ARTICLE DOI: 10.1038/s41467-017-00154-x OPEN A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate Evangelos C. Tatsis 1, Inês Carqueijeiro 2, Thomas Dugé de Bernonville 2, Jakob Franke1, Thu-Thuy T. Dang1, Audrey Oudin2, Arnaud Lanoue2, Florent Lafontaine2, Anna K. Stavrinides 1, Marc Clastre2, Vincent Courdavault 2 & Sarah E. O’Connor 1 Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism—an alcohol dehydrogenase and a cytochrome P450—produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-β- carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products. 1 John Innes Centre, Department of Biological Chemistry, Norwich Research Park, Norwich NR4 7UH, UK. 2 Université François-Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, Parc de Grandmont 37200 Tours, France. Evangelos C. Tatsis and Inês Carqueijeiro contributed equally to this work. Correspondence and requests for materials should be addressed to V.C. (email: [email protected])orto S.E.O. (email: [email protected]) NATURE COMMUNICATIONS | 8: 316 | DOI: 10.1038/s41467-017-00154-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00154-x he medicinal plant Catharanthus roseus synthesizes a on 4,21-dehydrogeissoschizine (strictosidine aglycone) to yield Tvariety of biologically active monoterpene indole alkaloids, akuammicine, a Strychnos alkaloid. These enzymes are char- most notably the anti-cancer agents vinblastine and acterized by both in vitro and in planta silencing approaches. vincristine. Vinblastine and vincristine, along with many other Akuammicine is known to arise from deformylation of – monoterpene indole alkaloids, are derived from the biosynthetic preakuammicine8, 10 12, which is the central biosynthetic intermediate, preakuammicine, as evidenced by extensive feeding precursor for the anti-cancer agents vinblastine and vincristine, as studies and isolation of biosynthetic intermediates from plant well as hundreds of other biologically active compounds. There- material1, 2. However, discovery of the enzymes that are fore, these enzymes open a gateway into a rich family of natural responsible for biosynthesis of the preakuammicine skeleton has products, and will facilitate metabolic engineering efforts to make proven elusive. high-value monoterpene indole alkaloids. Nearly all monoterpene indole alkaloid pathways begin with deglycosylation of strictosidine by a dedicated glucosidase, stric- tosidine glucosidase (SGD)3. Strictosidine aglycone, which can Results exist in numerous isomers such as 4,21-dehydrogeissoschizine, is Identification of gene candidates. To identify genes responsible then enzymatically transformed into the various monoterpene for generating Strychnos scaffolds such as preakuammicine and indole alkaloid skeletons. However, the enzymes responsible for akuammicine (Fig. 1a), we mined a transcriptomic database of the synthesis of preakuammicine, or any preakuammicine- C. roseus tissues13, 14, which also included data from plants with derived product from strictosidine aglycone remain unknown. increased alkaloid levels due to folivory15. Gene candidates iden- While the enzymatic reactions remain cryptic, extensive isolation tified were initially screened in C. roseus by virus-induced gene of reaction intermediates as well as model chemical studies silencing (VIGS), a transient silencing system in which qualitative suggest a sequence of reactions: 4,21-dehydrogeissoschizine perturbations in the metabolic profile between silenced and control (strictosidine aglycone isomer) is reduced to geissoschizine, which tissue provide clues to the metabolic function of the gene of is followed by oxidative rearrangement and reduction to pre- – interest. Transformation into preakuammicine requires both akuammicine4 10. Preakuammicine can non-enzymatically 4–10 – reduction and oxidation steps (Fig. 1b, Supplementary Fig. 1) . deformylate to produce the alkaloid akuammicine8, 10 12 or Therefore, we focused on alcohol dehydrogenases (reductase) and serve as a precursor for additional alkaloid scaffolds8. cytochromes P450 (oxidase); alcohol dehydrogenases have been Here we use a transcriptomic database for the medicinal plant implicated in other monoterpene indole alkaloid biosynthetic – Catharanthus roseus to identify two enzymes, an alcohol dehy- steps16 18, and cytochromes P450 are well known to play impor- drogenase (GS) and a cytochrome P450 (GO), that act in tandem tant roles in oxidative transformations in plant metabolism16, 19. a N H H N H N O N N H NH H β CH O C OH O- -Glc O 3 2 N H H Vincamine H H Strychnine O N OH H3CO2C CO CH N 2 3 N Strictosidine Preakuammicine N OH H N H OAc H H CO CH 2 3 N CO CH 2 3 H CO CH MeO N H OH 2 3 R R: CH3 vinblastine Akuammicine R: CHO vincristine b N N N SGD N H N H Strictosidine H Akuammicine H H H N OH H CO C H CO C CO CH 3 2 OH 3 2 OH 2 3 4,21-dehydrogeissoschizine Geissoschizine Preakuammicine (strictosidine aglycone) Fig. 1 Monoterpene indole alkaloid biosynthesis. a All ca. 2000 monoterpene indole alkaloids are derived from strictosidine, which is converted to preakuammicine through an unknown series of enzymatic reactions. Preakuammicine is hypothesized to be the precursor for many structurally divergent alkaloids. b A schematic overview of the proposed pathway that generates the Strychnos alkaloid akuammicine. Strictosidine is deglycosylated by the known enzyme, strictosidine glucosidase (SGD). An isomer of strictosidine aglycone, 4,21-dehydrogeissoschizine, can be reduced to form geissoschizine, which feeding studies suggest rearranges to form preakuammicine. Preakuammicine can deformylate to form the Strychnos alkaloid akuammicine, and is also hypothesized to be the precursor for many downstream alkaloids. See Supplementary Fig. 1 for a more detailed picture of monoterpene indole alkaloid biosynthesis 2 NATURE COMMUNICATIONS | 8: 316 | DOI: 10.1038/s41467-017-00154-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00154-x ARTICLE In planta and in vitro assay of enzyme candidates. Prioritization decreases in the production of the alkaloids catharanthine, of genes through Pearson’s correlation coefficient (PCC) deter- vindoline and vindorosine (Supplementary Fig. 2a), iboga and mination revealed a limited number of candidates displaying aspidosperma type alkaloids that are derived from pre- correlation with SGD (549 with PCC > 0.6), with only two can- akuammicine (Supplementary Fig. 1)23. didates predicted to encode cytochromes P450 (Supplementary All literature reports suggest geissoschizine (m/z 353), a Data 1). One was secologanin synthase, which is involved in reduction product of the 4,21-dehydrogeissoschizine isomer of upstream monoterpene indole alkaloid biosynthesis20, 21 and the strictosidine aglycone, is the precursor for preakuammicine second was CYP71D1V1, which was previously deposited as (Fig. 1b)4, 5, 10. As part of an effort to characterize alcohol JN613015 in a previous report, though not functionally char- dehydrogenases of C. roseus, we noted that silencing of one acterized22. Silencing CYP71D1V1 led to a statistically significant medium chain alcohol dehydrogenase gene (named GS1 for a Strictosidine 4,21-dehydrogeissoschizine (E )-geissoschizine Preakuammicine 21 6 5 5 N N 7 N 20 NH 2 3 14 O-β-Glc 3 20 6 SGD H GS H GO N H N N H 15 H H H 7 16 H O 15 16 17 N 2 17 OH H CO C H3CO2C H3CO2C 3 2 OH OH CO2CH3 GS GS spont. HCHO N N N N H N H H H H H N H H CO C H CO C CO2CH3 3 2 H OH 3 2 H OH (16R)-Z-isositsirikine (16R)-E-isositsirikine Akuammicine b c Akuammicine (16R)-E-isositsirikine (16R)-Z-isositsirikine GS1 + CYP71D1V1 (GO) GS1 GS1 GS1 + CYP71D1V1 (GO) GS2 + CYP71D1V1 (GO) GS2 GS2 THAS1 + CYP71D1V1 (GO) GS2 + CYP71D1V1 (GO) GS1 + microsomes EV 1.00 2.00 3.00 4.00Time 1.00 2.00 3.00 4.00 Time Fig. 2 Enzymatic activity of GS1 and GS2 and GO (CYP71D1V1). a Reaction of strictosidine with SGD, GS and GO. b Multiple reaction monitoring (MRM) on a LCMS showing compounds at m/z 355 in enzymatic assays of GS1/2 and GO. Isolation and NMR characterization of the two major compounds indicated that they are (16R)-E-isositsirikine and (16R)-Z-isositsirikine (Supplementary Figs 5–16). c MRM (m/z 323) of an akuammicine standard compared with enzymatic products of GS1/2 and GO. THAS1 is a medium chain alcohol dehydrogenase involved in the heteroyohimbine monoterpene indole alkaloid pathway (Supplementary Fig. 1). EV empty vector NATURE COMMUNICATIONS | 8: 316 | DOI: 10.1038/s41467-017-00154-x | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00154-x a 1 COOCH3 H-NMR of akuammicine H20 H15 H14 H13 H12 H6a H6b H9b H17 H8a H9a H19 H4 H8b H18a H18b 1H-NMR of enzyme reaction product CHCl3 CH3CN 6 8 N 19 12 9 1718 13 20 H 14 4 H 15 N 2 H CO2CH3 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 p.p.m. bc Synthetic akuammicine Enzymatic akuammicine 1.00 2.00 3.00 4.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Time Time Fig.