Repeated evolution of cytochrome P450- mediated spiroketal steroid biosynthesis in plants The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Christ, Bastien et al. "Repeated evolution of cytochrome P450- mediated spiroketal steroid biosynthesis in plants." Nature communications 10 (2019): 1038 © 2019 The Author(s) As Published 10.1038/s41467-019-11286-7 Publisher Springer Science and Business Media LLC Version Final published version Citable link https://hdl.handle.net/1721.1/124710 Terms of Use Creative Commons Attribution 4.0 International license Detailed Terms https://creativecommons.org/licenses/by/4.0/ ARTICLE https://doi.org/10.1038/s41467-019-11286-7 OPEN Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants Bastien Christ1,6,7, Chengchao Xu1,7, Menglong Xu1,7, Fu-Shuang Li1, Naoki Wada 2, Andrew J. Mitchell1, Xiu-Lin Han3, Meng-Liang Wen3, Makoto Fujita2,4 & Jing-Ke Weng 1,5 Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic 1234567890():,; occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris (Paris polyphylla), a monocot med- icinal plant with hemostatic and antibacterial properties, and fenugreek (Trigonella foenum–graecum), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6- spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the pro- duction of diosgenin and derived analogs in heterologous hosts. 1 Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA. 2 Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. 3 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Institute of Microbiology, School of Life Sciences, Yunnan University, Kunming, Yunnan 650091, China. 4 Division of Advanced Molecular Science, Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan. 5 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 6Present address: Agroscope, Route des Eterpys 18, 1964 Conthey, Switzerland. 7These authors contributed equally: Bastien Christ, Chengchao Xu, Menglong Xu. Correspondence and requests for materials should be addressed to J.-K. W. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:3206 | https://doi.org/10.1038/s41467-019-11286-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11286-7 terols are isoprene-derived tetracyclic triterpenoid lipids species, which are important defense compounds with a multitude that play essential roles in modulating membrane of antimicrobial and antiherbivory activities (Fig. 1a, b)9–11. For Sfl uidity, intracellular transport, and cell signaling in all instance, diosgenin is a major natural product of Paris polyphylla eukaryotes1–3. Sterols are also the precursors of a plethora of (a monocot) and Trigonella foenum–graecum (a eudicot), two specialized metabolites, such as steroid hormones, bile acids, plants with rich histories used by humans for medicinal and/or steroidal saponins, and steroidal glycoalkaloids, which serve culinary purposes12,13. Diosgenin holds particular significance for diverse functions ranging from defense to regulation of specific the modern pharmaceutical industry. Extracted in large quantities aspects of physiology in response to environmental cue4,5. The from Mexican yam (Dioscorea mexicana), diosgenin is used as the chemical diversity of specialized steroids can be largely attributed main precursor for the synthesis of most steroidal drugs, to the burgeoning catalytic activities from a number of enzyme including hormonal contraceptives and corticosteroid anti- families, including but not limited to 2,3-oxidosqualene cyclases, inflammatory agents, through the “Marker degradation” process cytochromes P450 (CYPs), oxidoreductases, and UDP-dependent developed by American chemist Russell Earl Marker in the 1940s glycosyltransferases6,7. The emergence and subsequent diversifi- (Fig. 1a)14. The characteristic 5,6-spiroketal moiety in diosgenin cation of specialized sterol metabolic systems driven by the is reminiscent of the 5,6- and 6,6-spiroketal moieties present in multifactorial selection pressures imposed by the ever-changing several polyketides isolated from soil bacteria15. In those systems, biotic and abiotic environments have shaped eukaryotic the spiroketal is formed via spontaneous cyclization of an evolution8. epoxide-ketone or a dihydroxy-ketone intermediate, the latter of Diosgenin is the aglycone of a class of spirostanol-type saponins which is guided by a unique cyclase to ensure production of a sporadically distributed in numerous monocot and eudicot plant single stereoisomer15,16. Considering these bacteria-specific a b c d Amborellales Paris Trigonella Nymphaeales De novo In planta biosynthesis Austrobaileyales polyphylla f.-graecum Early Chloranthales transcriptome Canellales (Liliales) (Fabales) angio- Piperales assembly H sperms Laurales H Magnoliales Acorales POOL 1 1.0E6POOL 1 1.2E6 H Alismatales H Petrosaviales Selection of Diosgenin Dioscoreales Diosgenin HO Pandanales 29 PpCYPs 5.0E4 5.0E3 Monocots Liliales and 33 TfCYPs All 29 PpCYPs All 33 TfCYPs Asparagales Cholesterol Arecales Poales Empty vector Empty vector Commelinales O Zingiberales SRM [415.3 -> 271.2] SRM [415.3 -> 271.2] Co-expression Time H Ceratophyllales 20 21 22 23 20 21 22 23 Ranunculales in tobacco (min) H O Proteales Trochodendrales Buxales PpCYPs: POOLS 2 & 3 TfCYPs: POOLS 2 & 3 H Eudicots Gunnerales H Dilleniales PpCYP76BM1 TfCYP76E24 HO Saxifragales PpCYP76BM2 TfCYP76F100 Vitales PpCYP76BM3 TfCYP706A21 Spiroketal steroids Zygophyllales PpCYP706C61 TfCYP75A95 Celastrales (e.g. diosgenin) Oxalidales PpCYP93A141 TfCYP93A140 Core Malpighiales PpCYP81B103 TfCYP93E1 Plant defense compounds eudicots Fabales POOL 1 PpCYP81BY1 TfCYP736A227 Rosales All PpCYPs PpCYP71EJ1 TfCYP71D545 Cucurbitales or PpCYP71EK1 TfCYP71AU101 Fagales TfCYP83E55 Geraniales All TfCYPs PpCYP71AQ18 Organic “Marker Myrtales PpCYP71DC3 TfCYP92A160 Crossosomatales PpCYP71AD8 TfCYP81E61 synthesis degradation” Picramniales PpCYP71AD9 TfCYP82A43 Sapindales POOL 2 PpCYP89A175 TfCYP82J17 O Huerteales TfCYP78A275 Malvales All PpCYPs PpCYP94D108 Brassicales PpCYP94D109 TfCYP89A174 Berberidopsidales or TfCYPs PpCYP704A178 TfCYP77A53 TfCYP77B41 H Santalales minus one PpCYP709C62 Caryophyllales at a time PpCYP72A617 TfCYP90B50 H Cornales PpCYP72A616 TfCYP90B51 H Ericales PpCYP72A614 TfCYP72A611 HO Icacinales Metteniusales POOL 3 PpCYP72A615 TfCYP72A612 Garryales PpCYP90G4 PpCYP728B66 TfCYP72A613 16-dehydropregnenolone Solanales PpCYP728U1 TfCYP714H7 Vahliales orTfCYP90B50 PpCYP87A68 TfCYP714E42 Gentianales plus each one PpCYP724B35 TfCYP714E43 Lamiales TfCYP704G23 Boraginales individually PpCYP90B53 Occurence Aquifoliales PpCYP90G4 TfCYP94A87 Asterales PpCYP90B52 TfCYP94A88 of spiroketal Escalloniales TfCYP96A151 steroids Bruniales Relative diosgenin abundance TfCYP86B46 Synthetic steroidal drugs Apiales TfCYP86A163 Paracryphiales Analysis by (e.g progesterone, cortisone) (log2 peak area) TfCYP86A164 Dipsacales LC-HRMS 0 5 10 15 Fig. 1 Identification of diosgenin-biosynthetic cytochrome P450s using pooled-screen approach. a Diosgenin is biosynthetically derived from cholesterol, and used as the precursor for the synthetic production of steroidal drugs via “Marker degradation”14. b Phylogenetic relationship of plant families that reportedly harbor spiroketal steroid chemotypes. P. polyphylla and the T. fenum–graecum, belonging to Liliales and Fabales respectively, are the two species investigated in this study. Angiosperm phylogeny was obtained from Theodor C.H. Cole, Hartmut H. Hilger, and Peter F. Stevens. c The stepwise pooled- screen approach employed in this study to identity diosgenin-biosynthetic CYPs from P. polyphylla and T. fenum–graecum. The screens were conducted using the combinatorial transient gene expression system in Nicotiana benthamiana. d Identification of the diosgenin-biosynthetic CYPs from P. polyphylla and T. fenum–graecum.29CYPs from P. polyphylla (PpCYPs) and 33 CYPs from T. foenum–graecum (TfCYPs) were selected for the screens. In the initial screen (POOL 1), co-expression of all PpCYPs or all TfCYPs resulted in diosgenin accumulation in N. benthamiana, shown in selected reaction monitoring chromatograms (SRM) from LC–MS data (peak intensity is indicated). In the second step (POOL 2), batches of CYPs omitting one CYP at a time were tested. PpCYP90G4 and TfCYP90B50 were identified as essential for reconstituting diosgenin production in N. benthamiana. In POOL 3, each PpCYP or TfCYP was co-expressed with PpCYP90G4 or TfCYP90B50, respectively.