Oxidation and cyclization of casbene in thebiosynthesis of Euphorbia factors from matureseeds of Euphorbia lathyris L.Dan Luoa, Roberta Callarib, Britta Hambergera, Sileshi Gizachew Wubshetc, Morten T. Nielsena,Johan Andersen-Ranberga,d, Björn M. Hallströme, Federico Cozzia, Harald Heiderb, Birger Lindberg Møllera,d,Dan Staerkc, and Björn Hambergera,d,1

aPlant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark; bEvolvaAS, CH-4153 Reinach, Switzerland; cDepartment of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,DK-2100 Copenhagen, Denmark; dCenter for Synthetic Biology “bioSYNergy,” DK-1871 Frederiksberg C, Denmark; and eScience for Life Laboratory, Schoolof Biotechnology, Kungliga Tekniska Högskolan Royal Institute of Technology, SE-171 21 Stockholm, Sweden

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved July 6, 2016 (received for review May 10, 2016)

The seed oil of Euphorbia lathyris L. contains a series of macrocy-clic diterpenoids known as Euphorbia factors. They are the currentindustrial source of ingenol mebutate, which is approved for thetreatment of actinic keratosis, a precancerous skin condition. Here,we report an alcohol dehydrogenase-mediated cyclization step inthe biosynthetic pathway of Euphorbia factors, illustrating theorigin of the intramolecular carbon–carbon bonds present in lath-yrane and ingenane diterpenoids. This unconventional cyclizationdescribes the ring closure of the macrocyclic diterpene casbene.Through transcriptomic analysis of E. lathyris L. mature seedsand in planta functional characterization, we identified three en-zymes involved in the cyclization route from casbene to jolkinol C,a lathyrane diterpene. These enzymes include two cytochromesP450 from the CYP71 clan and an alcohol dehydrogenase (ADH).CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and5-oxidation of casbene, respectively. When coupled with theseP450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes de-hydrogenation of the hydroxyl groups, leading to the subsequentrearrangement and cyclization. The discovery of this nonconven-tional cyclization may provide the key link to complete elucidationof the biosynthetic pathways of ingenol mebutate and other bio-active macrocyclic diterpenoids.

terpenoid biosynthesis | transcriptomic analysis | regio-specific oxidation |nonconventional cyclization

Species in the genus Euphorbia have a long history of use asmedicinal plants for traditional treatments. Their bioactivity

is largely due to the rich production of chemically diverse iso-prenoids, the most characteristic of which are the macrocyclicditerpenoids. Some of these have shown considerable potentialas lead compounds in drug discovery and have been the focus ofmany medicinal chemistry and therapeutic studies (1) (Fig. 1).Euphorbia factor L10 and euphodendroidin D show powerfulinhibition of the transport activity of P-glycoprotein, a multidrugtransporter overexpressed in cancer cell plasma membranes asan efflux pump conferring cellular resistance to anticancer che-motherapy (2, 3). Prostratin is in phase I human clinical trials forthe treatment of HIV, as it was shown to activate viral reservoirsin latently infected T cells, as well as to inhibit viral replication(4). Ingenol mebutate, first isolated from Euphorbia peplus, isone of the most well-studied macrocyclic diterpenoids for itsremarkable antitumor and antileukemic activity (5). In 2012,ingenol mebutate gel was approved for the treatment of actinickeratosis, a precondition of squamous cell carcinoma (6). Thecurrent supply of ingenol mebutate is limited to direct isolationfrom E. peplus, which yields only 1.1 mg/kg from the aerial tissue(7). Due to the increasing demand and low isolation yields ofingenol mebutate from the native plant, there is a strong need forthe development of a more economical and efficient method for

its production and for a robust supply chain. The latest 14-stepchemical synthesis from the chiral monoterpene (+)-3-careneobtained an overall yield of around 1% and relied on expensivecatalysts (8).Ingenol was identified as a constituent in Euphorbia ingens in

1968 (9). Subsequently, a wide range of macrocyclic diterpenoidshave been isolated from Euphorbia exhibiting a high diversity inbackbone structures, oxygenation levels, stereoisomerism, andesterification patterns (1). According to different stages of cy-clization of their C20 backbones, macrocyclic diterpenoids canbe classified into two groups: simple bicyclic casbene type andfurther cyclized types. The latter includes jatrophanes, lathyr-anes, tiglianes, daphnanes, and ingenanes ranked according totheir increased structural complexity (Fig. 1). Although jatro-phanes are widespread in Euphorbia, only very few Euphorbiaspecies have been reported to accumulate ingenanes (1).

Significance

Ingenol mebutate is a diterpene ester with a highly complexmacrocyclic structure that has been approved for the treatmentof actinic keratosis, a precondition of skin cancer. The currentproduction of ingenol mebutate through plant extraction orchemical synthesis is inefficient and costly. Here, we describethe discovery of a biosynthetic route in Euphorbia lathyris L.(caper spurge) in which regio-specific oxidation of casbene isfollowed by an unconventional cyclization to yield jolkinol C, aprobable key intermediate in the biosynthesis of macrocyclicditerpenes, including ingenol mebutate. These results can fa-cilitate the biotechnological production of this high-valuepharmaceutical and discovery of new biosynthetic intermedi-ates with important bioactivities.

Author contributions: Björn Hamberger designed research; D.L., R.C., M.T.N., J.A.-R., andBjörn Hamberger performed research; R.C., Britta Hamberger, S.G.W., F.C., H.H., and D.S.contributed new reagents/analytic tools; D.L., S.G.W., M.T.N., J.A.-R., B.M.H., B.L.M., andBjörn Hamberger analyzed data; D.L. and Björn Hamberger wrote the paper; and B.L.M. isresponsible for distributing materials and clones described in this work (blm@plen.ku.dk).

Conflict of interest statement: D.L., M.T.N., R.C., and Björn Hamberger have filed a patentapplication (PA 2015 70069) covering “Methods and materials for producing oxidizedmacrocyclic diterpenes” related to the manufacture of jolkinol C and precursors thereof.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The nucleotide sequences reported in this paper have been submitted tothe short read archive (SRA) at the NCBI [accession nos. SRP057971 (E. lathyris) andSRP058119 (E. peplus)]. Novel nucleotide sequences encoding enzymes functionally char-acterized during this work were deposited in the GenBank database (accession nos.KR350665 (ElADH1), KR350666 (ElADH2), KR350667 (ElCBS), KR350668 (CYP71D445),KR350669 (CYP726A27), KR350671 (EpADH1), and KX428471 (CYP71D365).1To whom correspondence should be addressed. Email: hamberge@msu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1607504113/-/DCSupplemental.

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Currently the commercially most interesting species is Euphorbialathyris L., a plant native to the Mediterranean area. E. lathyris L.contains both esterified lathyrane and ingenane derivatives(known as Euphorbia factors or L-factors) in the seed oil (10). Asemisynthesis approach to ingenol mebutate using hydrolysisproducts of ingenol-based Euphorbia factors as raw material hasbeen developed (11). However, access to the starting materialfrom E. lathyris L. seeds is limited due to natural fluctuations anda biennial life cycle.Recent successes in metabolic engineering and synthetic bi-

ology approaches have received wide attention as economicallycompetitive approaches for the production of structurally com-plex terpenoids (12, 13). However, as a prerequisite to reconstructpathways of high-value diterpenoids in biotechnological pro-duction hosts, the steps involved in their biosynthesis must beelucidated. Despite numerous chemical and pharmacological re-ports, biosynthetic pathways of macrocyclic diterpenoids remainpoorly understood. The simple bicyclic diterpene, casbene, hasbeen suggested as the first committed intermediate toward themore complex multicyclic diterpenoids found in Euphorbia spe-cies (14). Casbene synthase (CBS) was first identified in Ricinuscommunis (15), and cyclization of the general C20 precursor,geranylgeranyl diphosphate (GGDP), to the bicyclic casbene hasbeen well established. Although previously suggested to proceedvia a route as outlined in Fig. 1, the intramolecular cyclizationand rearrangements from the potential casbene precursor tomulticyclic diterpenes remain hypothetical. An examination ofthe vast structural complexity of many types of macrocyclicditerpenoids found in nature indicates that multicyclic diterpe-noids carry a higher degree of oxygenation compared withbicyclic casbenes. This relationship between cyclization and ox-ygenation stages may provide the key for formation of multicyclicditerpenoids in which intramolecular cyclizations are likely tooccur in a combined pathway controlled by cytochrome P450monooxygenases (P450s). Recently, a Euphorbiaceae-specificexpansion of P450s in the CYP71 clan was reported (16). Al-though the founding members of the subfamily were shown tocatalyze epoxidation of fatty acids in Euphorbia lagascae Spreng(17), several P450s of the CYP726 subfamily in castor bean hadactivity toward C5 of casbene, which is a characteristic of the

casbene-type diterpenoids present in castor bean and relatedspecies (18). In addition to P450s, members of the short-chaindehydrogenase∕reductase superfamily are also involved in ter-penoid metabolism in many plants. For example, ZSD1, a short-chain alcohol dehydrogenase (ADH) from Zingiber zerumbet,catalyzes the final step of zerumbone biosynthesis (19).To identify biosynthetic enzymes involved in the formation of

multicyclic diterpenoids, we integrated metabolomic analysestargeted for diterpenoids with an interrogation of a deep tran-scriptome library generated from mature seeds of E. lathyris L., aseed development stage where we detected the presence of bothcasbene and ingenane diterpenoids. We focused our search ongenes encoding enzymes with putative oxygenation or oxido-reductase activity, such as P450s and members of the ADH family.Here, we report the discovery of two P450s, CYP71D445

and CYP726A27, capable of regio-specific oxygenation of cas-bene, and an alcohol dehydrogenase, ADH1, which catalyzes de-hydrogenation of the hydroxyl groups of the resulting trioxidizedproducts. Specifically, results from both in vivo and in vitro ex-periments show that ADH1 catalyzes a nonconventional cyclizationof oxidized casbene to produce jolkinol C, a lathyrane diterpenoid,which is a potential key intermediate in ingenol biosynthesis. Theobserved patterns of regio-specific oxygenations and cyclizationcarried out by these enzymes reflect those found in the naturallyoccurring diterpenoids. These biosynthetic steps pave the way forbiotechnological production of multicyclic intermediates of highvalue and structurally complex natural products.

ResultsAccumulation of Casbene and Euphorbia Factors in Mature Seeds ofE. lathyris L. Although proposed to serve as a general precursor,the presence of casbene has not been described previously inEuphorbia plants accumulating multicyclic diterpenoids. To testfor casbene in the plant species being the most important com-mercial source of ingenol, mature seeds of E. lathyris L. wereextracted with n-hexane and analyzed by GC-MS. A traceamount of casbene matching retention time and mass spectra ofthe authentic standard was detected (SI Appendix, Fig. S1A).Accumulation of lathyrane and ingenane diterpenoids in thesame tissue was confirmed by LC coupled with high-resolution

Fig. 1. Distribution and structures of bioactive Euphorbia diterpenes and their carbon skeletons.

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MS (LC-HRMS). In the methanol extract, ingenane Euphorbiafactors L4 and L5 were detected (SI Appendix, Fig. S2D) to-gether with lathyrane Euphorbia factors L1–L3 and L7a (SIAppendix, Fig. S2 A–C). Co-occurrence of casbene with the morecomplex multicyclic diterpenoids in mature seeds may imply atissue-specific and active biosynthetic pathway for the efficientconversion of casbene in biosynthesis of Euphorbia factors.

Identification of Transcripts of CBS, P450s, and ADHs Expressed in E.lathyris Seeds. To probe the seeds of E. lathyris L. for enzymespotentially involved in the formation of multicyclic diterpenoids,a RNA-Seq transcriptome library was prepared from matureseeds. A CBS ortholog was identified in this library based on itshigh sequence identity to the previously identified gene fromE. peplus (16). The E. lathyris CBS ortholog was highly expressedin mature seeds (SI Appendix, Table S1). Based on this result,combined with the concentrated accumulation of potentiallycasbene derived ingenane esters, we hypothesized that matureseeds of E. lathyris represent the most specialized tissue for theirformation and prioritized the search for candidate genes by theirexpression level in this library. Based on transcriptomic datafrom leaf tissue, expansions of CYP71D and CYP726 subfamilieswere previously reported in E. peplus (16). To identify E. lathyrishomologs, we selected these subfamilies as starting points tointerrogate the mature seed transcriptome. A comprehensive listof P450 enzymes from subfamilies of CYP71D and CYP726 wasgenerated and candidates were prioritized based on their ex-pression level in the E. lathyris seed library (SI Appendix, TableS1). According to gene abundance data, E. lathyris CYP71D445and CYP726A27 were found to be the highest expressed P450s in

the CYP71D and CYP726 subfamilies. In addition, two highlyexpressed ADH candidates, E. lathyris ADH1 and ADH2, wereidentified in the transcriptome library using known ADHs in-volved in terpene metabolism as queries (19). Sequence com-parison and phylogenetic analysis implied ADH1 and ADH2 asmembers of the short chain dehydrogenase/reductase familySDR110C (ABA2 xanthoxin dehydrogenase family; SI Appendix,Fig. S3). SDR110C is expanded in vascular plants and catalyticactivities were reported in oxidiation of various phenolics orterpenoids, including monoterpenes isopiperitenol and borneol,the macrocyclic sesquiterpene zerumbone and the diterpenemomilactone (19–22).

Two P450s Expressed in Nicotiana benthamiana Function as Regio-Specific Casbene Monooxygenases. To test the capacity of theidentified P450s to oxygenate casbene, E. lathyris CYP71D445and CYP726A27 were coexpressed with CBS in N. benthamianausing the Agrobacterium-mediated transient expression system,engineered for optimized production of diterpenoids by thestrategy of coexpression with Coleus forskohlii 1-deoxy-D-xylulose5-phosphate synthase (DXS) and geranylgeranyl diphosphatesynthase (GGPPS) as described earlier (23). The production ofcasbene (1) in the infiltrated N. benthamiana leaves was con-firmed by GC-MS analysis (Fig. 2 A, b and SI Appendix, Fig. S1).The LC-MS total ion chromatograms of the N. benthamianacoexpressing CBS and CYP71D445 showed the appearance of anew constituent 2, which, based on the parental ion (m/z287.2376, [M+H]+), could represent an oxygenated casbenederivative with an additional degree of unsaturation (Fig. 2 B, b).Coexpression of CBS and CYP726A27 resulted in formation of

Fig. 2. Functional characterization of E. lathyris CBS, P450s, and ADH1 via in vivo expression. (A) GC-MS profiles of N. benthamiana transiently expressingE. lathyris CBS, internal standard: fluoranthene, 1 ppm. (B) LC-HRMS total ion chromatograms of N. benthamiana transiently expressing E. lathyris CBS,CYP71D445, CYP726A27, and ADH1. (C) Structures of isolated intermediates and products. (D) LC-HRMS total ion chromatograms of S. cerevisiae expressingCBS, CYP71D445, CYP726A27, and ADH1.

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compound 3 with a parental ion (m/z 289.2513, [M+H]+) cor-responding to a hydroxylated casbene (Fig. 2 B, c). Moreover,when CBS was coexpressed with CYP71D445 and CYP726A27,compound 4 (m/z 303.2317, [M+H]+), a casbene derivativecontaining two oxygen atoms, accumulated as the major chro-matographically detectable product together with trace amountsof compounds with molecular masses indicating multioxygenatedderivatives of casbene (Fig. 2 B, d). Compounds 2, 3, and 4 wereisolated from infiltrated N. benthamiana and purified by com-bined chromatography. The structures were elucidated by NMRanalyses, establishing 2, 3, and 4 as 9-ketocasbene, 5-hydrox-ycasbene, and 5-hydroxy-9-ketocasbene, respectively (Fig. 2Cand SI Appendix, Fig. S5 and Table S2).

Transcript Profiles of E. lathyris CBS, CYP71D445, CYP726A27, andADH1 Indicate Specific Expression in Mature Seeds. To discoverdownstream enzymes catalyzing sequential steps in the pathwaysof Euphorbia factors, transcript levels of CBS, CYP71D445, andCYP726A27 were investigated by quantitative RT-PCR (qPCR)and compared with the two putative ADH genes. qPCR analy-sis was performed using cDNA templates derived from totalRNA extracted from young and mature leaves, stems, fruits,developing seeds, mature seeds, and roots of E. lathyris L. Inthe transcript profiles obtained (SI Appendix, Fig. S4A), CBS,CYP71D445, CYP726A27, and ADH1 shared similar expressionpatterns across all tested tissues, with high transcript accumula-tion in mature seeds. In contrast, the transcript level of ADH2 inmature seeds was relatively low compared with the other tissues.The transcript profiles of E. lathyris CBS, CYP71D445, andCYP726A27 supported their involvement in the formation ofEuphorbia factors, which were only detected in mature seeds (SIAppendix, Fig. S4 C and D). The specific expression of ADH1 inmature seeds indicated a potential function downstream of theP450-based oxygenations of casbene in the biosynthesis of Eu-phorbia factors, whereas the expression pattern of ADH2 impliedlimited relevance for the pathway.

Coexpression of E. lathyris ADH1 with CBS, CYP71D445, andCYP726A27 Resulted in Cyclization Affording a Lathyrane-TypeDiterpene. To investigate the function of E. lathyris ADH1 andADH2, the enzymes were individually coexpressed with CBS,CYP71D445, and CYP726A27 in N. benthamiana. Comparedwith the combination producing 4 (Fig. 2 B, d), LC-MS total ionchromatograms of N. benthamiana leaves that in additionexpressed ADH1 showed an obvious difference in product for-mation (Fig. 2 B, e), whereas coexpression of ADH2 did notresult in any detectable activity (SI Appendix, Fig. S4B). Thecombined expression of CBS, CYP71D445, CYP726A27, andADH1 (Fig. 2 B, e) resulted in formation of a new constituent 5with m/z of 317.2104, possibly corresponding to a cyclizedproduct containing an additional oxygen atom. Compound 5 wasisolated from N. benthamiana transiently expressing the specificenzymes and identified using HPLC-HRMS with solid-phaseextraction (SPE) and NMR spectroscopy, i.e., HPLC-HRMS-SPE-NMR (24). Structure elucidation by NMR analysis estab-lished 5 as jolkinol C (SI Appendix, Fig. S5 and Table S2B), alathyrane type diterpene naturally found in Euphorbia jolkiniBoiss (25).

Production of Oxidized Derivatives of Casbene in Saccharomycescerevisiae. To confirm the catalytic activities observed in theN. benthamiana transient expression system, CYP71D445,CYP726A27, and ADH1, combined with the NADPH-dependentcytochrome P450 oxidoreductase (POR) from Ricinus communis,were cloned and expressed in a S. cerevisiae strain engineered forcasbene production. Consistent with the results in planta, com-pounds 2 and 3 were found when CYP71D445 and CYP726A27were independently expressed with CBS (Fig. 2 D, b and c).

In addition, compound 6 (m/z 289.2522, [M+H]+), the parentalion of which matches the hydroxylated casbene, was obtained asan extra product in the strain expressing CBS and CYP71D445(Fig. 2 D, b). In the profiles obtained from coexpression of CBS,CYP71D445, and CYP726A27 in the S. cerevisiae strain, the di-oxygenated derivative 4 was observed as the main product (Fig. 2D, d). However, when ADH1 was coexpressed with CBS and thetwo P450s, no accumulation of compound 5 was detected (Fig. 2D, e). Instead, the detectable diterpenoid products in this strainwere compounds 7 (m/z 319.2261, [M+H]+) and 8 (m/z 319.2263,[M+H]+), with the masses suggesting that these were casbenederivatives with trioxygenation. Compounds 6, 7, and 8 were pu-rified from the corresponding S. cerevisiae strains by combinedchromatography. The structures of 6, 7, and 8 were determined as9-hydroxycasbene, 5-keto-6,9-casbene diol, and 6-keto-5,9-casbenediol, respectively, based on NMR analysis (Fig. 2C and SI Ap-pendix, Fig. S5 and Table S2 B and C).

Characterization of Catalytic Features of ADH1 via in Vitro EnzymeAssays. To assign the role of ADH1 in the cyclization processyielding compound 5, a series of in vitro assays was carried outusing various isolated casbene derivatives as substrates. Thepurified His6-tagged recombinant enzyme of ADH1 was in-cubated with compounds 3, 4, 6, 7, and 8 individually in thepresence of NAD+, and the formation of products was analyzedby LC-HRMS (Fig. 3A). In the resulting profiles, efficient con-version of 6 to 2 was achieved by ADH1 (Fig. 3 A, a and b),indicating its capability of catalyzing dehydrogenation of the9-hydroxyl group. When ADH1 was incubated with 3, compound9 (m/z 287.2374, [M+H]+) was detected as the product formed(Fig. 3 A, c and d). The spectral data of 9 (SI Appendix, Fig. S1B)matches the previously reported 5-ketocasbene (18, 26). Cor-roborating its ability to catalyze dehydrogenation of the 5-hydroxylgroup, ADH1 also dehydrogenated 4 yielding the casbene dionederivative 10 (m/z 301.2157, [M+H]+) (Fig. 3 A, e and f). Re-markably, the trioxygenated casbene derivatives, 7 and 8, werefound to be converted to the cyclized product 5 by ADH1 (Fig. 3A, g–j).

Reproduction of the Lathyrane-Type Diterpene by in Vitro AssayedCYP71D445, CYP726A27, and ADH1. Although the trioxygenatedderivatives 7 and 8 were obtained, the cyclized diterpene 5 wasnot detected in the recombinant S. cerevisiae strains. A previousreport has shown that an acidified environment, such as in cul-tured yeast, can affect terpene cyclization (27). Therefore, wespeculate that the cyclization reaction affording 5 might beinhibited by the acidity of yeast culture. To reconstruct theproduction of 5, we performed the combined in vitro assays withmicrosomal fractions from yeast strains expressing the P450s andthe purified ADH1, expressed as His6-tagged recombinant en-zyme in Escherichia coli. First of all, the catalytic activities ofmicrosomes were confirmed by in vitro assays supplied with 1 asthe substrate. LC-MS total ion chromatograms of the in vitromicrosomal assays were highly consistent with the resultsobtained in vivo (Fig. 3 B, a–d). Specifically, when the micro-somal assays expressing both CYP71D445 and CYP726A27 werecoupled with ADH1, reproduction of the lathyrane-type diter-pene 5 was achieved in vitro (Fig. 3 B, e), demonstrating that thecharacterized enzymes are truly responsible for the cyclization.In these combined assays, the casbene dione 10, which is thedirect dehydrogenation product of 4, was obtained as well. Be-cause accumulation of 10 was not detected in vivo, this resultindicated that the catalytic abilities of the two CYPs and ADH1were not perfectly balanced at the tested in vitro conditions.Compared with the structural features of 4, an additional ox-

ygenation on C-6 was found in 5, 7, and 8, representing either6-hydroxylation or 6-ketonization. The results from in vitro en-zyme assays showed that the oxygenation of C-6 was not directly

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catalyzed by ADH1 (Fig. 3). Therefore, we hypothesized that itwas catalyzed by a multifunctional P450. To both elucidate theorigin of C-6 oxygenation and establish the order of multipleoxygenations during the ring closure process, we investigatedconversion of isolated casbene derivatives with regio-specificoxygenations (2, 3, 4, and 6) in the microsomal assays. No con-version was found when the ketone derivatives 2 and 4 weresupplied as the substrate (SI Appendix, Fig. S6). On the otherhand, the monohydroxyl derivatives 3 and 6 were converted to 5in the combined assays of CYP71D445, CYP726A27, and ADH1(Fig. 4 B, e and 4 A, e), demonstrating that both 3 and 6 can actas early intermediates in the pathway. When we supplied the 9-hydroxycasbene derivative 6 with microsomes of CYP726A27,which is the casbene 5-oxidase, a new peak 11 (m/z 305.2476,[M+H]+; 327.2286, [M+Na]+) corresponding to a putative cas-bene diol was detected by LC-HRMS (Fig. 4 A, b). Despite theunavailability of an authentic standard, 11 most likely repre-sents the 5,9-casbene diol based on the catalytic features ofCYP726A27. Although limited stability precluded isolation andstructural identification of 11, the hypothetical structure wasexemplified by feeding compound 6 with the microsomesexpressing CYP71D445 and CYP726A27. Compound 4 wasidentified instead of 11 in these assays (Fig. 4 A, c), suggestingthat CYP71D445 further oxidized the 9-hydroxyl group of 11into the 9-ketone group. To be noticed, trace amounts of 5 weredetected when compound 6 was incubated with the microsomesexpressing CYP726A27 and the recombinant enzymes of ADH1(Fig. 4 A, d). The generation of 6-oxygenation of 5 in the absenceof CYP71D445 demonstrates that CYP726A27 is a bifunctionalenzyme catalyzing hydroxylation on the adjacent positions C-5and C-6. Additionally, when tested with the 5-hydroxyl pre-inserted substrate 3, the formation of 5 was recognized in theassays coupling CYP71D445 with ADH1 (Fig. 4 B, d). Thisfinding indicates that CYP71D445 shares the role of insertingoxygen at C-6.

Activity of the Functional Orthologs from E. peplus. Comparativetranscriptomics of E. lathyris L. with E. peplus, a related Euphorbiaspecies accumulating the most complex multicyclic ingenol diter-penoids, revealed homologs of the four enzymes described in thisstudy in the stem derived cDNA library of E. peplus. E. peplusCBS (KC702397.1) has been reported together with CYP726A4(KF986823.1), found in the largely expanded tribe CYP71D, car-rying 23 members of E. peplus (16). The corresponding full-lengthcDNA sequences were cloned and tested functionally in the N.benthamiana system. Direct comparison indicated that E. peplusCBS, CYP71D365, CYP726A4, and ADH1 shared the activity withE. lathyris CBS, CYP71D445, CYP726A27, and ADH1, respectively(SI Appendix, Fig. S7 A and C), and hence are functional orthologswith the same catalytic features. Jolkinol C (5) was obtained fromthe combination of E. peplus CBS, CYP71D365, CYP726A4, andADH1, despite the lack of reported lathyrane-type diterpenoids inthis plant.

DiscussionMetabolite profiling of mature seeds of E. lathyris revealed theco-occurrence of casbene together with Euphorbia factors(lathyrane and ingenane diterpenoids). Extended metaboliteprofiling including several other plant tissues confirmed thespecific accumulation of casbene and the Euphorbia factors inmature seeds (SI Appendix, Figs. S1, S2, and S4 C and D). Thisfinding suggested that the mature seed of E. lathyris constituted ahighly specialized tissue for the biosynthesis of lathyrane andingenane diterpenoids, uniquely suited for pathway discovery. Itmay follow an emerging theme, where colocalization of thescaffold and final functionalized diterpene in a specific cell typeenabled discovery of the biosynthetic route (28).

Fig. 3. Investigation of in vitro assayed CYP71D445, CYP726A27, and ADH1by LC-HRMS analysis. (A) LC-HRMS total ion chromatograms of the productsgenerated from recombinant ADH1 when supplied with 3, 4, 6, 7, and 8.(B) LC-HRMS total ion chromatograms of the products generated by com-bined assays of microsomal fractions expressing CYP71D445, CYP726A27,and the recombinant ADH1, supplied with casbene.

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The observed polyoxygenation of the multicyclic Euphorbiafactors suggested that P450 catalyzed oxygenations might serveto activate casbene, a relatively inert hydrocarbon precursor, andthereby enable a subsequent cyclization reaction to proceed.Casbene 5-oxidation has previously been described in R. com-munis (18), followed by 6-hydroxylation or 7,8-epoxidation,matching natural casbene type diterpenoids present in this spe-cies (29). However, none of these oxidations led to a ring closuregenerating lathyrane or multicyclic backbones. This lack of ac-tivity indicated that a specific oxygenation of casbene at a particularcarbon position was crucial for the formation of the intramolecularC-C bonds present in lathyrane and ingenane diterpenoids.Transcriptome analysis of E. lathyris L. mature seeds and

functional characterization of candidate genes identified twocytochromes P450, CYP71D445, and CYP726A27, as casbenemonooxygenases. Among these, CYP71D445 catalyzed oxidationat the C-9 position of casbene. Surveying reported macrocyclicditerpenoids with different carbon backbones (1), we found thatcasbene 9-oxidation is characteristic of multicyclic diterpenetypes shared across all lathyrane, jatrophane, tigliane, daphnane,and ingenanes (Fig. 1). Based on these observations, we suggestthat C-9 oxidation defines early bifurcation steps in the bio-synthesis of macrocyclic diterpenoids. A lack of the essentialoxygenation at C-9 may, as a consequence, lead to the formationof casbene-type diterpenoids, whereas di-oxygenation at bothC-9 and C-5 constitutes the subsequent step en route to multi-cyclic diterpenes derived from casbene.Cloning and coexpression of the set of orthologous genes from

the relative E. peplus in the transient N. benthamiana systemresulted also in formation of jolkinol C (5). In the absence ofreported lathyrane-type diterpenes in E. peplus, these resultsimply that either jatrophane or ingenane diterpenoids, or pos-sibly both types accumulating in this species, are biosynthesizedvia lathyrane intermediates. Thus, it can be assumed that evo-lution of the pathway to jolkinol C (5) and presumably themost complex ingenanes predates speciation of E. lathyris andE. peplus.The results from ADH1 expression in N. benthamiana revealed

an unconventional pathway from casbene to the lathyrane diter-penoid jolkinol C (5) proceeding via an ADH catalyzed cyclizationreaction (Fig. 5). By testing the recombinant enzymes in vitro, twoimportant features of this pathway were demonstrated: the regio-

specific oxidations of casbene by the P450s initiate and precede theintramolecular cyclization and this cyclization reaction is dependenton the catalytic activity of the E. lathyris ADH1. The combined invitro assays of CYP71D445 and CYP726A27 demonstrated that thetwo P450s act as bifunctional enzymes, thereby clarifying theirshared responsibility with a partial functional redundancy for cat-alyzing oxidation at C-6. Similar bifunctional features have pre-viously been found in CYP726A14, a casbene monooxygenase fromR. communis (26). The in vitro assayed ADH1 displayed the activityof catalyzing dehydrogenation of various substrates with a casbenebackbone carrying hydroxyl groups at the C-5 and C-9 positions.In particular, the presented conversion of the trioxygenated in-termediate 7 to 5 fully supported the hypothesis that ADH1 is di-rectly catalyzing the cyclization reaction.The above results enabled us to identify individual steps and

intermediates in the conversion of casbene to the lathyranediterpenoid 5 (Fig. 5): CYP71D445 and CYP726A27 initially cat-alyze the C-9 and C-5 hydroxylation of casbene, respectively. Thisconversion is then followed by positional crossover hydroxylationsmediated by the two P450s resulting in the formation of the5,9-casbene diol 11. The resulting diol 11 is further oxidized byone of the bifunctional monooxygenases, either CYP726A27 orCYP71D445, resulting in the formation of a proposed triol in-termediate. The existence of the triol intermediate was impliedby the observed formation of 7 and 8. Subsequently, ADH1 ac-tivates this intermediate or the interconvertible pair of 7 and 8through dehydrogenation leading, plausibly, via a series of keto-enol tautomerisms to the formation of a new C-C bond betweenC-6 and C-10 (SI Appendix, Fig. S8). Instability and rapid furtherconversion may explain why the triol intermediate did not ac-cumulate. The ketocasbene derivatives 2 and 4 are consideredside products further oxidized by CYP71D445. Their high ac-cumulation in the in vivo assays may be favored by structuralstabilization via the conjugated double bonds.The results obtained document the origin of the intra-

molecular carbon-carbon bonds present in lathyrane, jatrophane,tigliane and ingenane diterpenoids. Knowledge on how to es-tablish these carbon backbones offers the possibility to identifythe subsequent enzymatic steps and to ultimately reconstruct thebiosynthetic pathways of pharmaceutically active diterpenoids andnew bioactive intermediates for biotechnological production. Therecently reported option to redirect P450-dependent biosynthetic

Fig. 4. Characterization of catalytic features of CYP71D445 and CYP726A27 by combined in vitro assays. (A) LC-HRMS total ion chromatograms of theproducts generated by combined assays of microsomes expressing CYP71D445, CYP726A27, and the recombinant ADH1 supplied with 6. (B) LC-HRMS total ionchromatograms of the products generated by combined assays of microsomes expressing CYP71D445, CYP726A27, and the recombinant ADH1 supplied with 3.

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pathways into tobacco chloroplasts and drive the P450s directlybased on light-driven photosynthetic electron transport using re-duced ferredoxin as the direct electron donor to the P450s offersopportunities to redirect the production capacity of photosyntheticcells toward production of high levels of structurally complexditerpenoids (30, 31).

Materials and MethodsPlant Material, RNA Extraction, and Quantitative Real-Time PCR. Plants weregrown in a greenhouse at the University of Copenhagen under ambientphotoperiod and 24 °C day/17 °C night temperatures. Total RNA fromE. lathyris L. young and mature leaves, fruits, stems, developing seeds (whiteand soft), mature seeds (black and hard), and roots was extracted accordingto a previously described method (32), followed by DNase I digestion. Thesame method was used to extract total RNA from E. peplus leaves, stems,and roots. Quantitative real-time PCR reactions were performed with gene-specific primers and the SensiFAST SYBR No-ROX Kit from Bioline on a Rotor-Gene Q cycler (Qiagen). Details of the qPCR reactions are provided in SIAppendix, SI Materials and Methods.

Transcriptome Sequencing and de Novo Assembly. RNA was prepared for se-quencing using Illumina TruSeq preparation kit v2 using poly-A selection. Thefragmentswere clustered on cBot and sequencedwith paired ends (2× 100bp)on an Illumina HiSeq 2500, according to the manufacturer’s instructions. Intotal, 166.6 million read-pairs were generated from E. lathyris L. matureseeds RNA and 55.7 million read-pairs from E. peplus stem RNA, respectively.Adaptor sequences were removed from raw reads, and reads were trimmedat the ends to a minimum phred score of 20, using the fastq-mcf tool from

ea-utils (https://github.com/ExpressionAnalysis/ea-utils). Processed readswere assembled using Trinity (33) resulting in a total of 145,990 and 89,163assembled putative transcripts from E. lathyris L. mature seeds and E. peplusstem RNA-seq, respectively. Transcript abundance estimation was performedusing RSEM and the scripts provided with Trinity. Likewise, the putativecoding sequences were predicted using TransDecoder scripts from Trinity.

Diterpenoid Profiling of E. lathyris Mature Seeds. For information, please seeSI Appendix, Materials and Methods.

Construction of Uracil-Specific Excision Reagent-Compatible Expression Vector.The GATEWAY-compatible pEAQ-DEST1 vector (34), for high level transient ex-pression in N. benthamiana was obtained as a gift from George Lomonosoff,John Innes Centre, Norwich, UK. By the removal of a PacI restriction site and theintroduction of a uracil-specific excision reagent (USER)-PacI site in the GATEWAYsite of pEAQ-DEST, the USER compatible (35) vector pEAQ-USER was constructed.Details of vector construction are provided in SI Appendix, SI Materialsand Methods.

Generation of Expression Constructs. Total RNA from E. lathyris L. seedsextracted as described above was used for cDNA synthesis. First-strandcDNAs were synthesized using the Thermo Scientific RevertAid First StrandcDNA Synthesis Kit and oligo(dT) primer. Full-length cDNAs of identifiedcandidate genes were obtained by PCR amplification using gene-specificprimers with desired USER cassettes (SI Appendix, Table S3). PCR productswere cloned into pEAQ-USER vectors for plant transformation as describedpreviously (35, 36) and verified by sequencing.

Transient Expression in N. benthamiana. Transient expression of full-lengthgenes in N. benthamiana leaves and extraction of diterpenes were per-formed as recently described (37). For details, see SI Appendix, Materialsand Methods.

Classification of E. lathyris ADH Sequences. For information, please see SIAppendix, Materials and Methods.

Heterologous Expression in S. cerevisiae. The N-terminally truncated casbenesynthase from R. communis (XP_002513340) was transformed together withthe full-length genes encoding CYP71D445, CYP726A27, and E. lathyrisADH1 into S. cerevisiae. All genes were synthetized as yeast codon opti-mized variants and expressed in yeast on plasmid construction by an in vivoDNA assembler technique. Details are provided in SI Appendix, SI Materialsand Methods.

ADH Enzyme Assays. For expression in E. coli, full-length cDNAs of E. lathyrisADH1 and E. peplus ADH1 were cloned into pET28b+ (Novagen) expressionvectors and sequence verified. Recombinant proteins were expressed inE. coli BL21DE3-C41 and Ni2+-affinity purified as described elsewhere (19).Coupled in vitro enzyme assays were conducted with 200 μg enzymes and100 μM of the substrate in a buffer containing 20 mM KH2PO4, 10 mM EDTA,and 1 mM nicotinamide adenine dinucleotide (NAD) in a total assay volumeof 150 μL. The reactions were incubated at 28 °C overnight and extractedwith 500 μL ethyl acetate. After removal of the solvent, the residues wereresuspended in 50 μL methanol and analyzed by LC-HRMS.

Microsomal Assays. Yeast cultures were grown and microsomes prepared asdescribed in previous reports (38). Microsomal assays were conducted in atotal volume of 200 μL of 50 mM potassium phosphate (pH 7.5), containing1 mM NADPH, 500 μg microsomal protein, and 100 μM substrates. The assayswere performed on a shaking incubator (Eppendorf Thermomixer C; EppendorfNordic A/S, Denmark) for 1 h at 30 °C, 300 rpm. For the combined assays, thereaction mixture was supplied with 200 μg ADH enzymes, 10 mM EDTA, and1 mM NAD and incubated at 28 °C overnight. The reactions were terminated byextraction with 500 μL ethyl acetate before LC-HRMS analysis.

Metabolite Analysis from in Vivo and in Vitro Assays. For information, pleasesee SI Appendix, Materials and Methods.

Isolation and NMR Experiments for Compounds 2–8. For information, pleasesee SI Appendix, Materials and Methods.

ACKNOWLEDGMENTS. We thank Drs. Irini Pateraki and Allison Maree Heskesfor critical reading of the manuscript and Dr. Irini Pateraki for providing out-standing mentorship and guidance for D.L.; Drs. Carl Erik Olsen, Mohammed

Fig. 5. Proposed pathway for the conversion of casbene (1) to jolkinol C (5)through enzymatic reactions catalyzed by CYP71D445, CYP726A27, andE. lathyris ADH1.

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Saddik Motawia (University of Copenhagen, Department of Plant and Environ-mental Sciences, Plant Biochemistry Laboratory), and Nils Nyberg (University ofCopenhagen, Department of Drug Design and Pharmacology) for excellenttechnical assistance; and the greenhouse service personnel for handling ourplants (University of Copenhagen, Department of Plant and EnvironmentalSciences, Plant Biochemistry Laboratory). We gratefully acknowledge Dr. DavidNelson (University of Tennessee) for P450 naming and helpful discussions, Dr.Aparajita Banerjee for fruitful discussions of the mechanism of the cyclization,and Dr. Gunnar Grue-Sørensen (LEO Pharma A/S) for providing seeds of Euphor-bia lathyris L. We acknowledge support from Science for Life Laboratory, theNational Genomics Infrastructure (NGI), and Uppmax for providing assistance inmassive parallel sequencing and computational infrastructure. This work was

supported by the Investment Capital for University Research Center for SyntheticBiology “bioSYNergy” at the University of Copenhagen (to B.L.M.), EuropeanResearch Council Advanced Grant ERC-2012-ADG_20120314 (to B.L.M.), the NovoNordisk Foundation (Björn Hamberger), and “Plant Power: Light-driven synthesisof complex terpenoids using cytochrome P450s” (12-131834) funded by the Dan-ish Innovation Foundation [project lead, Dr. Poul Erik Jensen (University ofCopenhagen, Department of Plant and Environmental Sciences, Plant Biochem-istry Laboratory); partners B.L.M. and Björn Hamberger). Björn Hamberger iscurrently in part funded by the Department of Energy (DOE) Great Lakes Bio-energy Research Center (DOE Office of Science BER DE-FC02-07ER64494) andgratefully acknowledges startup funding from the Department of MolecularBiology and Biochemistry, Michigan State University.

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