Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures Fainmarinat S

Biochemistry, Biophysics and Molecular Biology Biochemistry, Biophysics and Molecular Biology Publications

9-2017 Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures Fainmarinat S. Inabuy Washington State University

Justin T. Fischedick Washington State University

Iris Lange Washington State University

Michael Hartmann Washington State University

Narayanan Srividya Washington State University

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Abstract Adventitious root cultures were developed from Tripterygium regelii Sprague & Takeda and growth conditions optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f and four to the TPS-b subfamily. These genes were characterized by heterologous expression in a modular metabolic engineering system in E. coli. Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases and those belonging to the TPS-e/ f family catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The trs uctural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the significant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.

Disciplines Biochemistry, Biophysics, and Structural Biology | Genetics and Genomics | Molecular Biology | Molecular Genetics

Comments This article is published as Inabuy, F.X., Fischedick, J.T., Lange, I., Hartmann, M., Srividya, N., Parrish, A.N., Xu, M., Peters, R.J., Lange, B.M. (2017) “Biosynthesis of diterpenoids in Tripterygium adventitious root cultures”, Plant Physiol., 175(1):92-103. doi: 10.1104/pp.17.00659. Posted with permission.

Authors Fainmarinat S. Inabuy, Justin T. Fischedick, Iris Lange, Michael Hartmann, Narayanan Srividya, Amber N. Parrish, Meimei Xu, Reuben J. Peters, and B. Markus Lange

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/bbmb_ag_pubs/184 Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures1[OPEN]

Fainmarinat S. Inabuy,a Justin T. Fischedick,a,2 Iris Lange,a Michael Hartmann,a,3 Narayanan Srividya,a Amber N. Parrish,a Meimei Xu,b Reuben J. Peters,b and B. Markus Langea,4 aInstitute of Biological Chemistry and M.J. Murdock Metabolomics Laboratory, Washington State University, Pullman, Washington 99164-6340 bRoy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011-1079 ORCID IDs: 0000-0003-4828-958X (M.H.); 0000-0001-7934-7987 (N.S.); 0000-0002-4080-8608 (A.N.P.); 0000-0003-4691-8477 (R.J.P.); 0000-0001-6565-9584 (B.M.L.).

Adventitious root cultures were developed from Tripterygium regelii, and growth conditions were optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f, and four to the TPS-b subfamilies. These genes were characterized by heterologous expression in a modular metabolic engineering system in Escherichia coli. Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases, and those belonging to the TPS-e/f subfamily catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The structural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the signiﬁcant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.

Tripterygium wilfordii, also known as léi gōng téng in Chinese medicine for the treatment of fever, chills, Mandarin Chinese (generally translated as “thunder edema, and carbuncles (Helmstädter, 2013). The genus god vine”), has a long history of use in traditional Tripterygium (Celastraceae) is known to be a rich source of specialized metabolites, of which more than 400 have 1 This work was supported by the National Institutes of Health been isolated, structurally characterized, and assessed (award nos. RC2GM092561 to B.M.L. and GM076324 to R.J.P.) and in cell-based assays (Brinker et al., 2007). Root extracts McIntire-Stennis formula funds from the Agricultural Research Cen- have been evaluated as a medication for rheumatoid ter at Washington State University (to B.M.L.). arthritis, cancer, hepatitis, nephritis, ankylosing spon- 2 Current address: Excelsior Analytical, Union City, CA 94587. dylitis, polycystic kidney disease, and obesity; more 3 Current address: Institute of Molecular Ecophysiology of Plants, than a dozen clinical trials with such extracts (often Heinrich Heine Universität Düsseldorf, 40225 Duesseldorf, Germany. 4 referred to as Tripterygium glycoside) have been com- Address correspondence to [email protected]. pleted, but, in part due to shortcomings in study de- The author responsible for distribution of materials integral to the signs, the efficacy has remained a matter of debate findings presented in this article in accordance with the policy de- (Chen et al., 2010; Liu et al., 2013; Zhu et al., 2013). More scribed in the Instructions for Authors (www.plantphysiol.org) is: fi B. Markus Lange ([email protected]). promising results have been obtained with puri ed B.M.L. conceived of the project; F.S.I. cloned candidate genes, constituents, which are usually extracted from Trip- subcloned them into appropriate modular expression vectors, and terygium roots. Semisynthetic chemical derivatives of analyzed diterpenoids produced in E. coli cells harboring these con- triptolide, a diterpenoid epoxide, have been evaluated structs; J.T.F. established the methods for the isolation, purification, in phase I clinical trials (Zhou et al., 2012; Meng et al., identification, and quantitation of diterpenoids; I.L. isolated RNA from 2014). Minnelide, a water-soluble prodrug analog of adventitious root cultures, cloned candidate genes, and performed the triptolide, has shown very promising activity in mul- scale-up and selection of adventitious root cultures for high diterpenoid tiple animal models of pancreatic cancer (Chugh et al., production; M.H. established and A.N.P. maintained the adventitious 2012) and, in 2013, was advanced to phase I clinical root cultures; N.S. performed the functional characterization of mono- fi terpene synthase genes; M.X. and R.J.P. supplied modular vectors and trials in the United States (clinicaltrials.gov identi er carried out diterpenoid characterizations; B.M.L. and F.S.I. wrote the NCT01927965). article, with contributions from all authors. One of the critical challenges for clinical evaluations [OPEN] Articles can be viewed without a subscription. is a supply shortage for triptolide and other diterpe- www.plantphysiol.org/cgi/doi/10.1104/pp.17.00659 noids. The most abundant specialized metabolites in

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Tripterygium roots are quinone methide triterpenoids adventitious root cultures accumulated diterpenoids and sesquiterpene pyridine alkaloid macrolides, abundantly in the medium and triterpenoids (in par- whereas triptolide and other diterpenoids occur only at ticular celastrol) in roots (Fig. 1, A and B). By comparing very low concentrations ranging from 0.0001% to adjusted peak areas from HPLC-QTOF-MS runs, we 0.002% of dry weight biomass (Zhou et al., 2012; Zeng estimated that diterpenoids constitute approximately et al., 2013; Guo et al., 2014). Accordingly, the extraction 77% of all detected metabolites in an organic extract of yields of diterpenoids from Tripterygium roots are poor the culture medium (Supplemental Fig. S1). and alternative, sustainable production methods need An accurate quantitation of all diterpenoids was not to be developed. Tissue cultures represent a promising possiblebecausewedidnothaveacompletesetof alternative for the production of high-value plant me- authentic standards, but we performed absolute tabolites. Early studies with T. wilfordii suspension quantitations for signature metabolites (triptolide and cultures were designed to unravel the structures of celastrol). Various common reagents and elicitors were small-molecule constituents (Kutney et al., 1981, 1992, tested (methyl jasmonate, methyl salicylate, chitosan, 1993; Kutney and Han, 1996; Nakano et al., 1997, 1998). yeast extract, and cold exposure), but only the methyl Similarly, hairy root cultures were initially employed salicylate treatment resulted in a significant increase in for phytochemical investigations (Nakano et al., 1998). triptolide accumulation (2.2-fold; P value of 0.01) and a It was recognized only recently that triptolide concen- concomitant decrease in celastrol concentration trations produced by tissue cultures (up to 0.15% of dry (Supplemental Table S1). The disadvantage of methyl weight; Miao et al., 2013, 2014; Zhu et al., 2014) far ex- salicylate treatments was that root color darkened and ceed those reported for roots. growth ceased, often for months. Therefore, we focused Surprisingly, despite considerable pharmaceutical our efforts on further selecting cultures that were high interest, the biosynthesis of triptolide has only recently diterpenoid producers under noninducing conditions attracted the deserved attention. Several diterpene and could be maintained for extended periods of time. synthases, some of which are relevant to triptolide These cultures were gradually scaled up to 2.8-L flasks formation, have now been characterized in T. wilfordii (500 mL of medium; Fig. 1C). Triptolide accumulated at 2 (Zerbe et al., 2013; Andersen-Ranberg et al., 2016; 4.7 mg L 1 (Fig. 1D) and was readily extractable from Hansen et al., 2017). However, the remaining genes the culture medium every 2 weeks (when adventitious involved in the biosynthesis of the highly functional- roots were transferred to fresh medium). ized triptolide structure have remained enigmatic. In Hundreds of structurally diverse metabolites have this article, we describe the development of Triptery- been isolated and characterized from various Triptery- gium adventitious root cultures in which diterpenoids gium organs and tissues (Lange et al., 2017). Our ad- are produced as principal metabolites that can be har- ventitious root cultures, in contrast, secreted primarily vested sustainably from the medium. Based on the diterpenoids into the culture medium and, therefore, structures of these metabolites, we can now postulate are an excellent experimental model system in which to the biochemical steps leading to the main diterpenoid study the biosynthesis of these clinically relevant me- metabolites. In addition, analysis of transcriptome data tabolites. As a first step to further assess the potential of sets, acquired with diterpenoid-producing adventitious these tissue cultures, it was important to identify the root cultures, enabled the selection and subsequent major constituents of medium extracts. Following functional characterization of genes involved in HPLC fractionation of medium extracts of T. regelii the early steps of diterpenoid biosynthesis. Our results adventitious root cultures, fractions containing metab- indicate that Tripterygium tissue cultures offer oppor- olites that corresponded to prominent peaks in HPLC- tunities to both unravel diterpenoid biosynthetic QTOF-MS chromatograms were characterized by mass pathways and produce target diterpenoids, such as spectrometry (MS), tandem mass spectrometry (MS/ triptolide, at larger scale. MS), and 1H-NMR spectroscopy (Supplemental Fig. S2; Supplemental Methods and Data File S1), and metabolites were identified by searches against the Spektraris RESULTS database (Fischedick et al., 2015). We ascertained the Development of Diterpenoid-Secreting Adventitious identities of five diterpenoids, were able to annotate Root Cultures three additional metabolites with high confidence, and acquired tentative identifications for an additional two Tripterygium regelii adventitious root cultures were metabolites (Table I and Fig. 2). Three metabolites showed initiated based on standard protocols. Over a 1-year the mass fragmentation patterns typical of diterpenoids, period, during which medium was extracted with ethyl but their identities remained unknown (Table I). acetate and hydrophobic metabolites were analyzed by HPLC-quadrupole time of flight-mass spectrometry (QTOF-MS), the highest triptolide producers were se- Functional Characterization of Candidate lected for further propagation. In contrast to roots of Diterpene Synthases mature Tripterygium plants, where triterpenoids occur at low concentrations (less than 1%) and diterpenoids To obtain full-length candidate genes, we deemed it are trace constituents (less than 0.01%), our advantageous to employ all available T. regelii sequences.

Figure 1. Accumulation of abietane diterpenoids in T. regelii adventitious root cultures. A and B, HPLC-QTOF-MS traces (total ion current) obtained with ethanolic extracts of cultured roots (A) and culture medium (B). The identities of the metabolite peaks 1 to 13 are given in Table I (peak 13 [celastrol] is highlighted in blue because it is the only metabolite that does not belong to the abietane diterpenoids). C, Fernbach ﬂask with root culture photographed from below. The arm of the person holding the 4-L ﬂask is visible at the bottom right and serves as a size comparison. D, Concentrations of triptolide and celastrol in cultured roots and culture medium.

Therefore, we downloaded several publicly available characterization. Finally, nine members of the TPS-a transcriptome data sets obtained with roots and root family (TrTPS9, TrTPS10, TrTPS11, TrTPS12, TrTPS16, cultures and then generated a consensus assembly. TrTPS17, TrTPS18, TrTPS19, and TrTPS20) clustered with tBLASTn searches with peptide sequences of diterpene known genes that encode linear diterpene cyclases and synthases of dicotyledons were performed against our macrocyclases, which were not of direct interest to this assembly data. A phylogenetic analysis indicated that study and, therefore, were not further characterized. translated peptide sequences of two T. regelii genes Diterpene synthase candidate genes were character- (TrTPS2 and TrTPS1) clustered with functionally charac- ized by functional expression in engineered Escherichia terized class II diterpene synthases of the TPS-c family coli strains that provide appropriate precursors (Cyr (copalyl diphosphate synthases; Fig. 3; Supplemental et al., 2007). Appropriately truncated cDNAs of class II Table S2). Full-length cDNAs were cloned (KX533964 and diterpene synthase candidates from T. regelii (lacking KX533965, respectively; for primer sequences, see the plastidial targeting sequence; Supplemental Fig. S3) Supplemental Table S3), and their biochemical functions were introduced into E. coli harboring a recombinant were evaluated (see below). Translated peptide sequences geranylgeranyl diphosphate synthase (GGPPS) of three genes (TrTPS13, TrTPS14, and TrTPS15) clustered gene from Abies grandis (Burke and Croteau, 2002), in with functionally characterized class I ent-kaurene syn- combination with either the ent-kaurene synthase thases of the TPS-e/f family (Fig. 3). Full-length clones (eKS) gene from Arabidopsis (Arabidopsis thaliana; (KX533966, KX533967, and KX533968, respectively) were Yamaguchi et al., 1998) or the miltiradiene synthase obtained by PCR and subjected to functional characteri- (MDS) gene from Saliva miltiorrhiza (Gao et al., 2009). zation (see below). Five additional genes (TrTPS19, The expression of TrTPS2 (a candidate diterpene syn- TrTPS17, TrTPS20, TrTPS18, and TrTPS16) clustered with thase gene from the TPS-c family) led to the production linear-type diterpene synthases of the TPS-a family but, of miltiradiene when combined with MDS but did not because of the signiﬁcant separation from sequences of generate detectable products in other combinations, previously characterized diterpene synthases, also might indicating that the gene encodes a (+)-copalyl diphos- catalyze the formation of nonlinear diterpenes of as yet phate synthase [(+)-CPS; Fig. 4A]. The combination of unknown structure. While this was an interesting ﬁnding, TrTPS1 (also a TPS-c family member) with eKS yielded it was not of direct interest to this investigation (involve- ent-kaurene and small amounts of ent-isokaurene, but ment in the biosynthesis of kauranes or abietanes no products were detected when it was expressed in unlikely); therefore, we did not proceed with further other gene combinations, indicating that this gene en- characterizations. The translated sequences of two codes an ent-copalyl diphosphate synthase (ent-CPS; cDNAs (TrTPS8 [KY856995] and TrTPS7 [KY856996]) Fig. 4B). clustered with a recently discovered class I diterpene A similar modular approach was taken for the char- synthase of the TPS-b family from T. wilfordii (Hansen acterization of class I diterpene synthase candidates et al., 2017; Fig. 3) and were selected for functional char- (once again lacking the plastidial targeting sequence; acterization (see below). Other members from the TPS-b Supplemental Fig. S4), involving coexpression with family were TrTPS3 (KY856993) and TrTPS4 (KY856994), GGPPS (from A. grandis) and either ent-CPS (from which were also functionally characterized (see below), Arabidopsis), (+)-CPS [a mutant version of abietadiene and two additional transcripts (TrTPS5 and TrTPS6) that synthase from A. grandis that produces (+)-copalyl di- were quite short (less than 600 bp) and could not be ex- phosphate], or syn-CPS (from rice [Oryza sativa]; Cyr tended to a length that would have allowed functional et al., 2007). ent-Kaurene was the most prominent

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Table I. Metabolites detected by HPLC-QTOF-MS in the medium of T. regelii adventitious root cultures MS/MS spectra were acquired with a fragmentation voltage of 30 eV.

reaction product (with ent-isokaurene as a minor side TrTPS3 and TrTPS4, members of the TPS-b subfamily product) when TrTPS13 or TrTPS14 (TPS-e/f family (Supplemental Fig. S6), did not form products in any members) were expressed in combination with ent-CPS combination with CPSs. Instead, both TrTPS3 and (Fig. 5, A and B), indicating ent-kaurene synthase TrTPS4 produced mixtures of monoterpenes upon re- functions for these isoforms. When combined with action with geranyl diphosphate (substrate for mono- (+)-CPS, TrTPS13 expression produced small amounts terpene synthases), with (2)-linalool and (+)-linalool as of sandaracopimaradiene and isopimaradiene, while major products (Table II; Supplemental Fig. S7). When coexpression of TrTPS13 with syn-CPS led to the for- reacted with linalyl diphosphate (a reaction interme- mation of syn-pimara-7,15-diene and syn-stemod- diate of monoterpene synthases), TrTPS3 and TrTPS4 13(17)-ene (Fig. 5, C and D). The combination of both both formed (2)-terpineol and (+)-terpineol. In assays class II and class I diterpene synthases from T. regelii with linalyl diphosphate, TrTPS3, but not TrTPS4, also (TrTPS2 and TrTPS13) in one modular construct resul- generated appreciable amounts (2)-limonene (;20% of ted in the production of ent-kaurene (with smaller total products; Table II). TrTPS7 was expressed in the quantities of ent-isokaurene; Supplemental Fig. S5), recombinant E. coli strains but was not active in any thus conﬁrming the function of these genes in ent- combination with copalyl diphosphate synthases. It kaurane diterpenoid biosynthesis. TrTPS15 (also a also did not generate products from geranyl diphos- member of the TPS-e/f family) produced small quan- phate as a substrate in in vitro assays. tities of ent-manool, syn-manool, or (+)-manool when coupled with ent-CPS, syn-CPS, or (+)-CPS, respectively (Fig. 5, E–G). When coupled with (+)-CPS, TrTPS8 DISCUSSION (from the TPS-b family; Supplemental Fig. S6) pro- Proposing a Diterpenoid Biosynthetic Pathway Based on duced primarily miltiradiene (Fig. 6A) along with small Metabolites That Accumulate in Tripterygium amounts of abietatriene, presumably arising from a Adventitious Root Cultures previously noted autoxidation (Zi and Peters, 2013). Both products also were found to be present in the medium of In contrast to roots of mature Tripterygium plants, T. regelii adventitious root cultures (Supplemental Fig. where diterpenoids are only very minor constituents S1). When combined with ent-CPS or syn-CPS, TrTPS8 (less than 0.01%), our adventitious root cultures se- generated small quantities of ent-manool or syn-manool, creted these metabolites at very high levels (approxi- respectively (Fig. 6, B and C). mately 77% of the area under all peaks detected by

Figure 2. Putative biosynthetic pathway toward abietane diterpenoids in Tripterygium. Metabolites found in this study are boxed. The numbering of the carbons forming the abietane skeleton is indicated using dehydroabietic acid as an example. Reactions requiring multiple steps are indicated by dotted arrows. The numbering of metabolites is the same as in Figure 1.

HPLC-QTOF-MS in organic extracts of the culture tripdiolide, as the primary end products of the bio- medium). The structure and abundance of the principal synthetic conversions detected in our adventitious root diterpenoids found in our tissue cultures can thus be cultures. employed to develop hypotheses regarding the bio- Schemes for abietane diterpenoid biosynthesis in the synthesis of abietane-type diterpenoids of Tripterygium genus Tripterygium have been proposed before (Kutney (Fig. 2). It has been proposed previously that the two- et al., 1981; Kutney and Han, 1996), but these were step cyclization of the universal diterpenoid precursor based on the available phytochemical evidence at the geranylgeranyl diphosphate, via (+)-copalyl diphos- time and did not incorporate information about relative phate, yields miltiradiene as an abietane hydrocarbon metabolite abundance (Fig. 1; Supplemental Fig. S1). By intermediate (Hansen et al., 2017), and our data agree adding this new dimension, we are increasing the fi with this suggestion. Oxidation at C18, like that de- con dence in predictions regarding the design princi- scribed for CYP720 in conifers (Ro et al., 2005; ples of the pathways leading to structurally unusual Hamberger et al., 2011; Geisler et al., 2016), would diterpene triepoxides. Because of the high abundance of produce dehydroabietic acid (which we detected in diterpenoids in the medium of T. regelii adventitious root cultures, we have been able to employ simple pu- T. regelii root cultures; Fig. 2). Demethylation at C4, fi carboxylation at C3, and hydroxylation at C14 would ri cation protocols for intermediates (triptophenolide) result in the formation of triptinin B. This metabolite and end products (triptolide and tripdiolide). This was not detected in our root cultures, but its methyl provides commercially unavailable substrates and ether, triptinin A, was identified tentatively as a sig- products for in vitro enzyme assays and, therefore, enables the functional characterization of candidate nificant constituent. The tentatively identified neo- genes. triptophenolide and its glycoside, both likely derived from triptinin B and/or triptinin A, accumulated as significant by-products in our root cultures. The lacto- Identifying Genes with Roles in Generating Diterpenoid nization of triptinin B would generate triptophenolide, Structural Diversity which accumulated as the most abundant abietane diterpenoid in our root cultures (Fig. 2). The function- One T. regelii clone (TrTPS1) was characterized as alization of the aromatic ring would then produce encoding an ent-CPS that, in combination with eKSs triptolide and, following an additional hydroxylation, (TrTPS13 and TrTPS14), generates ent-kaurene (and

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Figure 3. Molecular phylogeny of dicot (di)terpene synthases, with an emphasis on those from the genus Tripterygium. Functionally characterized terpene synthases from T. regelii are highlighted by gray boxes. The analysis was carried out using the maximum likelihood method based on a matrix-based model (Jones et al., 1992). The bootstrap consensus tree inferred from 1,000 replications (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates are collapsed. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances and then selecting the topology with superior log likelihood value. The analysis involved 77 amino acid sequences. All positions containing gaps and missing data were elimi- nated. There were a total of 94 positions in the final data set. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016). The filled triangle indicates the position of the sequence chosen as an outgroup for rooting the phylogenetic tree. very small amounts of ent-isokaurene; Fig. 5). These five-ring oxygen bridge-containing ent-kauranes (Duan reactions are common to all vascular plants, which et al., 1999, 2001; Tanaka et al., 2004). In the closely re- require endogenous production of the derived GA lated species T. wilfordii, recent studies also identified hormones (Zi et al., 2014). However, ent-kaurane an ent-CPS (TwTPS3) and several isoforms of ent- diterpenoids also occur as specialized metabolites in kaurene/ent-isokaurene synthase (TwTPS2, TwTPS16, some plant families, including the Celastraceae (which TwTPS17, and TwTPS18; Zerbe et al., 2013; Hansen contains the genus Tripterygium). The previously et al., 2017). Various biological activities of Tripterygium reported metabolites can be grouped into two major ent-kauranes have been documented in in vitro assays classes: four-ring ent-kauranoic acids/alcohols and (for review, see Brinker et al., 2007), but the in vivo

Figure 4. Modular in vivo assay results obtained with T. regelii class II diterpene synthase candidates of the TPS-c subfamily. From left to right, constructs (selectable markers in gray boxes; Chlor, chloramphenicol; Carb, carbenicillin; MDS [from Salvia miltiorrhiza]; eKS [from Arabidopsis]), gas chromatography (GC)-MS chromatograms, and mass spectra of assay products and authentic standards. TrTPS2 was identified as a (+)-CPS (A), while TrTPS1 showed activity as an ent-CPS (B). functions remain to be elucidated. When combined ThegenecodingforT. regelii TrTPS8 [converts with a (+)-CPS, TrTPS13 formed sandaracopimaradiene (+)-copalyl diphosphate to miltiradiene, a likely inter- and isopimaradiene; in combination with a syn-CPS, mediate in triptolide and tripdiolide biosynthesis] is an TrTPS13 showed activity for the production of syn- ortholog of the recently characterized TwTPS27 gene pimara-7,15-diene and syn-stemod-13(17)-ene (Fig. 5). from T. wilfordii (Hansen et al., 2017). Based on phylo- We did not find a syn-CPS in T. regelii,nordoesthere genetic analyses, Hansen et al. (2017) concluded that appear to be such an activity among the class II TwTPS27 diversified from members of the TPS-b sub- diterpene synthases of T. wilfordii (Hansen et al., 2017); family, more specifically from terpene synthases that therefore, the biological relevance of this finding is catalyze the formation of acyclic monoterpenes. The unknown at this time. location of TwTPS27 on the phylogenetic tree of an- Genes from T. wilfordii were characterized previously giosperm terpene synthases was a surprise because as coding for ent-copal-8-ol diphosphate synthase all angiosperm diterpene synthases involved in abie- (termed TwTPS21) or kolavenyl diphosphate synthase tane/labdane biosynthesis characterized until then (TwTPS10, TwTPS14, and TwTPS28; Andersen- belonged to the TPS-e/f subfamily (Chen et al., 2011). Ranberg et al., 2016; Hansen et al., 2017). However, This evolutionary history also applies to TrTPS8, which the stereochemistry at C8 and C9 (8S,9S) is the opposite is very closely related to TwTPS27. of that of all labdane-type diterpenoids characterized Hansen et al. (2017) expressed TwTPS23, TwTPS24, from Tripterygium thus far (8R,9R; Duan et al., 1999, and TwTPS26 transiently in Nicotiana benthamiana and 2001). In our functional assays, TrTPS15 produced then subjected volatiles to a head-space analysis. ent-manool, (+)-manool, and syn-manool when com- The authors did not have authentic standards for bined with ent-CPS, (+)-CPS, or syn-CPS, respectively. monoterpenes and, therefore, could only use spectral Interestingly, (+)-manool has the correct stereochemis- comparisons for tentative identification. We are in the try (8R,9R) to serve as a precursor for the known lab- fortunate position to host a sizable repository of mon- dane diterpenoids of Tripterygium (Duan et al., 1999, oterpenes and, therefore, were able to perform in vitro 2001). The other manools and manoyl oxides, generated assays with unequivocal results. TrTPS3 and TrTPS4 by diterpene synthases of T. regelii and T. wilfordii, re- catalyzed the formation of a mixture of (2)-linalool and spectively, may only be produced in vivo under specific (+)-linalool from geranyl diphosphate as a substrate. environmental conditions or may simply reflect enzy- These results from early termination (by water capture) matic substrate promiscuity. However, the generation immediately following the formation of a linalyl cation of structural diversity by combinations of class II and (Supplemental Fig. S6). To test if these enzymes would be class I diterpene synthases, whether with recognizable capable of catalyzing cyclization if initial water capture in vivo relevance, does provide biosynthetic access to was avoided, we also performed assays with linalyl di- these and derived diterpenoids. phosphate as a substrate. Indeed, in these assays, TrTPS3

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Figure 5. Modular in vivo assay results obtained with T. regelii class I diterpene synthase candidates of the TPS-e/f family. From left to right: constructs (selectable markers in gray boxes; Chlor, chloramphenicol; Carb, carbenicillin), GC-MS chromatograms, and mass spectra of assay products and authentic standards. The following gene combinations were tested: ent-CPS/ TrTPS13 (A), ent-CPS/TrTPS14 (B), (+)-CPS/TrTPS13 (C), syn-CPS/TrTPS13 (D), ent-CPS/TrTPS15 (E), (+)-CPS/TrTPS15 (F), and syn-CPS/TrTPS15 (G).

Figure 6. Modular in vivo assay results obtained with TrTPS8, a class I diterpene synthase candidate of the TPS-a subfamily. From left to right: constructs (selectable markers in gray boxes; Chlor, chloramphenicol; Carb, carbenicillin), GC-MS chromatograms, and mass spectra of assay products and authentic standards. The following gene combinations were tested: ent-CPS/TrTPS8 (A), (+)-CPS/TrTPS8 (B), and syn-CPS/TrTPS8 (C). and TrTPS4 formed (2)-terpineol and (+)-terpineol as In summary, the high abundance of diterpene syn- products [with TrTPS3 also releasing (2)-limonene], thase transcripts in T. regelii adventitious root cultures indicating that, although water capture is still the main facilitated the rapid cloning of candidate genes. These means of terminating the catalyzed reaction, these were functionally characterized using a suitable mod- TPSs can catalyze cyclization. To the best of our ular expression system (Cyr et al., 2007). Thus, our tis- knowledge, these are the ﬁrst monoterpene synthases sue cultures are an excellent experimental system for to be unequivocally identiﬁed from Tripterygium. further pathway elucidation.

Table II. Product distribution in in vitro assays with TrTPS3 and TrTPS4 Dashes indicate quantities lower than 0.1% (v/v) of total detected volatiles. Geranyl Diphosphate as Linalyl Diphosphate Monoterpene Product Retention Time Substrate as Substrate TrTPS3 TrTPS4 TrTPS3 TrTPS4 min % of total Myrcene 17.135 – 4.39 1.45 1.57 (2)-Limonene 20.567 7.77 – 2.05 2.06 (+)-Limonene 20.823 – – 1.87 1.85 Terpinolene 23.734 – – 2.18 2.22 (2)-Linalool 32.171 35.60 40.38 21.88 23.81 (+)-Linalool 32.327 44.77 46.04 28.32 29.93 (2)-Terpineol 40.083 7.07 4.83 20.63 20.22 (+)-Terpineol 40.359 4.78 4.35 21.62 18.33

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Can Adventitious Root Cultures Serve as Sustainable spectrometry grade (Sigma-Aldrich). CDCl3 was obtained from Cambridge Resources for the Production of Pharmaceutically Isotope Laboratories. All other reagents were purchased from Sigma-Aldrich. Relevant Diterpenoids? Adventitious Root Cultures Cultures of Tripterygium hairy roots, adventitious roots, or cell suspensions all have the advantage of ac- Young Tripterygium regelii plants were purchased from Woodlanders and maintained under greenhouse conditions (illumination, 16-h day/8-h night cumulating abietane diterpenoids at high concentra- [250-500 mE]; temperature, 24°C day/20°C night; relative humidity, 45%–55%). tions (Miao et al., 2013; Zhu et al., 2014) and, in To initiate tissue cultures, leaf material was harvested, rinsed with sterile water, comparison with the unsustainable harvest of roots and surface sterilized by soaking in 20% (v/v) commercial bleach. Sterilized (where the concentrations of these specialized metab- leaves were cut into 1-cm2 pieces and placed onto Murashige and Skoog medium with macronutrients and micronutrients plus Gamborg’s vitamins olites are very low), should be considered as commer- 2 2 (Caisson), Suc (20 g L 1; Sigma-Aldrich), 1-naphthaleneacetic acid (0.2 mg L 1; 2 cial sources. We demonstrate here that diterpenoids are Caisson), 6-benzylaminopurine (1 mg L 1; Sigma-Aldrich), and phytagel 2 secreted into the culture medium of T. regelii adventi- (2.4 g L 1; Sigma-Aldrich), with pH adjusted to 5.8. After 4 to 6 weeks, callus tious root cultures, which significantly reduces the that developed along the leaf square edges was moved to plates containing root induction medium (Murashige and Skoog medium with macronutrients and complexity of the matrix for extraction. Triptolide is 2 micronutrients plus Gamborg’s vitamins, Suc [20 g L 1], 1-naphthaleneacetic detected as the peak with the second highest intensity 2 2 2 acid [1 mg L 1], 6-benzylaminopurine [0.2 mg L 1], and phytagel [2.4 g L 1], (16% of the total peak area) in our HPLC-QTOF-MS with pH adjusted to 5.8). Developing adventitious roots were transferred to chromatograms of culture medium extracts. Further- fresh plates every 4 to 6 weeks until they appeared strong enough for transfer more, it is well separated from other medium constit- to liquid medium (Murashige and Skoog medium with macronutrients and 2 micronutrients plus Gamborg’s vitamins, Suc [20 g L 1], and 1-naphthaleneacetic uents, which enabled a one-step HPLC processing to 2 acid [1 mg L 1], pH 5.8). Adventitious roots were partially submerged in medium greater than 95% purity (as judged by HPLC-QTOF-MS fl 1 (50 mL in a 250-mL Erlenmeyer ask) and maintained at 25°C, by shaking at and H-NMR). In our hands, the production of tripto- 80 rpm, in the dark, with transfer to fresh medium every 2 weeks. As adventitious lide in adventitious root cultures also has been reliable roots grew, the size of the flask and the volume of liquid medium were adjusted (grown continuously for more than 2 years) and readily up to 500 mL in a 2-L Fernbach flask. scalable (from 50- to 500-mL volumes within weeks). Others have scaled up Tripterygium cultures to 10- to Metabolite Extraction from Adventitious Root Cultures 20-L bioreactors (Kutney et al., 1992; Miao et al., 2014); therefore, it appears that typical shortcomings of tissue Solid materials from adventitious root cultures were washed with water (volume equivalent to the wet weight of material) and freeze dried for 3 d (Lyph- culture (reliability and scalability) have already been Lock 12L; LabConco), and the resulting dry matter was stored at 280°C until addressed. further use. Aliquots of 50 6 0.5 mg were transferred to receptacles of an MM01 It is probable that triptolide levels could be enhanced Dry Mill (Retsch) and further homogenized by ball shaking for 45 s at a rate of further if flux through the abietane diterpenoid path- 20 shakes per second. The homogenate was then transferred to glass test tubes with Teflon-lined caps. Each sample was extracted four times at 23°C with 5 mL way was increased in transgenic tissue cultures, where of acetone for 30 min (Ultrasound Bath FS30H; Fisher Scientific). After each genes involved in diterpenoid biosynthesis are already extraction step, samples were centrifuged at 3,000g for 5 min, and the combined expressed at fairly high levels (Supplemental Fig. S8). supernatants were collected in a separate glass test tube. The solvent was re- One approach would be the overexpression of genes moved under reduced pressure (EZ Bio; GeneVac), and the remaining residue 21 involved in providing diterpenoid precursors. A dif- was redissolved in 1 mL of 90% acetonitrile containing 10 mgmL 9-anthracene carboxylic acid as an internal standard. Prior to further analysis, samples were ferent and potentially complementary approach would passed through syringe filters (polytetrafluoroethylene; 0.22-mm pore size) and be the down-regulation of genes involved in competing stored at 4°C for a maximum of 2 d. pathways (e.g. reducing triterpenoid formation). A medium aliquot of adventitious root cultures (generally 5 mL) was We are currently investigating methods for the trans- extracted twice with ethyl acetate (2 mL each time) by thorough mixing for 1 min formation of Tripterygium to enable such endeavors. at 23°C. The combined organic extracts (4 mL) were extracted against water (2 mL) and transferred to a new glass vial, and the solvent was removed under Alternatively, the genes required for triptolide biosyn- reduced pressure (EZ Bio; GeneVac). The residue was redissolved in 200 mLof 2 thesis, once discovered, could be transferred to an 90% aqueous acetonitrile containing 10 mgmL 1 9-anthracene carboxylic acid engineered microbial strain, akin to the successful ef- as an internal standard. Prior to further analysis, samples were passed through forts to produce another diterpenoid, forskohlin syringe filters (polypropylene; 0.22-mm pore size) and stored at 4°C for a (Pateraki et al., 2017). At this point in time, yields of maximum of 2 d. highly functionalized plant diterpenoids in synthetic hosts have been relatively low; therefore, plant tissue HPLC-QTOF-MS and MS/MS Data Acquisition and cultures would seem to be a competitive option for Method Validation triptolide production. The separation of metabolites was almost identical to that described previously (Fischedick et al., 2015). The conditions for metabolite separation were modified slightly: the initial conditions were 70% solvent A (water with 0.1% [v/v] formic acid) and 30% solvent B (acetonitrile with 0.1% [v/v] formic acid). MATERIALS AND METHODS 2 A linear gradient (flow rate, 0.6 mL min 1) was used to increase solvent B to Chemicals 80% over 35 min, followed by a more rapid gradient to 95% solvent B at 40 min. The diode array detector was set to record at 219, 254, and 424 nm, with UV/ Triptolide and celastrol were purchased from Cayman Chemical. Dehy- visible spectra being recorded from 200 to 500 nm. Mass spectral data were droabietic acid was synthesized according to a literature protocol (González obtained based on the protocols outlined previously (Fischedick et al., 2015) et al., 2010). Acetone was of HPLC grade (Fisher Scientific), ethyl acetate was with the following differences: the electrospray ionization source was operated ACS quality (Avantor Performance Materials), ethanol was Omnisolv (EMD in positive polarity and MS/MS data were acquired with a collision energy of Serono), and acetonitrile and water were liquid chromatography-mass 30 eV. Data analysis was performed using the MassHunter software version

B.03.01 (Agilent Technologies). The approach for validating the quantitation of Supplemental Figure S3. Sequences of T. regelii diterpene synthases of the triptolide and celastrol was reported previously for various analytes in plant TPS-c family. matrices (Cuthbertson et al., 2013), and only the relevant values are given here: Supplemental Figure S4. Sequences of T. regelii diterpene synthases of recovery from adventitious roots (n = 3): 85.6% 6 5.6% for triptolide, 96.8% 6 the TPS-e/f family. 2.8% for celastrol; recovery from culture medium (n = 3): 106.6% 6 3.9% for triptolide, 86.1% 6 2.9% for celastrol; reproducibility of extraction/detection (as Supplemental Figure S5. Sequences of T. regelii diterpene synthases of the relative SD; n = 3): 6.5% (interday) and 0.9% (intraday) for triptolide, 0.5% TPS-b family. (interday) and 0.6% (intraday) for celastrol; limit of detection at signal-to-noise ratio of 1: 5 (n = 3): 0.01 ng for triptolide (MS detection) and 1 ng for celastrol Supplemental Figure S6. Modular in vivo combination of T. regelii class II (diode array detection at 424 nm); limit of quantitation at signal-to-noise ratio of (TrTPS2) and class I (TrTPS13) diterpene synthases. 1: 10 (n = 3): 0.05 for triptolide (MS detection) and 10 ng for celastrol (diode array Supplemental Figure S7. Proposed mechanism for T. regelii monoterpene detection at 424 nm); regression equation for calibration curve (n = 3): y = synthases. 25,938x + 16,475x (R2 = 0.989) for triptolide (corrected for matrix effects) and y = 0.9077x (R2 = 0.999) for celastrol (matrix effects irrelevant for diode array de- Supplemental Figure S8. Comparison of expression levels of candidate tection); and linear range, 0.05 to 50 ng for triptolide and 1 to 2,500 ng for diterpene synthase genes in T. regelii adventitious root cultures and roots celastrol. based on RNA sequence by expectation-maximization analysis. Supplemental Figure S9. Alignment of diterpene synthase sequences in- cluded in the phylogenetic analysis. Cloning and Functional Characterization of Terpene Synthases Supplemental Table S1. Changes in triptolide and celastrol concentrations in T. regelii adventitious root cultures following various elicitor treat- Raw data from several transcriptome sequencing projects performed pre- ments. viously with T. regelii tissues (National Center for Biotechnology Information Supplemental Table S2. List of dicot diterpene synthase protein sequences Short Read Archive accession no. SRP075639 [adventitious root cultures]) and used in the phylogenetic analysis. data sets available at http://www.medplantrnaseq.org/ (roots and root cultures) were downloaded, and a consensus assembly was generated using the Supplemental Table S3. List of primers used in T. regelii terpene synthase Trinity (Haas et al., 2013) and TransABySS (Robertson et al., 2010) assemblers. gene cloning. tBLASTn searches with peptide sequences of characterized class I and class II Supplemental Methods and Data File S1. Purification and characteriza- diterpene synthases (Supplemental Table S2) were performed against the tion of diterpenoids from T. regelii adventitious root cultures. T. regelii consensus assembly. RNA was isolated from T. regelii adventitious root cultures (harvested at 10 d after transfer to fresh medium) using the Plant RNA Purification reagent (Invitrogen) and the RNEasy Mini Kit (Qiagen) according to each manufacturer’s instructions, converted to cDNA using Maxima Reverse ACKNOWLEDGMENTS Transcriptase (Thermo Fisher Scientific), and PCR with gene-specific primers was then employed to clone candidate diterpene synthase sequences (details in We thank Sean R. Johnson for generating sequence assemblies with publicly ’ Supplemental Table S3). RACE-PCR was performed according to the manu- available transcriptome data sets, Washington State University s NMR Core facturer’s instructions (SMARTer-RACE 59/39 kit; Clontech/TaKaRa). The Facility for access to instruments, and Dr. Greg Helms for expert support. Gateway cloning system (Invitrogen) was used to insert TrTPS1 and TrTPS2 Received May 17, 2017; accepted July 18, 2017; published July 27, 2017. into a previously described pGG-DEST vector, and the other putative diterpene synthases into pDEST15, for functional expression in the Escherichia coli C41 (DE3) (Cyr et al., 2007). Diterpenoids were extracted directly from 50-mL cul- LITERATURE CITED tures with an equal volume of n-hexanes (Fisher Scientific). The extract was then run through silica gel 60 and magnesium sulfate columns as described else- Andersen-Ranberg J, Kongstad KT, Nielsen MT, Jensen NB, Pateraki I, where (Cyr et al., 2007). The eluents were dried under a flow of nitrogen, and Bach SS, Hamberger B, Zerbe P, Staerk D, Bohlmann J, et al (2016) the residue was dissolved in 200 mLofn-hexanes. Aliquots (1 mL) were injected Expanding the landscape of diterpene structural diversity through onto an HP-5MS column (30-m length 3 0.25-mm diameter, 0.25-mm film stereochemically controlled combinatorial biosynthesis. Angew Chem thickness; J&W Scientific, distributed through Agilent) of a 6890N gas chro- Int Ed Engl 55: 2142–2146 matograph (operated in splitless mode) coupled to a 5973 inert mass selective Brinker AM, Ma J, Lipsky PE, Raskin I (2007) Medicinal chemistry and detector (Agilent). Settings were as follows: helium as carrier gas at a flow rate pharmacology of genus Tripterygium (Celastraceae). Phytochemistry 21 of 1 mL min ; inlet temperature set to 250°C; oven program with 50°C for 68: 732–766 21 1 min, first linear gradient to 300°C at 7°C min , second linear gradient to Burke C, Croteau R (2002) Interaction with the small subunit of geranyl 21 330°C at 20°C min ,andfinal hold of 5 min; transfer line set to 180°C; electron diphosphate synthase modifies the chain length specificity of ger- impact spectra recorded at 70 eV with MS data collection from mass-to-charge anylgeranyl diphosphate synthase to produce geranyl diphosphate. J ratio of 50 to 450. Peaks were identified based on comparisons of retention times Biol Chem 277: 3141–3149 and MS fragmentation patterns with those of authentic standards. The func- Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene tional evaluation of monoterpene synthases was performed according to synthases in plants: a mid-size family of genes for specialized metabo- Srividya et al. (2015). lism that is highly diversified throughout the kingdom. Plant J 66: 212– 229 Chen YZ, Gao Q, Zhao XZ, Chen XM, Zhang F, Chen J, Xu CG, Sun LL, Accession Numbers Mei CL (2010) Meta-analysis of Tripterygium wilfordii Hook F in the immunosuppressive treatment of IgA nephropathy. Intern Med 49: All genes characterized as part of this study have been deposited in GenBank 2049–2055 with the following accession numbers: KX533964 (TrTPS2), KX533965 (TrTPS1), Chugh R, Sangwan V, Patil SP, Dudeja V, Dawra RK, Banerjee S, KX533966 (TrTPS13), KX533967 (TrTPS14), KX533968 (TrTPS15), KY856993 Schumacher RJ, Blazar BR, Georg GI, Vickers SM, et al (2012) A (TrTPS3), KY856994 (TrTPS4), KY856995 (TrTPS8), and KY856996 (TrTPS7). preclinical evaluation of Minnelide as a therapeutic agent against pan- creaticcancer.SciTranslMed4: 156ra139 Supplemental Data Cuthbertson DJ, Johnson SR, Piljac-Zegarac J, Kappel J, Schäfer S, Wüst M, Ketchum RE, Croteau RB, Marques JV, Davin LB, et al (2013) Ac- The following supplemental materials are available. curate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants. Phytochemistry 91: 187–197 Supplemental Figure S1. Accumulation of abietane diterpenoids in the Cyr A, Wilderman PR, Determan M, Peters RJ (2007) A modular approach medium of T. regelii adventitious root cultures. for facile biosynthesis of labdane-related diterpenes. J Am Chem Soc Supplemental Figure S2. 1H-NMR spectra of abietane diterpenoids. 129: 6684–6685

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