the policy de- process (Tian and Eriksson, 2012) that terminates by scribed in the Instructions for Authors (www.plantphysiol.org) is: deprotonation of the carbocation intermediate, result- Søren Bak ([email protected]). ing in the formation of a neutral compound. Some OSCs A.A., S.B., and G.A. conceived the original research plan; A.A. deprotonate a single carbocation intermediate and, performed most of the experiments, analyzed the data, and drafted thus, form a single cyclized product, while others have the article; J.-E.B. assisted in the design and performance of FLIM the plasticity to deprotonate several carbocations at experiments; B.K. provided the GC-MS analysis; T.M. and A.G. were involved in the yeast expression experiments; F.L. generated the different positions, rendering them multifunctional and hairy root lines; L.D. and S.B. supervised and, together with A.A., capable of releasing multiple cyclized products complemented the writing. (Lodeiro et al., 2007; Wu et al., 2008). Thus, OSCs in- [OPEN] Articles can be viewed without a subscription. crease triterpenoid diversity by producing distinct tri- www.plantphysiol.org/cgi/doi/10.1104/pp.17.01369 terpenoid skeletons, with over 100 triterpenoid Ò Plant Physiology , February 2018, Vol. 176, pp. 1469–1484, www.plantphysiol.org Ó 2018 American Society of Plant Biologists. All Rights Reserved. 1469 Downloaded from on June 24, 2020 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. Almeida et al. skeletons estimated to originate from their action (Xu second interesting step in a-onocerin biosynthesis is the et al., 2004). cyclization reaction. Recently, it was shown that, in L. The seco-triterpenoids are characterized by the pres- clavatum, the biosynthesis of a-onocerin is performed in ence of an open ring, a structural element that can be two steps by two substrate-specific OSCs acting se- connected directly to the cyclization mechanism or re- quentially. LcLCC cyclizes SDO to the two-ring struc- sult from postcyclization bond cleavage. In this study, ture pre-a-onocerin, after which LcLCD initiates we focused on the seco-triterpenoid a-onocerin (Fig. 1), cyclization from the remaining epoxide bond of pre- a symmetrical tetracyclic triterpenoid first isolated in a-onocerin to finally produce a-onocerin (Araki et al., 1855 from Ononis spinosa (Hlasiwetz, 1855) but only 2016). However L. clavatum and O. spinosa are phylo- structurally elucidated a century later by Barton and genetically very distant, with the former being a lyco- Overton (1955). More than a decade after this work, pod and the latter an angiosperm. Therefore, it is likely a-onocerin was isolated from the club moss Lycopodium that the a-onocerin pathways evolved convergently in clavatum (Ageta et al., 1962) as well. these two plant species. The occurrence of a-onocerin in only two phyloge- Another aspect of triterpenoid biosynthesis that has netically distant branches of the plant kingdom makes been overlooked in the literature is the uptake of SQE the biosynthesis of a-onocerin intriguing from an evo- products by OSCs. It has been suggested that the lutionary standpoint. In fact, the occurrence of a-ono- sterol pathway proceeds by the channeling of sub- cerin is inconsistent even within the Ononis genus. In a strates between each step through microdomains of survey of a-onocerin accumulation in Ononis spp., ex- the endoplasmic reticulum (ER; Benveniste, 2004). tracts from three species were recorded as lacking However, it is known that active SQE and OSC en- a-onocerin: Ononis reclinata, Ononis viscosa, and Ononis zymes can localize to related but nevertheless distinct pusilla (Rowan and Dean, 1972). Decades later, Ononis cell compartments (e.g. in yeast, active SQE is found rotundifolia was reported as another non-a-onocerin on/in the ER, whereas active lanosterol synthase is producer (Hayes, 2012). The Ononis genus is mono- found in lipid droplets along with the inactive form of phyletic and splits into five major clades (Turini et al., SQE;Leberetal.,1998;Athenstaedtetal.,1999).Aside 2010); however, there is no clear clustering of a-ono- from the probable close proximity of lipid droplets to cerin-producing species, as the nonproducing species the ER membrane network, another force driving tri- are found in the four major clades that have been tested terpenoid biosynthesis could be protein-protein in- for a-onocerin accumulation. The simultaneous occur- teractions. Surprisingly, protein-protein interactions
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