Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

Dan Luo, Roberta Callari, Britta Hamberger, Sileshi Gizachew Wubshet, Morten T. Nielsen, Johan Andersen-Ranberg, Björn M. Hallström, Federico Cozzi, Harald Heider, Birger Lindberg Møller, Dan Staerk, and Björn Hamberger
PNAS August 23, 2016 113 (34) E5082-E5089; first published August 9, 2016 https://doi.org/10.1073/pnas.1607504113

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

Significance

Ingenol mebutate is a diterpene ester with a highly complex macrocyclic structure that has been approved for the treatment of actinic keratosis, a precondition of skin cancer. The current production of ingenol mebutate through plant extraction or chemical synthesis is inefficient and costly. Here, we describe the discovery of a biosynthetic route in Euphorbia lathyris L. (caper spurge) in which regio-specific oxidation of casbene is followed by an unconventional cyclization to yield jolkinol C, a probable key intermediate in the biosynthesis of macrocyclic diterpenes, including ingenol mebutate. These results can facilitate the biotechnological production of this high-value pharmaceutical and discovery of new biosynthetic intermediates with important bioactivities.

Abstract

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon–carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

Species in the genus Euphorbia have a long history of use as medicinal plants for traditional treatments. Their bioactivity is largely due to the rich production of chemically diverse isoprenoids, the most characteristic of which are the macrocyclic diterpenoids. Some of these have shown considerable potential as lead compounds in drug discovery and have been the focus of many medicinal chemistry and therapeutic studies (1) (Fig. 1). Euphorbia factor L10 and euphodendroidin D show powerful inhibition of the transport activity of P-glycoprotein, a multidrug transporter overexpressed in cancer cell plasma membranes as an efflux pump conferring cellular resistance to anticancer chemotherapy (2, 3). Prostratin is in phase I human clinical trials for the treatment of HIV, as it was shown to activate viral reservoirs in latently infected T cells, as well as to inhibit viral replication (4). Ingenol mebutate, first isolated from Euphorbia peplus, is one of the most well-studied macrocyclic diterpenoids for its remarkable antitumor and antileukemic activity (5). In 2012, ingenol mebutate gel was approved for the treatment of actinic keratosis, a precondition of squamous cell carcinoma (6). The current supply of ingenol mebutate is limited to direct isolation from E. peplus, which yields only 1.1 mg/kg from the aerial tissue (7). Due to the increasing demand and low isolation yields of ingenol mebutate from the native plant, there is a strong need for the development of a more economical and efficient method for its production and for a robust supply chain. The latest 14-step chemical synthesis from the chiral monoterpene (+)-3-carene obtained an overall yield of around 1% and relied on expensive catalysts (8).

Fig. 1.
Fig. 1.

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

Ingenol was identified as a constituent in Euphorbia ingens in 1968 (9). Subsequently, a wide range of macrocyclic diterpenoids have been isolated from Euphorbia exhibiting a high diversity in backbone structures, oxygenation levels, stereoisomerism, and esterification patterns (1). According to different stages of cyclization of their C20 backbones, macrocyclic diterpenoids can be classified into two groups: simple bicyclic casbene type and further cyclized types. The latter includes jatrophanes, lathyranes, tiglianes, daphnanes, and ingenanes ranked according to their increased structural complexity (Fig. 1). Although jatrophanes are widespread in Euphorbia, only very few Euphorbia species have been reported to accumulate ingenanes (1). Currently the commercially most interesting species is Euphorbia lathyris 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). A semisynthesis approach to ingenol mebutate using hydrolysis products of ingenol-based Euphorbia factors as raw material has been developed (11). However, access to the starting material from E. lathyris L. seeds is limited due to natural fluctuations and a biennial life cycle.

Recent successes in metabolic engineering and synthetic biology approaches have received wide attention as economically competitive approaches for the production of structurally complex terpenoids (12, 13). However, as a prerequisite to reconstruct pathways of high-value diterpenoids in biotechnological production hosts, the steps involved in their biosynthesis must be elucidated. Despite numerous chemical and pharmacological reports, biosynthetic pathways of macrocyclic diterpenoids remain poorly understood. The simple bicyclic diterpene, casbene, has been suggested as the first committed intermediate toward the more complex multicyclic diterpenoids found in Euphorbia species (14). Casbene synthase (CBS) was first identified in Ricinus communis (15), and cyclization of the general C20 precursor, geranylgeranyl diphosphate (GGDP), to the bicyclic casbene has been well established. Although previously suggested to proceed via a route as outlined in Fig. 1, the intramolecular cyclization and rearrangements from the potential casbene precursor to multicyclic diterpenes remain hypothetical. An examination of the vast structural complexity of many types of macrocyclic diterpenoids found in nature indicates that multicyclic diterpenoids carry a higher degree of oxygenation compared with bicyclic casbenes. This relationship between cyclization and oxygenation stages may provide the key for formation of multicyclic diterpenoids in which intramolecular cyclizations are likely to occur in a combined pathway controlled by cytochrome P450 monooxygenases (P450s). Recently, a Euphorbiaceae-specific expansion of P450s in the CYP71 clan was reported (16). Although the founding members of the subfamily were shown to catalyze epoxidation of fatty acids in Euphorbia lagascae Spreng (17), several P450s of the CYP726 subfamily in castor bean had activity toward C5 of casbene, which is a characteristic of the casbene-type diterpenoids present in castor bean and related species (18). In addition to P450s, members of the short-chain dehydrogenase∕reductase superfamily are also involved in terpenoid 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 analyses targeted for diterpenoids with an interrogation of a deep transcriptome library generated from mature seeds of E. lathyris L., a seed development stage where we detected the presence of both casbene and ingenane diterpenoids. We focused our search on genes 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 casbene, and an alcohol dehydrogenase, ADH1, which catalyzes dehydrogenation of the hydroxyl groups of the resulting trioxidized products. Specifically, results from both in vivo and in vitro experiments show that ADH1 catalyzes a nonconventional cyclization of oxidized casbene to produce jolkinol C, a lathyrane diterpenoid, which is a potential key intermediate in ingenol biosynthesis. The observed patterns of regio-specific oxygenations and cyclization carried out by these enzymes reflect those found in the naturally occurring diterpenoids. These biosynthetic steps pave the way for biotechnological production of multicyclic intermediates of high value and structurally complex natural products.

Results

Accumulation of Casbene and Euphorbia Factors in Mature Seeds of E. lathyris L.

Although proposed to serve as a general precursor, the presence of casbene has not been described previously in Euphorbia plants accumulating multicyclic diterpenoids. To test for casbene in the plant species being the most important commercial source of ingenol, mature seeds of E. lathyris L. were extracted with n-hexane and analyzed by GC-MS. A trace amount of casbene matching retention time and mass spectra of the authentic standard was detected (SI Appendix, Fig. S1A). Accumulation of lathyrane and ingenane diterpenoids in the same tissue was confirmed by LC coupled with high-resolution MS (LC-HRMS). In the methanol extract, ingenane Euphorbia factors L4 and L5 were detected (SI Appendix, Fig. S2D) together with lathyrane Euphorbia factors L1–L3 and L7a (SI Appendix, Fig. S2 A–C). Co-occurrence of casbene with the more complex multicyclic diterpenoids in mature seeds may imply a tissue-specific and active biosynthetic pathway for the efficient conversion 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 enzymes potentially involved in the formation of multicyclic diterpenoids, a RNA-Seq transcriptome library was prepared from mature seeds. A CBS ortholog was identified in this library based on its high sequence identity to the previously identified gene from E. peplus (16). The E. lathyris CBS ortholog was highly expressed in mature seeds (SI Appendix, Table S1). Based on this result, combined with the concentrated accumulation of potentially casbene derived ingenane esters, we hypothesized that mature seeds of E. lathyris represent the most specialized tissue for their formation and prioritized the search for candidate genes by their expression level in this library. Based on transcriptomic data from leaf tissue, expansions of CYP71D and CYP726 subfamilies were previously reported in E. peplus (16). To identify E. lathyris homologs, we selected these subfamilies as starting points to interrogate the mature seed transcriptome. A comprehensive list of P450 enzymes from subfamilies of CYP71D and CYP726 was generated and candidates were prioritized based on their expression level in the E. lathyris seed library (SI Appendix, Table S1). According to gene abundance data, E. lathyris CYP71D445 and CYP726A27 were found to be the highest expressed P450s in the CYP71D and CYP726 subfamilies. In addition, two highly expressed ADH candidates, E. lathyris ADH1 and ADH2, were identified in the transcriptome library using known ADHs involved in terpene metabolism as queries (19). Sequence comparison and phylogenetic analysis implied ADH1 and ADH2 as members of the short chain dehydrogenase/reductase family SDR110C (ABA2 xanthoxin dehydrogenase family; SI Appendix, Fig. S3). SDR110C is expanded in vascular plants and catalytic activities were reported in oxidiation of various phenolics or terpenoids, including monoterpenes isopiperitenol and borneol, the macrocyclic sesquiterpene zerumbone and the diterpene momilactone (19⇓⇓–22).

Two P450s Expressed in Nicotiana benthamiana Function as Regio-Specific Casbene Monooxygenases.

To test the capacity of the identified P450s to oxygenate casbene, E. lathyris CYP71D445 and CYP726A27 were coexpressed with CBS in N. benthamiana using the Agrobacterium-mediated transient expression system, engineered for optimized production of diterpenoids by the strategy of coexpression with Coleus forskohlii 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) as described earlier (23). The production of casbene (1) in the infiltrated N. benthamiana leaves was confirmed by GC-MS analysis (Fig. 2 A, b and SI Appendix, Fig. S1). The LC-MS total ion chromatograms of the N. benthamiana coexpressing CBS and CYP71D445 showed the appearance of a new constituent 2, which, based on the parental ion (m/z 287.2376, [M+H]+), could represent an oxygenated casbene derivative with an additional degree of unsaturation (Fig. 2 B, b). Coexpression of CBS and CYP726A27 resulted in formation of compound 3 with a parental ion (m/z 289.2513, [M+H]+) corresponding 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 derivative containing two oxygen atoms, accumulated as the major chromatographically detectable product together with trace amounts of compounds with molecular masses indicating multioxygenated derivatives of casbene (Fig. 2 B, d). Compounds 2, 3, and 4 were isolated from infiltrated N. benthamiana and purified by combined chromatography. The structures were elucidated by NMR analyses, establishing 2, 3, and 4 as 9-ketocasbene, 5-hydroxycasbene, and 5-hydroxy-9-ketocasbene, respectively (Fig. 2C and SI Appendix, Fig. S5 and Table S2).

Fig. 2.
Fig. 2.

Functional characterization of E. lathyris CBS, P450s, and ADH1 via in vivo expression. (A) GC-MS profiles of N. benthamiana transiently expressing E. 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 expressing CBS, CYP71D445, CYP726A27, and ADH1.

Transcript Profiles of E. lathyris CBS, CYP71D445, CYP726A27, and ADH1 Indicate Specific Expression in Mature Seeds.

To discover downstream enzymes catalyzing sequential steps in the pathways of Euphorbia factors, transcript levels of CBS, CYP71D445, and CYP726A27 were investigated by quantitative RT-PCR (qPCR) and compared with the two putative ADH genes. qPCR analysis was performed using cDNA templates derived from total RNA extracted from young and mature leaves, stems, fruits, developing seeds, mature seeds, and roots of E. lathyris L. In the transcript profiles obtained (SI Appendix, Fig. S4A), CBS, CYP71D445, CYP726A27, and ADH1 shared similar expression patterns across all tested tissues, with high transcript accumulation in mature seeds. In contrast, the transcript level of ADH2 in mature seeds was relatively low compared with the other tissues. The transcript profiles of E. lathyris CBS, CYP71D445, and CYP726A27 supported their involvement in the formation of Euphorbia factors, which were only detected in mature seeds (SI Appendix, Fig. S4 C and D). The specific expression of ADH1 in mature seeds indicated a potential function downstream of the P450-based oxygenations of casbene in the biosynthesis of Euphorbia factors, whereas the expression pattern of ADH2 implied limited relevance for the pathway.

Coexpression of E. lathyris ADH1 with CBS, CYP71D445, and CYP726A27 Resulted in Cyclization Affording a Lathyrane-Type Diterpene.

To investigate the function of E. lathyris ADH1 and ADH2, the enzymes were individually coexpressed with CBS, CYP71D445, and CYP726A27 in N. benthamiana. Compared with the combination producing 4 (Fig. 2 B, d), LC-MS total ion chromatograms of N. benthamiana leaves that in addition expressed ADH1 showed an obvious difference in product formation (Fig. 2 B, e), whereas coexpression of ADH2 did not result in any detectable activity (SI Appendix, Fig. S4B). The combined expression of CBS, CYP71D445, CYP726A27, and ADH1 (Fig. 2 B, e) resulted in formation of a new constituent 5 with m/z of 317.2104, possibly corresponding to a cyclized product containing an additional oxygen atom. Compound 5 was isolated from N. benthamiana transiently expressing the specific enzymes and identified using HPLC-HRMS with solid-phase extraction (SPE) and NMR spectroscopy, i.e., HPLC-HRMS-SPE-NMR (24). Structure elucidation by NMR analysis established 5 as jolkinol C (SI Appendix, Fig. S5 and Table S2B), a lathyrane type diterpene naturally found in Euphorbia jolkini Boiss (25).

Production of Oxidized Derivatives of Casbene in Saccharomyces cerevisiae.

To confirm the catalytic activities observed in the N. benthamiana transient expression system, CYP71D445, CYP726A27, and ADH1, combined with the NADPH-dependent cytochrome P450 oxidoreductase (POR) from Ricinus communis, were cloned and expressed in a S. cerevisiae strain engineered for casbene production. Consistent with the results in planta, compounds 2 and 3 were found when CYP71D445 and CYP726A27 were independently expressed with CBS (Fig. 2 D, b and c). In addition, compound 6 (m/z 289.2522, [M+H]+), the parental ion of which matches the hydroxylated casbene, was obtained as an 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. 2 D, d). However, when ADH1 was coexpressed with CBS and the two P450s, no accumulation of compound 5 was detected (Fig. 2 D, e). Instead, the detectable diterpenoid products in this strain were compounds 7 (m/z 319.2261, [M+H]+) and 8 (m/z 319.2263, [M+H]+), with the masses suggesting that these were casbene derivatives with trioxygenation. Compounds 6, 7, and 8 were purified from the corresponding S. cerevisiae strains by combined chromatography. The structures of 6, 7, and 8 were determined as 9-hydroxycasbene, 5-keto-6,9-casbene diol, and 6-keto-5,9-casbene diol, respectively, based on NMR analysis (Fig. 2C and SI Appendix, Fig. S5 and Table S2 B and C).

Characterization of Catalytic Features of ADH1 via in Vitro Enzyme Assays.

To assign the role of ADH1 in the cyclization process yielding compound 5, a series of in vitro assays was carried out using various isolated casbene derivatives as substrates. The purified His6-tagged recombinant enzyme of ADH1 was incubated with compounds 3, 4, 6, 7, and 8 individually in the presence of NAD+, and the formation of products was analyzed by LC-HRMS (Fig. 3A). In the resulting profiles, efficient conversion of 6 to 2 was achieved by ADH1 (Fig. 3 A, a and b), indicating its capability of catalyzing dehydrogenation of the 9-hydroxyl group. When ADH1 was incubated with 3, compound 9 (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). Corroborating its ability to catalyze dehydrogenation of the 5-hydroxyl group, ADH1 also dehydrogenated 4 yielding the casbene dione derivative 10 (m/z 301.2157, [M+H]+) (Fig. 3 A, e and f). Remarkably, the trioxygenated casbene derivatives, 7 and 8, were found to be converted to the cyclized product 5 by ADH1 (Fig. 3 A, g–j).

Fig. 3.
Fig. 3.

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

Reproduction of the Lathyrane-Type Diterpene by in Vitro Assayed CYP71D445, CYP726A27, and ADH1.

Although the trioxygenated derivatives 7 and 8 were obtained, the cyclized diterpene 5 was not detected in the recombinant S. cerevisiae strains. A previous report has shown that an acidified environment, such as in cultured yeast, can affect terpene cyclization (27). Therefore, we speculate that the cyclization reaction affording 5 might be inhibited by the acidity of yeast culture. To reconstruct the production of 5, we performed the combined in vitro assays with microsomal fractions from yeast strains expressing the P450s and the purified ADH1, expressed as His6-tagged recombinant enzyme in Escherichia coli. First of all, the catalytic activities of microsomes were confirmed by in vitro assays supplied with 1 as the substrate. LC-MS total ion chromatograms of the in vitro microsomal assays were highly consistent with the results obtained in vivo (Fig. 3 B, a–d). Specifically, when the microsomal assays expressing both CYP71D445 and CYP726A27 were coupled with ADH1, reproduction of the lathyrane-type diterpene 5 was achieved in vitro (Fig. 3 B, e), demonstrating that the characterized enzymes are truly responsible for the cyclization. In these combined assays, the casbene dione 10, which is the direct dehydrogenation product of 4, was obtained as well. Because accumulation of 10 was not detected in vivo, this result indicated that the catalytic abilities of the two CYPs and ADH1 were not perfectly balanced at the tested in vitro conditions.

Compared with the structural features of 4, an additional oxygenation on C-6 was found in 5, 7, and 8, representing either 6-hydroxylation or 6-ketonization. The results from in vitro enzyme assays showed that the oxygenation of C-6 was not directly catalyzed by ADH1 (Fig. 3). Therefore, we hypothesized that it was catalyzed by a multifunctional P450. To both elucidate the origin of C-6 oxygenation and establish the order of multiple oxygenations during the ring closure process, we investigated conversion of isolated casbene derivatives with regio-specific oxygenations (2, 3, 4, and 6) in the microsomal assays. No conversion was found when the ketone derivatives 2 and 4 were supplied as the substrate (SI Appendix, Fig. S6). On the other hand, the monohydroxyl derivatives 3 and 6 were converted to 5 in the combined assays of CYP71D445, CYP726A27, and ADH1 (Fig. 4 B, e and 4 A, e), demonstrating that both 3 and 6 can act as 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 casbene diol was detected by LC-HRMS (Fig. 4 A, b). Despite the unavailability of an authentic standard, 11 most likely represents the 5,9-casbene diol based on the catalytic features of CYP726A27. Although limited stability precluded isolation and structural identification of 11, the hypothetical structure was exemplified by feeding compound 6 with the microsomes expressing CYP71D445 and CYP726A27. Compound 4 was identified instead of 11 in these assays (Fig. 4 A, c), suggesting that CYP71D445 further oxidized the 9-hydroxyl group of 11 into the 9-ketone group. To be noticed, trace amounts of 5 were detected when compound 6 was incubated with the microsomes expressing CYP726A27 and the recombinant enzymes of ADH1 (Fig. 4 A, d). The generation of 6-oxygenation of 5 in the absence of CYP71D445 demonstrates that CYP726A27 is a bifunctional enzyme catalyzing hydroxylation on the adjacent positions C-5 and C-6. Additionally, when tested with the 5-hydroxyl preinserted substrate 3, the formation of 5 was recognized in the assays coupling CYP71D445 with ADH1 (Fig. 4 B, d). This finding indicates that CYP71D445 shares the role of inserting oxygen at C-6.

Fig. 4.
Fig. 4.

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

Activity of the Functional Orthologs from E. peplus.

Comparative transcriptomics of E. lathyris L. with E. peplus, a related Euphorbia species accumulating the most complex multicyclic ingenol diterpenoids, revealed homologs of the four enzymes described in this study in the stem derived cDNA library of E. peplus. E. peplus CBS (KC702397.1) has been reported together with CYP726A4 (KF986823.1), found in the largely expanded tribe CYP71D, carrying 23 members of E. peplus (16). The corresponding full-length cDNA sequences were cloned and tested functionally in the N. benthamiana system. Direct comparison indicated that E. peplus CBS, CYP71D365, CYP726A4, and ADH1 shared the activity with E. lathyris CBS, CYP71D445, CYP726A27, and ADH1, respectively (SI Appendix, Fig. S7 A and C), and hence are functional orthologs with the same catalytic features. Jolkinol C (5) was obtained from the combination of E. peplus CBS, CYP71D365, CYP726A4, and ADH1, despite the lack of reported lathyrane-type diterpenoids in this plant.

Discussion

Metabolite profiling of mature seeds of E. lathyris revealed the co-occurrence of casbene together with Euphorbia factors (lathyrane and ingenane diterpenoids). Extended metabolite profiling including several other plant tissues confirmed the specific accumulation of casbene and the Euphorbia factors in mature seeds (SI Appendix, Figs. S1, S2, and S4 C and D). This finding suggested that the mature seed of E. lathyris constituted a highly specialized tissue for the biosynthesis of lathyrane and ingenane diterpenoids, uniquely suited for pathway discovery. It may follow an emerging theme, where colocalization of the scaffold and final functionalized diterpene in a specific cell type enabled discovery of the biosynthetic route (28).

The observed polyoxygenation of the multicyclic Euphorbia factors suggested that P450 catalyzed oxygenations might serve to activate casbene, a relatively inert hydrocarbon precursor, and thereby enable a subsequent cyclization reaction to proceed. Casbene 5-oxidation has previously been described in R. communis (18), followed by 6-hydroxylation or 7,8-epoxidation, matching natural casbene type diterpenoids present in this species (29). However, none of these oxidations led to a ring closure generating lathyrane or multicyclic backbones. This lack of activity indicated that a specific oxygenation of casbene at a particular carbon position was crucial for the formation of the intramolecular C-C bonds present in lathyrane and ingenane diterpenoids.

Transcriptome analysis of E. lathyris L. mature seeds and functional characterization of candidate genes identified two cytochromes P450, CYP71D445, and CYP726A27, as casbene monooxygenases. Among these, CYP71D445 catalyzed oxidation at the C-9 position of casbene. Surveying reported macrocyclic diterpenoids with different carbon backbones (1), we found that casbene 9-oxidation is characteristic of multicyclic diterpene types shared across all lathyrane, jatrophane, tigliane, daphnane, and ingenanes (Fig. 1). Based on these observations, we suggest that C-9 oxidation defines early bifurcation steps in the biosynthesis of macrocyclic diterpenoids. A lack of the essential oxygenation at C-9 may, as a consequence, lead to the formation of casbene-type diterpenoids, whereas di-oxygenation at both C-9 and C-5 constitutes the subsequent step en route to multicyclic diterpenes derived from casbene.

Cloning and coexpression of the set of orthologous genes from the relative E. peplus in the transient N. benthamiana system resulted also in formation of jolkinol C (5). In the absence of reported lathyrane-type diterpenes in E. peplus, these results imply that either jatrophane or ingenane diterpenoids, or possibly both types accumulating in this species, are biosynthesized via lathyrane intermediates. Thus, it can be assumed that evolution of the pathway to jolkinol C (5) and presumably the most complex ingenanes predates speciation of E. lathyris and E. peplus.

The results from ADH1 expression in N. benthamiana revealed an unconventional pathway from casbene to the lathyrane diterpenoid jolkinol C (5) proceeding via an ADH catalyzed cyclization reaction (Fig. 5). By testing the recombinant enzymes in vitro, two important features of this pathway were demonstrated: the regio-specific oxidations of casbene by the P450s initiate and precede the intramolecular cyclization and this cyclization reaction is dependent on the catalytic activity of the E. lathyris ADH1. The combined in vitro assays of CYP71D445 and CYP726A27 demonstrated that the two P450s act as bifunctional enzymes, thereby clarifying their shared responsibility with a partial functional redundancy for catalyzing oxidation at C-6. Similar bifunctional features have previously been found in CYP726A14, a casbene monooxygenase from R. communis (26). The in vitro assayed ADH1 displayed the activity of catalyzing dehydrogenation of various substrates with a casbene backbone carrying hydroxyl groups at the C-5 and C-9 positions. In particular, the presented conversion of the trioxygenated intermediate 7 to 5 fully supported the hypothesis that ADH1 is directly catalyzing the cyclization reaction.

Fig. 5.
Fig. 5.

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

The above results enabled us to identify individual steps and intermediates in the conversion of casbene to the lathyrane diterpenoid 5 (Fig. 5): CYP71D445 and CYP726A27 initially catalyze the C-9 and C-5 hydroxylation of casbene, respectively. This conversion is then followed by positional crossover hydroxylations mediated by the two P450s resulting in the formation of the 5,9-casbene diol 11. The resulting diol 11 is further oxidized by one of the bifunctional monooxygenases, either CYP726A27 or CYP71D445, resulting in the formation of a proposed triol intermediate. The existence of the triol intermediate was implied by the observed formation of 7 and 8. Subsequently, ADH1 activates this intermediate or the interconvertible pair of 7 and 8 through dehydrogenation leading, plausibly, via a series of keto-enol tautomerisms to the formation of a new C-C bond between C-6 and C-10 (SI Appendix, Fig. S8). Instability and rapid further conversion may explain why the triol intermediate did not accumulate. The ketocasbene derivatives 2 and 4 are considered side products further oxidized by CYP71D445. Their high accumulation in the in vivo assays may be favored by structural stabilization via the conjugated double bonds.

The results obtained document the origin of the intramolecular carbon-carbon bonds present in lathyrane, jatrophane, tigliane and ingenane diterpenoids. Knowledge on how to establish these carbon backbones offers the possibility to identify the subsequent enzymatic steps and to ultimately reconstruct the biosynthetic pathways of pharmaceutically active diterpenoids and new bioactive intermediates for biotechnological production. The recently reported option to redirect P450-dependent biosynthetic pathways into tobacco chloroplasts and drive the P450s directly based on light-driven photosynthetic electron transport using reduced ferredoxin as the direct electron donor to the P450s offers opportunities to redirect the production capacity of photosynthetic cells toward production of high levels of structurally complex diterpenoids (30, 31).

Materials and Methods

Plant Material, RNA Extraction, and Quantitative Real-Time PCR.

Plants were grown in a greenhouse at the University of Copenhagen under ambient photoperiod and 24 °C day/17 °C night temperatures. Total RNA from E. lathyris L. young and mature leaves, fruits, stems, developing seeds (white and soft), mature seeds (black and hard), and roots was extracted according to a previously described method (32), followed by DNase I digestion. The same 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 SI Appendix, SI Materials and Methods.

Transcriptome Sequencing and de Novo Assembly.

RNA was prepared for sequencing using Illumina TruSeq preparation kit v2 using poly-A selection. The fragments were clustered on cBot and sequenced with paired ends (2 × 100 bp) on an Illumina HiSeq 2500, according to the manufacturer's instructions. In total, 166.6 million read-pairs were generated from E. lathyris L. mature seeds RNA and 55.7 million read-pairs from E. peplus stem RNA, respectively. Adaptor sequences were removed from raw reads, and reads were trimmed at the ends to a minimum phred score of 20, using the fastq-mcf tool from ea-utils (https://github.com/ExpressionAnalysis/ea-utils). Processed reads were assembled using Trinity (33) resulting in a total of 145,990 and 89,163 assembled putative transcripts from E. lathyris L. mature seeds and E. peplus stem RNA-seq, respectively. Transcript abundance estimation was performed using RSEM and the scripts provided with Trinity. Likewise, the putative coding sequences were predicted using TransDecoder scripts from Trinity.

Diterpenoid Profiling of E. lathyris Mature Seeds.

For information, please see SI Appendix, Materials and Methods.

Construction of Uracil-Specific Excision Reagent-Compatible Expression Vector.

The GATEWAY-compatible pEAQ-DEST1 vector (34), for high level transient expression 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 the introduction of a uracil-specific excision reagent (USER)-PacI site in the GATEWAY site of pEAQ-DEST, the USER compatible (35) vector pEAQ-USER was constructed. Details of vector construction are provided in SI Appendix, SI Materials and Methods.

Generation of Expression Constructs.

Total RNA from E. lathyris L. seeds extracted as described above was used for cDNA synthesis. First-strand cDNAs were synthesized using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit and oligo(dT) primer. Full-length cDNAs of identified candidate genes were obtained by PCR amplification using gene-specific primers with desired USER cassettes (SI Appendix, Table S3). PCR products were cloned into pEAQ-USER vectors for plant transformation as described previously (35, 36) and verified by sequencing.

Transient Expression in N. benthamiana.

Transient expression of full-length genes in N. benthamiana leaves and extraction of diterpenes were performed as recently described (37). For details, see SI Appendix, Materials and Methods.

Classification of E. lathyris ADH Sequences.

For information, please see SI Appendix, Materials and Methods.

Heterologous Expression in S. cerevisiae.

The N-terminally truncated casbene synthase from R. communis (XP_002513340) was transformed together with the full-length genes encoding CYP71D445, CYP726A27, and E. lathyris ADH1 into S. cerevisiae. All genes were synthetized as yeast codon optimized variants and expressed in yeast on plasmid construction by an in vivo DNA assembler technique. Details are provided in SI Appendix, SI Materials and Methods.

ADH Enzyme Assays.

For expression in E. coli, full-length cDNAs of E. lathyris ADH1 and E. peplus ADH1 were cloned into pET28b+ (Novagen) expression vectors and sequence verified. Recombinant proteins were expressed in E. coli BL21DE3-C41 and Ni2+-affinity purified as described elsewhere (19). Coupled in vitro enzyme assays were conducted with 200 µg enzymes and 100 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 volume of 150 µL. The reactions were incubated at 28 °C overnight and extracted with 500 µL ethyl acetate. After removal of the solvent, the residues were resuspended in 50 µL methanol and analyzed by LC-HRMS.

Microsomal Assays.

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

Metabolite Analysis from in Vivo and in Vitro Assays.

For information, please see SI Appendix, Materials and Methods.

Isolation and NMR Experiments for Compounds 2–8.

For information, please see SI Appendix, Materials and Methods.

Acknowledgments

We thank Drs. Irini Pateraki and Allison Maree Heskes for critical reading of the manuscript and Dr. Irini Pateraki for providing outstanding mentorship and guidance for D.L.; Drs. Carl Erik Olsen, Mohammed Saddik Motawia (University of Copenhagen, Department of Plant and Environmental Sciences, Plant Biochemistry Laboratory), and Nils Nyberg (University of Copenhagen, Department of Drug Design and Pharmacology) for excellent technical assistance; and the greenhouse service personnel for handling our plants (University of Copenhagen, Department of Plant and Environmental Sciences, Plant Biochemistry Laboratory). We gratefully acknowledge Dr. David Nelson (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 Euphorbia lathyris L. We acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure (NGI), and Uppmax for providing assistance in massive parallel sequencing and computational infrastructure. This work was supported by the Investment Capital for University Research Center for Synthetic Biology “bioSYNergy” at the University of Copenhagen (to B.L.M.), European Research Council Advanced Grant ERC-2012-ADG_20120314 (to B.L.M.), the Novo Nordisk Foundation (Björn Hamberger), and “Plant Power: Light-driven synthesis of complex terpenoids using cytochrome P450s” (12-131834) funded by the Danish Innovation Foundation [project lead, Dr. Poul Erik Jensen (University of Copenhagen, Department of Plant and Environmental Sciences, Plant Biochemistry Laboratory); partners B.L.M. and Björn Hamberger). Björn Hamberger is currently in part funded by the Department of Energy (DOE) Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) and gratefully acknowledges startup funding from the Department of Molecular Biology and Biochemistry, Michigan State University.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: hamberge{at}msu.edu.

  • Author contributions: Björn Hamberger designed research; D.L., R.C., M.T.N., J.A.-R., and Björn 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., and Björn Hamberger analyzed data; D.L. and Björn Hamberger wrote the paper; and B.L.M. is responsible for distributing materials and clones described in this work (blm{at}plen.ku.dk).

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

  • This article is a PNAS Direct Submission.

  • Data deposition: The nucleotide sequences reported in this paper have been submitted to the short read archive (SRA) at the NCBI [accession nos. SRP057971 (E. lathyris) and SRP058119 (E. peplus)]. Novel nucleotide sequences encoding enzymes functionally characterized 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).

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

Freely available online through the PNAS open access option.

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Unconventional cyclization of Euphorbia diterpenes
Dan Luo, Roberta Callari, Britta Hamberger, Sileshi Gizachew Wubshet, Morten T. Nielsen, Johan Andersen-Ranberg, Björn M. Hallström, Federico Cozzi, Harald Heider, Birger Lindberg Møller, Dan Staerk, Björn Hamberger
Proceedings of the National Academy of Sciences Aug 2016, 113 (34) E5082-E5089; DOI: 10.1073/pnas.1607504113
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