biosynthesis of diterpenoids in tripterygium …...2017/07/27 · 97 triptolide, a diterpenoid...

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Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures1 1

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Fainmarinat S. Inabuy, Justin T. Fischedick2, Iris Lange, Michael Hartmann3, Narayanan 4

Srividya, Amber N. Parrish, Meimei Xu, Reuben J. Peters, and B. Markus Lange* 5

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Institute of Biological Chemistry and M.J. Murdock Metabolomics Laboratory, 8

Washington State University, Pullman, WA 99164-6340, USA (A.M.P., B.M.L., F.S.I., 9

J.T.F., M.H., N.S.); Roy J. Carver Department of Biochemistry, Biophysics and 10

Molecular Biology, Iowa State University, Ames, IA 50011-1079, USA (M.X., R.J.P.) 11

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1This work was supported by the National Institutes of Health (award numbers 15

RC2GM092561 to B.M.L. and GM076324 to R.J.P.) and McIntire-Stennis formula funds 16

from the Agricultural Research Center at Washington State University (to B.M.L.). 17

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2Present address: Excelsior Analytical, Union City, CA 94587, USA. 19

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3Present address: Institute of Molecular Ecophysiology of Plants, Heinrich Heine 21

Universität Düsseldorf, 40225 Düsseldorf, Germany. 22

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*Correspondence (Tel +1 509 335 3794; fax +1 509 335 3794; email lange-26

[email protected]) 27

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Plant Physiology Preview. Published on July 27, 2017, as DOI:10.1104/pp.17.00659

Copyright 2017 by the American Society of Plant Biologists

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ONE SENTENCE SUMMARY 30

Adventitious root cultures provide insights to elucidating the biosynthesis of 31

pharmaceutically relevant diterpenoids in the model genus Tripterygium 32

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AUTHOR CONTRIBUTIONS 35

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B.M.L. conceived of the project. F.S.I. cloned candidate genes, subcloned them into 37

appropriate modular expression vectors and analyzed diterpenoids produced in E. coli 38

cells harboring these constructs. J.T.F. established the methods for the isolation, 39

purification, identification and quantitation of diterpenoids. I.L. isolated RNA from 40

adventitious root cultures and cloned candidate genes. In addition, I.L. performed the 41

scale-up and selection of adventitious root cultures for high diterpenoid production. 42

M.H. established and A.N.P. maintained the adventitious root cultures. N.S. performed 43

the functional characterization of monoterpene synthase genes. M.X. and R.J.P. 44

supplied modular vectors and carried out diterpenoid characterizations. B.M.L. and 45

F.S.I. wrote the manuscript, with contributions from all authors. 46

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Key words: diterpene synthase, diterpenoid, monoterpene synthase, tissue culture, 49

triterpenoid 50

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Abbreviations and Acronyms: CPP, copalyl diphosphate; CPS, copalyl diphosphate 53

synthase; eKS, ent-kaurene synthase; GGPPS, geranylgeranyl diphosphate synthase; 54

HPLC-QTOF-MS, high performance liquid chromatography-quadrupole time of flight-55

mass spectrometry; MS, mass spectrometry; MS/MS, tandem mass spectrometry; 56

NMR, nuclear magnetic resonance; TPS, terpene synthase. 57

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ABSTRACT 59

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Adventitious root cultures were developed from Tripterygium regelii Sprague & Takeda 61

and growth conditions optimized for the abundant production of diterpenoids, which can 62

be collected directly from the medium. An analysis of publicly available transcriptome 63

data sets collected with T. regelii roots and root cultures indicated the presence of a 64

large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate 65

diterpene synthase genes were selected for follow-up functional evaluation, of which 66

two belonged to the TPS-c, three to the TPS-e/f and four to the TPS-b subfamily. 67

These genes were characterized by heterologous expression in a modular metabolic 68

engineering system in E. coli. Members of the TPS-c subfamily were characterized as 69

copalyl diphosphate (diterpene) synthases and those belonging to the TPS-e/f family 70

catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily 71

encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis 72

and others with activities as monoterpene synthases. The structural characterization of 73

diterpenoids accumulating in the medium of T. regelii adventitious root cultures, 74

facilitated by searching the Spektraris online spectral database, enabled us to formulate 75

a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with 76

pharmaceutical potential. Considering the significant enrichment of diterpenoids in the 77

culture medium, fast-growing adventitious root cultures may hold promise as a 78

sustainable resource for the large-scale production of triptolide. 79

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INTRODUCTION 82

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Tripterygium wilfordii Hook. f., also known as léi gōng téng in Mandarin Chinese 84

(generally translated as "thunder god vine"), has a long history of use in traditional 85

Chinese Medicine for the treatment of fever, chills, edema, and carbuncles 86

(Helmstädter, 2013). The genus Triptergyium (Celastraceae) is known to be a rich 87

source of specialized metabolites, of which more than 400 have been isolated, 88

structurally characterized, and assessed in cell-based assays (Brinker et al., 2007). 89

Root extracts have been evaluated as a medication for rheumatoid arthritis, cancer, 90

hepatitis, nephritis, ankylosing spondylitis, polycystic kidney disease, and obesity; more 91

than a dozen clinical trials with such extracts (often referred to as “Tripterygium 92

glycoside”) have been completed but, in part due to shortcomings in study designs, the 93

efficacy has remained a matter of debate (Chen et al., 2010; Liu et al., 2013; Zhu et al., 94

2013). More promising results have been obtained with purified constituents, which are 95

usually extracted from Tripterygium roots. Semi-synthetic chemical derivatives of 96

triptolide, a diterpenoid epoxide, have been evaluated in phase I clinical trials (Meng et 97

al., 2014; Zhou et al., 2012). Minnelide, a water soluble pro-drug analogue of triptolide, 98

has shown very promising activity in multiple animal models of pancreatic cancer 99

(Chugh et al., 2012) and, in 2013, was advanced to phase I clinical trials in the United 100

States (clinicaltrials.gov identifier NCT01927965). 101

One of the critical challenges for clinical evaluations is a supply shortage for 102

triptolide and other diterpenoids. The most abundant specialized metabolites in 103

Tripterygium roots are quinone methide triterpenoids and sesquiterpene pyridine 104

alkaloid macrolides, whereas triptolide and other diterpenoids occur only at very low 105

concentrations ranging from 0.0001 to 0.002 % of dry weight biomass (Guo et al., 2014; 106

Zeng et al., 2013; Zhou et al., 2012). The extraction yields of diterpenoids from 107

Tripterygium roots are accordingly poor and alternative, sustainable production methods 108

need to be developed. Tissue cultures represent a promising alternative for the 109

production of high value plant metabolites. Early studies with T. wilfordii suspension 110

cultures were designed to unravel the structures of small molecule constituents (Kutney 111

and Han, 1996; Kutney et al., 1981; Kutney et al., 1992; Kutney et al., 1993; Nakano et 112



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al., 1997; Nakano et al., 1998). Similarly, hairy root cultures were initially employed for 113

phytochemical investigations (Nakano et al., 1998). It was recognized only recently that 114

triptolide concentrations produced by tissue cultures (up to 0.15 % of dry weight) (Miao 115

et al., 2013; Miao et al., 2014; Zhu et al., 2014) far exceed those reported for roots. 116

Surprisingly, despite considerable pharmaceutical interest, the biosynthesis of 117

triptolide has only recently attracted the deserved attention. Several diterpene 118

synthases, some of which are relevant to triptolide formation, have now been 119

characterized in T. wilfordii (Andersen-Ranberg et al., 2016; Hansen et al., 2017; Zerbe 120

et al., 2013). However, the remaining genes involved in the biosynthesis of the highly 121

functionalized triptolide structure have remained enigmatic. In this manuscript, we 122

describe the development of Tripterygium adventitious roots cultures in which 123

diterpenoids are produced as principal metabolites that can be harvested sustainably 124

from the medium. Based on the structures of these metabolites, we can now postulate 125

the biochemical steps leading to the main diterpenoid metabolites. In addition, analysis 126

of transcriptome data sets, acquired with diterpenoid-producing adventitious root 127

cultures, enabled the selection and subsequent functional characterization of genes 128

involved in the early steps of diterpenoid biosynthesis. Our results indicate that 129

Tripterygium tissue cultures offer opportunities to both unravel diterpenoid biosynthetic 130

pathways and produce target diterpenoids – such as triptolide – at larger scale. 131

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RESULTS 133

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Development of Diterpenoid-Secreting Adventitious Root Cultures 135

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Tripterygium regelii Sprague & Takeda adventitious roots cultures were initiated based 137

on standard protocols. Over a one-year period, during which medium was extracted 138

with ethyl acetate and hydrophobic metabolites were analyzed by High Performance 139

Liquid Chromatography – Quadrupole Time-Of-Flight – Mass Spectrometry (HPLC-140

QTOF-MS), the highest triptolide producers were selected for further propagation. In 141

contrast to roots of mature Tripterygium plants, where triterpenoids occur at low 142

concentrations (< 1 %) and diterpenoids are trace constituents (< 0.01 %), our 143



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adventitious root cultures accumulated diterpenoids abundantly in the medium and 144

triterpenoids (in particular celastrol) in roots (Fig. 1A, B). By comparing adjusted peak 145

areas from HPLC-QTOF-MS runs, we estimated that diterpenoids constitute 146

approximately 77 % of all detected metabolites in an organic extract of the culture 147

medium (Supplemental Fig. S1). 148

An accurate quantitation of all diterpenoids was not possible because we did not 149

have a complete set of authentic standards, but we performed absolute quantitations for 150

signature metabolites (triptolide and celastrol). Various common reagents and elicitors 151

were tested (methyl jasmonate; methyl salicylate, chitosan, yeast extract, and cold 152

exposure), but only the methyl salicylate treatment resulted in a significant increase in 153

triptolide accumulation (2.2-fold; P-value 0.01) and concomitant decrease in celastrol 154

concentration (Supplemental Table S1). The disadvantage of methyl salicylate 155

treatments was that root color darkened and growth ceased, often for months. We 156

therefore focused our efforts on further selecting cultures that were high diterpenoid 157

producers under non-inducing conditions, and could be maintained for extended periods 158

of time. These cultures were gradually scaled up to 2.8 L flasks (500 mL medium) (Fig. 159

1C). Triptolide accumulated at 4.7 mg L-1 (Fig. 1D), and was readily extractable from 160

the culture medium every two weeks (when adventitious roots were transferred to fresh 161

media). 162

Hundreds of structurally diverse metabolites have been isolated and characterized 163

from various Tripterygium organs and tissues (Lange et al., 2017). Our adventitious 164

root cultures, in contrast, secreted primarily diterpenoids into the culture medium and 165

are therefore an excellent experimental model system to study the biosynthesis of these 166

clinically relevant metabolites. As a first step to further assess the potential of these 167

tissue cultures, it was important to identify the major constituents of medium extracts. 168

Following HPLC fractionation of medium extracts of Tripterygium adventitious root 169

cultures, fractions containing metabolites that corresponded to prominent peaks in 170

HPLC-QTOF-MS chromatograms were characterized by MS, MS/MS and 1H-NMR 171

spectroscopy (Supplemental Fig. S1 and Supplemental Methods and Data File S1), and 172

metabolites identified by searches against the Spektraris database (Fischedick et al., 173

2015). We ascertained the identify of five diterpenoids, were able to annotate three 174



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additional metabolites with high confidence, and acquired tentative identifications for an 175

additional two metabolites (Table 1). Three metabolites showed the mass 176

fragmentation patterns typical of diterpenoids but their identity remained unknown 177

(Table1). 178

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Functional Characterization of Candidate Diterpene Synthases 181

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For obtaining full-length candidate genes, we deemed it advantageous to employ all 183

available T. regelii sequences. We therefore downloaded several publicly available 184

transcriptome data sets obtained with roots and root cultures, and then generated a 185

consensus assembly. tBLASTn searches with peptide sequences of diterpene 186

synthases of dicotyledons were performed against our assembly data. A phylogenetic 187

analysis indicated that translated peptide sequences of two T. regelii genes (TrTPS2 188

and TrTPS1) clustered with functionally characterized class II diterpene synthases of 189

the TPS-c family (copalyl diphosphate synthases) (Fig. 3 and Supplemental Table S2). 190

Full-length cDNAs were cloned (KX533964 and KX533965, respectively) (see 191

Supplemental Table S3 for primer sequences) and their biochemical function evaluated 192

(see below). Translated peptide sequences of three genes (TrTPS13, TrTPS14 and 193

TrTPS15) clustered with functionally characterized class I ent-kaurene synthases of the 194

TPS-e/f family (Fig. 3). Full-length clones (KX533966, KX533967 and KX533968, 195

respectively) were obtained by PCR and subjected to functional characterization (see 196

below). Five additional genes (TrTPS19, TrTPS17, TrTPS20, TrTPS18 and TrTPS16) 197

clustered with linear-type diterpene synthases of the TPS-a family, but, because of the 198

significant separation from sequences of previously characterized diterpene synthases, 199

might also catalyze the formation of non-linear diterpenes of as yet unknown structure. 200

While this was an interesting finding, it was not of direct interest to this investigation 201

(involvement in the biosynthesis of kauranes or abietanes unlikely) and were therefore 202

did not proceed with further characterizations. The translated sequences of two cDNAs 203

(TrTPS8 (KY856995) and TrTPS7 (KY856996)) clustered with a recently discovered 204

class I diterpene synthase of the TPS-b family from T. wilfordii (Hansen et al., 2017) 205



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(Fig. 3), and were selected for functional characterization (see below). Other members 206

from the TPS-b family were TrTPS3 (KY856993) and TrTPS4 (KY856994), which were 207

functionally characterized (see below), and two additional transcripts (TrTPS5 and 208

TrTPS6) that were quite short (< 600 bp) and could not be extended to a length that 209

would have allowed functional characterization. Finally, nine members of the TPS-a 210

family (TrTPS9, TrTPS10, TrTPS11, TrTPS12, TrTPS16, TrTPS17, TrTPS18, TrTPS19, 211

and TrTPS20) clustered with known genes that encode linear diterpene cyclases and 212

macrocyclases, which were not of direct interest to this study and were therefore not 213

further characterized. 214

Diterpene synthase candidate genes were characterized by functional expression in 215

engineered E. coli strains that provide appropriate precursors (Cyr et al., 2007). 216

Appropriately truncated cDNAs of class II diterpene synthase candidates from T. regelii 217

(lacking the plastidial targeting sequence) (Supplemental Fig. S3) were introduced into 218

E. coli harboring a recombinant geranylgeranyl diphosphate synthase (GGPPS) gene 219

from Abies grandis (Croteau, 2002), in combination with either the ent-kaurene 220

synthase (eKS) gene from Arabidopsis thaliana (Yamaguchi et al., 1998) or miltiradiene 221

synthase (MDS) gene from Saliva miltiorrhiza (Gao et al., 2009). Expression of TrTPS2 222

(candidate diterpene synthase gene from the TPS-c family) led to the production of 223

miltiradiene when combined with MDS, but did not generate detectable products in 224

other combinations, indicating that the gene encodes a (+)-copalyl diphosphate 225

synthase ((+)-CPS) (Fig. 4A). The combination of TrTPS1 (also a TPS-c family 226

member) with eKS yielded ent-kaurene and small amounts of ent-isokaurene, but no 227

products were detected when it was expressed in other gene combinations, indicating 228

that this gene encodes an ent-copalyl diphosphate synthase (ent-CPS) (Fig. 4B). 229

A similar modular approach was taken for characterization of the class I diterpene 230

synthase candidates (once again lacking the plastidial targeting sequence) 231

(Supplemental Fig. S4), involving co-expression with GGPPS (from Abies grandis) and 232

either ent-CPS (from Arabidopsis thaliana), (+)-CPS (a mutant version of abietadiene 233

synthase from Abies grandis that produces (+)-copalyl diphosphate) or syn-CPS (from 234

Oryza sativa) (Cyr et al., 2007). ent-Kaurene was the most prominent reaction product 235

(with ent-isokaurene as a minor side product) when TrTPS13 or TrTPS14 (TPS-e/f 236



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family members) were expressed in combination with ent-CPS (Fig. 5A, B), indicating 237

ent-kaurene synthase functions for these isoforms. When combined with (+)-CPS, 238

TrTPS13 expression produced small amounts of sandaracopimaradiene and 239

isopimaradiene, while co-expression of TrTPS13 with syn-CPS led to the formation of 240

syn-pimara-7,15-diene and syn-stemod-13(17)-ene (Fig. 5C, D). The combination of 241

both class II and class I diterpene synthases from Tripterygium (TrTPS2 and TrTPS13) 242

in one modular construct resulted in the production of ent-kaurene (with smaller 243

quantities of ent-isokaurene) (Supplemental Fig. S5), thus confirming the function of 244

these genes in ent-kaurane diterpenoid biosynthesis. TrTPS15 (also a member of the 245

TPS-e/f family) produced small quantities of ent-manool, syn-manool or (+)-manool 246

when coupled with ent-CPS, syn-CPS or (+)-CPS, respectively (Fig. 5E-G). When 247

coupled with (+)-CPS, TrTPS8 (from the TPS-b family) (Supplemental Fig. S6) 248

produced primarily miltiradiene (Fig. 6A), along with small amounts of abietatriene 249

presumably arising from a previously noted autoxidation (Zi and Peters, 2013). Both 250

products were also found to be present in the medium of T. regelii adventitious root 251

cultures (Supplemental Fig. S1). When combined with ent-CPS or syn-CPS, TrTPS8 252

generated small quantities of ent-manool or syn-manool, respectively (Fig. 6B, C). 253

TrTPS3 and TrTPS4, members of the TPS-b subfamily (Supplemental Fig. S6), did 254

not form products in any combination with CPSs. Instead, both TrTPS3 and TrTPS4 255

produced mixtures of monoterpenes upon reaction with geranyl diphosphate (substrate 256

for monoterpene synthases), with (-)-linalool and (+)-linalool as major products (Table 2 257

and Supplemental Fig. S7). When reacted with linalyl diphosphate (reaction 258

intermediate of monoterpene synthases), TrTPS3 and TrTPS4 both formed (-)-terpineol 259

and (+)-terpineol. In assays with linalyl diphosphate, TrTPS3, but not TrTPS4, also 260

generated appreciable amounts (-)-limonene (~ 20 % of total products) (Table 2). 261

TrTPS7 was expressed in the recombinant E. coli strains but was not active in any 262

combination with copalyl diphosphate synthases. It also did not generate products from 263

geranyl diphosphate as a substrate in in vitro assays. 264

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DISCUSSION 267

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Proposing a Diterpenoid Biosynthetic Pathway Based On Metabolites that 269

Accumulate in Tripterygium Adventitious Root Cultures 270

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In contrast to roots of mature Tripterygium plants, where diterpenoids are only very 272

minor constituents (< 0.01 %), our adventitious root cultures secreted these metabolites 273

at very high levels (approximately 77 % of the area under all peaks detected by HPLC-274

QTOF-MS in organic extracts of the culture medium). The structure and abundance of 275

the principal diterpenoids found in our tissue cultures can thus be employed to develop 276

hypotheses regarding the biosynthesis of abietane-type diterpenoids of Tripterygium 277

(Fig. 2). It has previously been proposed that the two-step cyclization of the universal 278

diterpenoid precursor geranylgeranyl diphosphate, via (+)-copalyl diphosphate, yields 279

miltiradiene as an abietane hydrocarbon intermediate (Hansen et al., 2017), and our 280

data agree with this suggestion. Oxidation at C18 akin to that described for CYP720 in 281

conifers (Ro et al., 2005; Hamberger et al., 2011; Geisler et al., 2016) would produce 282

dehydroabietic acid (which we detected in Tripterygium root cultures) (Fig. 2). 283

Demethylation at C4, carboxylation at C3 and hydroxylation at C14 would result in the 284

formation of triptinin B. This metabolite was not detected in our root cultures but its 285

methyl ether, triptinin A, was tentatively identified as a significant constituent. The 286

tentatively identified neotriptophenolide and its glycoside, both likely derived from 287

triptinin B and/or triptinin A, accumulated as significant byproducts in our root cultures. 288

The lactonization of triptinin B would generate triptophenolide, which accumulated as 289

the most abundant abietane diterpenoid in our root cultures (Fig. 2). The 290

functionalization of the aromatic ring would then produce triptolide and, following an 291

additional hydroxylation, tripdiolide, as the primary end products of the biosynthetic 292

conversions detected in our adventitious root cultures. 293

Schemes for abietane diterpenoid biosynthesis in the genus Tripterygium have been 294

proposed before (Kutney and Han, 1996; Kutney et al., 1981), but these were based on 295

the available phytochemical evidence at the time and did not incorporate information 296

about relative metabolite abundance (Fig. 1 and Supplemental Fig. S1). By adding this 297



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new dimension, we are increasing the confidence in predictions regarding the design 298

principles of the pathways leading to structurally unusual diterpene triepoxides. 299

Because of the high abundance of diterpenoids in the medium of Tripterygium 300

adventitious root cultures, we have been able to employ simple purification protocols for 301

intermediates (triptophenolide) and end products (triptolide and tripdiolide). This 302

provides commercially unavailable substrates and products for in vitro enzyme assays 303

and, therefore, enables the functional characterization of candidate genes. 304

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Identifying Genes with Roles in Generating Diterpenoid Structural Diversity 306

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One T. regelii clone (TrTPS1) was characterized as encoding an ent-CPS that, in 308

combination with eKSs (TrTPS13 and TrTPS14), generates ent-kaurene (and very small 309

amounts of ent-isokaurene) (Fig. 5). These reactions are common to all vascular 310

plants, which require endogenous production of the derived gibberellin hormones (Zi et 311

al., 2014). However, ent-kaurane diterpenoids also occur as specialized metabolites in 312

some plant families, including the Celastraceae (comprising the genus Tripterygium). 313

The previously reported metabolites can be grouped into two major classes: four-ring 314

ent-kauranoic acids/alcohols and five-ring oxygen bridge-containing ent-kauranes (Duan 315

et al., 1999; Duan et al., 2001; Tanaka et al., 2004). In the closely related species T. 316

wilfordii, recent studies also identified an ent-CPS (TwTPS3) and several isoforms of 317

ent-kaurene/ent-isokaurene synthase (TwTPS2, TwTPS16, TwTPS17 and TwTPS18) 318

(Zerbe et al., 2013; Hansen et al., 2017). Various biological activities of Tripterygium 319

ent-kauranes have been documented in in vitro assays (reviewed in Brinker et al., 320

2007), but the in vivo functions remain to be elucidated. When combined with a (+)-321

CPS, TrTPS13 formed sandaracopimaradiene and isopimaradiene; in combination with 322

a syn-CPS, TrTPS13 showed activity for the production of syn-pimara-7,15-diene and 323

syn-stemod-13(17)-ene (Fig. 5). We did not find a syn-CPS in T. regelii, nor does there 324

appear to be such an activity among the class II diterpene synthases of T. wilfordii 325

(Hansen et al., 2017), and the biological relevance of this finding is therefore unknown 326

at this time. 327



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Genes from T. wilfordii were previously characterized as coding for ent-copal-8-ol 328

diphosphate synthase (termed TwTPS21) or kolavenyl diphosphate synthase 329

(TwTPS10, TwTPS14, TwTPS28) (Andersen-Ranberg et al., 2016; Hansen et al., 330

2017). However, the stereochemistry at C8 and C9 (8S, 9S) is the opposite of that of all 331

labdane-type diterpenoids characterized from Tripterygium thus far (8R, 9R) (Duan et 332

al., 1999; Duan et al., 2001). In our functional assays, TrTPS15 produced ent-manool, 333

(+)-manool and syn-manool when combined with ent-CPS, (+)-CPS or syn-CPS, 334

respectively. Interestingly, (+)-manool has the correct stereochemistry (8R, 9R) to 335

serve as a precursor for the known labdane diterpenoids of Tripterygium (Duan et al., 336

1999; Duan et al., 2001). The other manools and manoyl oxides, generated by 337

diterpenes synthases of T. regelii and T. wilfordii, respectively, may only be produced in 338

vivo under specific environmental conditions or simply reflect enzymatic substrate 339

promiscuity. However, the generation of structural diversity by combinations of class II 340

and class I diterpene synthases, whether or not with recognizable in vivo relevance, 341

does provide biosynthetic access to these and derived diterpenoids. 342

The gene coding for T. regelii TrTPS8 (converts (+)-copalyl diphosphate to 343

miltiradiene, a likely intermediate in triptolide and tripdiolide biosynthesis) is an ortholog 344

of the recently characterized TwTPS27 gene from T. wilfordii (Hansen et al., 2017). 345

Based on phylogenetic analyses, Hansen et al. (2017) concluded that TwTPS27 346

diversified from members of the TPS-b subfamily, more specifically from terpene 347

synthases that catalyze the formation of acyclic monoterpenes. The location of 348

TwTPS27 on the phylogenetic tree of angiosperm terpene synthases was a surprise 349

because all angiosperm diterpene synthases involved in abietane/labdane biosynthesis 350

characterized until then belonged to the TPS-e/f subfamily (Chen et al., 2011). This 351

evolutionary history also applies to TrTPS8, which is very closely related to TwTPS27. 352

Hansen et al. (2017) expressed TwTPS23, TwTPS24 and TwTPS26 transiently in 353

Nicotiana benthamiana, and then subjected volatiles to a headspace analysis. The 354

authors did not have authentic standards for monoterpenes and therefore could only 355

use spectral comparisons for tentative identification. We are in the fortunate position to 356

host a sizable repository of monoterpenes and therefore were able to perform in vitro 357

assays with unequivocal results. TrTPS3 and TrTPS4 catalyzed the formation of a 358



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mixture of (-)-linalool and (+)-linalool from geranyl diphosphate as a substrate. These 359

result from early termination (by water capture), immediately following the formation of a 360

linalyl cation (Supplemental Fig. S6). To test if these enzymes would be capable of 361

catalyzing cyclization if initial water capture was avoided, we also performed assays 362

with linalyl diphosphate as a substrate. Indeed, in these assays, TrTPS3 and TrTPS4 363

formed (-)-terpineol and (+)-terpineol as products (with TrTPS3 also releasing (-)-364

limonene), indicating that, although water capture is still the main means of terminating 365

the catalyzed reaction, these TPSs can catalyze cyclization. To the best of our 366

knowledge, these are the first monoterpene synthases to be unequivocally identified 367

from Tripterygium. 368

In summary, the high abundance of diterpene synthase transcripts in Tripterygium 369

adventitious root cultures facilitated the rapid cloning of candidate genes. These were 370

functionally characterized using a suitable modular expression system (Cyr et al., 2007). 371

Thus, our tissue cultures are an excellent experimental system for further pathway 372

elucidation. 373

374

Can Adventitious Root Cultures Serve as Sustainable Resources for the 375

Production of Pharmaceutically Relevant Diterpenoids? 376

377

Cultures of Tripterygium hairy roots, adventitious roots or cell suspensions, all have the 378

advantage of accumulating abietane diterpenoids at high concentrations (Miao et al., 379

2013; Zhu et al., 2014) and, in comparison to the unsustainable harvest of roots (where 380

the concentrations of these specialized metabolites are very low), should be considered 381

as commercial sources. We demonstrate here that diterpenoids are secreted into the 382

culture medium of Tripterygium adventitious root cultures, which significantly reduces 383

the complexity of the matrix for extraction. Triptolide is detected as the peak with the 384

second highest intensity (16 % of the total peak area) in our HPLC-QTOF-MS 385

chromatograms of culture medium extracts. Furthermore, it is well separated from other 386

medium constituents, which enabled a one-step HPLC processing to > 95 % purity (as 387

judged by HPLC-QTOF-MS and 1H-NMR). In our hands, the production of triptolide in 388

adventitious root cultures has also been reliable (grown continuously for more than two 389



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years) and readily scalable (from 50 to 500 mL volumes within weeks). Others have 390

scaled up Tripterygium cultures to 10-20 L bioreactors (Kutney et al., 1992; Miao et al., 391

2014), and it therefore appears that typical shortcoming of tissue culture (reliability and 392

scalability) have already been addressed. 393

It is probable that triptolide levels could be further enhanced if flux through the 394

abietane diterpenoid pathway was increased in transgenic tissue cultures, where genes 395

involved in diterpenoid biosynthesis are already expressed at fairly high levels 396

(Supplemental Fig. S8). One approach would be the over-expression of genes involved 397

in providing diterpenoid precursors. A different and potentially complimentary approach 398

would be the down-regulation of genes involved in competing pathways (e.g., reducing 399

triterpenoid formation). We are currently investigating methods for the transformation of 400

Tripterygium to enable such endeavors. Alternatively, the genes required for triptolide 401

biosynthesis, once discovered, could be transferred to an engineered microbial strain, 402

akin to the successful efforts to produce another diterpenoid, forskohlin (Pateraki et al., 403

2017). At this point in time, yields of highly functionalized plant diterpenoids in synthetic 404

hosts have been relatively low, and plant tissue cultures would therefore seem to be a 405

competitive option for triptolide production. 406

407

Experimental Procedures 408

409

Chemicals 410

411

Triptolide and celastrol were purchased from Cayman Chemical (Ann Arbor, MI, USA). 412

Dehydroabietic acid was synthesized according to a literature protocol (Gonzalez et al., 413

2010). Acetone was of HPLC grade (Fischer Scientific, Pittsburgh, PA, USA), ethyl 414

acetate was ACS quality (Avantor Performance Materials Inc, Center Valley, PA, USA), 415

ethanol was Omnisolv, (EMD Serono, Rockland, MA, USA), and acetonitrile and water 416

were LC-MS grade (Sigma-Aldrich, St. Louis, MO, USA). CDCl3 was obtained from 417

Cambridge Isotope Laboratories Inc. (Andover, MA, USA). All other reagents were 418

purchased from Sigma-Aldrich (St. Louis, MO, USA). 419

420



15

421



16

Adventitious Root Cultures 422

423

Young T. regelii Sprague & Takeda plants were purchased from Woodlanders Inc. 424

(Aiken, SC, USA) and maintained under greenhouse conditions (illumination: 16 h day, 425

8 h night (250-500 µE); temperature: 24 °C day, 20 °C night; relative humidity: 45-55 426

%). To initiate tissue cultures, leaf material was harvested, rinsed with sterile H2O, and 427

surface-sterilized by soaking in 20 % (v/v) commercial bleach. Sterilized leaves were 428

cut into 1 cm2 squares and placed onto with Murashige & Skoog (MS) media with macro 429

and micro-nutrients plus Gamborg's vitamins (Caisson, Logan, UT, USA), sucrose (20 g 430

L-1; Sigma-Aldrich, St. Louis, MO, USA), 1-naphthaleneacetic acid (0.2 mg L-1; Caisson, 431

Logan, UT, USA), 6-benzylaminopurine (1.0 mg L-1; Sigma-Aldrich, St. Louis, MO, 432

USA), phytagel (2.4 g L-1; Sigma-Aldrich, St. Louis, MO, USA), and a pH adjusted to 433

5.8. After 4-6 weeks, callus that developed along the leaf square edges was moved to 434

plates containing root induction medium (MS media with macro and micro-nutrients plus 435

Gamborg's vitamins, sucrose (20 g L-1), 1-naphthaleneacetic acid (1.0 mg L-1), 6-436

benzylaminopurine (0.2 mg L-1), phytagel (2.4 g L-1); pH adjusted to 5.8). Developing 437

adventitious roots were transferred to fresh plates every 4-6 weeks until they appeared 438

strong enough for transfer to liquid medium (MS media with macro and micro-nutrients 439

plus Gamborg's vitamins, sucrose (20 g L-1), 1-naphthaleneacetic acid (1.0 mg L-1), pH 440

5.8). Adventitious roots were partially submerged in medium (50 mL in a 250 mL 441

Erlenmeyer flask) and maintained at 25°C, by shaking at 80 revelations per minute, in 442

the dark, with transfer to fresh medium every two weeks. As adventitious roots grew, 443

the size of the flask and volume of liquid medium were adjusted up to 500 mL in a 2 L 444

Fernbach flask. 445

446

Metabolite Extraction from Adventitious Root Cultures 447

448

Solid materials from adventitious root cultures were washed with water (volume 449

equivalent to wet weight of material), freeze-dried for 3 d (Lyph-Lock 12L, LabConco, 450

Kansas City, MO, USA), and the resulting dry matter stored at -80°C until further use. 451

Aliquots of 50 ± 0.5 mg were transferred to receptacles of a MM01 Dry Mill (Retsch, 452



17

Newtown, PA, USA) and further homogenized by ball shaking for 45 s at a rate of 20 453

shakes per second. The homogenate was then transferred to glass test tubes with 454

teflon-lined caps. Each sample was extracted 4 times at 23°C with 5 mL acetone for 30 455

min (Ultrasound Bath FS30H, Fischer Scientific). After each extraction step, samples 456

were centrifuged at 3,000 x g for 5 min and the combined supernatants collected in a 457

separate glass test tube. The solvent was removed under reduced pressure (EZ Bio, 458

GeneVac, Stone Ridge, NY, USA) and the remaining residue re-dissolved in 1 mL 90 % 459

acetonitrile containing 10 μg/mL 9-anthracene carboxylic acid as internal standard. 460

Prior to further analysis, samples were passed through syringe filters 461

(polytetrafluoroethylene; 0.22 μM pore size) and stored at 4°C for a maximum of 2 d. 462

A medium aliquot of adventitious root cultures (generally 5 mL) was extracted twice 463

with ethyl acetate (2 mL each time), by thorough mixing for 1 min at 23°C. The 464

combined organic extracts (4 mL) were extracted against water (2 mL), transferred to a 465

new glass vial, and the solvent was removed under reduced pressure (EZ Bio, 466

GeneVac, Stone Ridge, NY, USA). The residue was re-dissolved in 200 µl 90 % 467

aqueous acetonitrile containing 10 μg/mL 9-anthracene carboxylic acid as internal 468

standard. Prior to further analysis, samples were passed through syringe filters 469

(polypropylene; 0.22 μM pore size) and stored at 4°C for a maximum of 2 d. 470

471

HPLC–QTOF–MS and MS/MS Data Acquisition and Method Validation 472

473

The separation of metabolites was almost identical to that described previously 474

(Fischedick et al., 2015). The conditions for metabolite separation were modified 475

slightly: the initial conditions were 70 % solvent A (water with 0.1 % (v/v) formic acid) 476

and 30 % solvent B (acetonitrile with 0.1 % (v/v) formic acid). A linear gradient (flow 477

rate 0.6 mL/min) was used to increase solvent B to 80 % over 35 min, followed by a 478

more rapid gradient to 95 % solvent B at 40 min. The diode array detector was set to 479

record at 219, 254, and 424 nm, with UV/VIS spectra being recorded from 200-500 nm. 480

Mass spectral data were obtained based on the protocols outlined previously 481

(Fischedick et al., 2015) with the following differences: the electrospray ionization 482

source was operated in positive polarity and MS/MS data were acquired with a collision 483



18

energy of 30 eV. Data analysis was performed using the MassHunter software version 484

B.03.01 (Agilent Technologies, Santa Clara, CA, USA). The approach for validating the 485

quantitation of triptolide and celastrol was reported previously for various analytes in 486

plant matrices (Cuthbertson et al., 2013) and only the relevant values are given here: 487

recovery from adventitious roots (n = 3): 85.6 ± 5.6 % for triptolide, 96.8 ± 2.8 % for 488

celastrol; recovery from culture medium (n = 3): 106.6 ± 3.9 % for triptolide, 86.1 ± 2.9 489

% for celastrol; reproducibility of extraction/detection (as relative standard deviation; n = 490

3): 6.5 % (interday) and 0.9 % (intraday) for triptolide, 0.5 % (interday) and 0.6 % 491

(intraday) for celastrol; limit of detection at signal-to-noise ratio of 1 : 5 (n = 3): 0.01 ng 492

for triptolide (MS detection) and 1.0 ng for celastrol (diode array detection at 424 nm); 493

limit of quantitation at signal-to-noise ratio of 1 : 10 (n = 3): 0.05 for triptolide (MS 494

detection) and 10 ng for celastrol (diode array detection at 424 nm); regression equation 495

for calibration curve (n = 3): y = 25938x + 16475 (R2 = 0.989) for triptolide (corrected for 496

matrix effects) and y = 0.9077 x (R2 = 0.999) for celastrol (matrix effects irrelevant for 497

diode array detection); and linear range: 0.05 – 50.0 ng for triptolide and 1.0 – 2,500 ng 498

for celastrol. 499

500

Cloning and Functional Characterization of Terpene Synthases 501

502

Raw data from several transcriptome sequencing projects previously performed with T. 503

regelii tissues (NCBI Short Read Archive accession number SRP075639 (adventitious 504

root cultures)) and data sets available at http://www.medplantrnaseq.org/ (roots and root 505

cultures) were downloaded and a consensus assembly generated using the Trinity 506

(Haas et al., 2013) and TransABySS (Robertson et al., 2010) assemblers. tBLASTn 507

searches with peptide sequences of characterized class I and class II diterpene 508

synthases (Supplemental Table S2) were performed against the T. regelii consensus 509

assembly. RNA was isolated from T. regelii adventitious root cultures (harvested at 10 510

d after transfer to fresh medium) using the Plant RNA Purification reagent (Invitrogen, 511

Carlsbad, CA, USA) and the RNEasy Mini kit (Qiagen, Valencia, CA, USA) according to 512

the manufacturer’s instructions, converted to cDNA using Maxima Reverse 513

Transcriptase (ThermoFisher Scientific, Waltham, MA, USA), and PCR reactions with 514


http://www.medplantrnaseq.org/


19

gene-specific primers were then employed to clone candidate diterpene synthase 515

sequences (details in Supplemental Table S3). RACE-PCR was performed according 516

to the manufacturer’s instructions (SMARTer-RACE 5’/3’ kit, Clontech/TaKaRa, 517

Mountain View, CA, USA). The Gateway® cloning system (Invitrogen, Carlsbad, CA, 518

USA) was used to insert candidate diterpene synthases into vectors of the pGGxC 519

series for functional expression in the E. coli C41 (DE3) (Cyr et al., 2007). Diterpenoids 520

were extracted directly from 50 mL cultures with an equal volume of n-hexanes (Fisher 521

Scientific, Fair Lawn, NJ, USA). The extract was then run through silica gel 60 and 522

magnesium sulfate columns as described elsewhere (Cyr et al., 2007). The eluents 523

were dried under a flow of nitrogen and the residue was dissolved in 200 µl n-hexanes. 524

Aliquots (1 µL) were injected onto an HP-5MS column (30 m length x 0.25 mm 525

diameter; 0.25 µm film thickness; J&W Scientific, distributed through Agilent, Santa 526

Clara, CA, USA) of a 6890N gas chromatograph (operated in splitless mode) coupled to 527

a 5973 inert mass selective detector (Agilent, Santa Clara, CA, USA). Settings: helium 528

as carrier gas at a flow rate of 1 mL min-1; inlet temperature set to 250°C; oven program 529

with 50°C for 1 min, first linear gradient to 300°C at 7°C min-1, second linear gradient to 530

330°C with 20°C min-1, and final hold of 5 min; transfer line set to 180°C; electron 531

impact spectra recorded at 70 eV with MS data collection from m/z 50 to 450. Peaks 532

were identified based on comparisons of retention time and MS fragmentation patterns 533

with those of authentic standards. The functional evaluation of monoterpene synthases 534

was performed according to Srividya et al. (2015). 535

All genes characterized as part of this study have been deposited in GenBank with 536

the following accession numbers: KX533964 (TrTPS2); KX533965 (TrTPS1); KX533966 537

(TrTPS13); KX533967 (TrTPS14); KX533968 (TrTPS15), KY856993 (TrTPS3), 538

KY856994 (TrTPS4), KY856995 (TrTPS8), and KY856996 (TrTPS7). 539

540

ACKNOWLEDGMENTS 541

542

This work was supported by the National Institutes of Health (award number 543

RC2GM092561 to B.M.L. and GM076324 to R.J.P.) and Hatch funds from the 544

Agricultural Research Center at Washington State University (to B.M.L.). The authors 545



20

thank Sean R. Johnson for generating sequence assemblies with publicly available 546

transcriptome data sets. The authors would also like to thank Washington State 547

University’s NMR Core Facility for access to instruments and expert support from Dr. 548

Greg Helms. 549

Legends to Tables, Figures and Supplemental Data 550

551

Table 1. Metabolites detected by HPLC-QTOF-MS in the medium of Tripterygium 552

adventitious root cultures. MS/MS spectra were acquired with a fragmentation voltage 553

of 30 eV. 554

555

Table 2. Product distribution in in vitro assays with TrTPS3 and TrTPS4. 556

557

Fig. 1. Accumulation of abietane diterpenoids in Tripterygium adventitious root cultures. 558

HPLC-QTOF-MS traces (total ion current) obtained with ethanolic extracts of cultured 559

roots (A) and culture medium (B). The identity of the metabolite peaks 1-13 is given in 560

Table 1 (peak #13 (celastrol) is highlighted in blue because it is the only metabolite that 561

does not belong to the abietane diterpenoids). Fernbach flasks with root culture 562

photographed from below (C); the arm of the person holding the 4 L flask is visible at 563

the bottom right and serves as a size comparison. Concentrations of triptolide and 564

celastrol in cultured roots and culture medium (D). 565

566

Fig. 2. Putative biosynthetic pathway toward abietane diterpenoids in Tripterygium. 567

Metabolites found in this study are boxed. The numbering of the carbons forming the 568

abietane skeleton is indicated using dehydroabietic acid as an example. Reactions 569

requiring multiple steps are indicated by dotted arrows. The numbering of metabolites 570

is the same as in Fig. 1. 571

572

Fig. 3. Molecular phylogeny of dicot (di)terpene synthases, with an emphasis on those 573

from the genus Tripterygium. Functionally characterized terpene synthases from T. 574

regelii are highlighted by grey boxes. The analysis was carried out using the Maximum 575

Likelihood method based on a matrix-based model (Jones et al., 1992). The bootstrap 576



21

consensus tree inferred from 1,000 replications (Felsenstein, 1985) is taken to 577

represent the evolutionary history of the taxa analyzed. Branches corresponding to 578

partitions reproduced in less than 50 % bootstrap replicates are collapsed. Initial tree(s) 579

for the heuristic search were obtained automatically by applying neighbor-joining and 580

BioNJ algorithms to a matrix of pairwise distances, and then selecting the topology with 581

superior log likelihood value. The analysis involved 77 amino acid sequences. All 582

positions containing gaps and missing data were eliminated. There were a total of 94 583

positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar 584

et al., 2016). 585

586

Fig. 4. Modular in vivo assay results obtained with Tripterygium class II diterpene 587

synthase candidates of the TPS-c subfamily. Left to right: constructs (selectable 588

markers in gray boxes; Chlor, chloramphenicol; Carb, carbenicillin; MDS, miltiradiene 589

synthase (from Salvia miltiorrhiza); eKS, ent-kaurene synthase (from Arabidopsis 590

thaliana)), GC-MS chromatograms, mass spectra of assay products and mass spectra 591

of authentic standards. TrTPS2 was identified as a (+)-copalyl diphosphate synthase 592

(A), while TrTPS1 showed activity as an ent-copalyl diphosphate synthase (B). 593

594

Fig. 5. Modular in vivo assay results obtained with Tripterygium class I diterpene 595

synthase candidates of the TPS-e/f family. Left to right: constructs (selectable markers 596

in gray boxes; Chlor, chloramphenicol; Carb, carbenicillin), GC-MS chromatograms, 597

mass spectra of assay products and mass spectra of authentic standards. The 598

following gene combinations were tested: (A) ent-CPS/TrTPS13, (B) ent-CPS/TrTPS14, 599

(C) (+)-CPS/TrTPS13, (D) syn-CPS/TrTPS13, (E) ent-CPS/TrTPS15, (F) (+)-600

CPS/TrTPS15, (G) syn-CPS/TrTPS15. 601

602

Fig. 6. Modular in vivo assay results obtained with TrTPS8, a class I diterpene 603

synthase candidate of the TPS-a subfamily. Left to right: constructs (selectable markers 604

in gray boxes; Chlor, chloramphenicol; Carb, carbenicillin), GC-MS chromatograms, 605

mass spectra of assay products and mass spectra of authentic standards. The 606



22

following gene combinations were tested: (A) ent-CPS/TrTPS8, (B) (+)-CPS/TrTPS8, 607

(C) syn-CPS/TrTPS8. 608

609

SUPPLEMENTAL DATA 610

Supplemental Fig. S1. Accumulation of abietane diterpenoids in the medium of T. 611

regelii adventitious root cultures. 612

Supplemental Fig. S2. 1H-NMR spectra of abietane diterpenoids. 613

Supplemental Fig. S3. Sequences of T. regelii diterpene synthases of the TPS-c 614

family. 615

Supplemental Fig. S4. Sequences of T. regelii diterpene synthases of the TPS-e/f 616

family. 617

Supplemental Fig. S5. Sequences of T. regelii diterpene synthases of the TPS-b 618

family. 619

Supplemental Fig. S6. Modular in vivo combination of T. regelii class II (TrTPS2) and 620

class I (TrTPS13) diterpene synthases. 621

Supplemental Fig. S7. Proposed mechanism for T. regelii monoterpene synthases. 622

OPP denotes the diphosphate moiety. 623

Supplemental Fig. S8. Comparison of expression levels of candidate diterpene 624

synthase genes in T. regelii adventitious root cultures (ARC) and roots based on RNA 625

Sequence by Expectation-Maximization analysis. 626

Supplemental Fig. S9. Alignment of diterpene synthase sequences included in the 627

phylogenetic analysis. 628

Supplemental Table S1. Changes in triptolide and celastrol concentrations in T. regelii 629

adventitious root cultures following various elicitor treatments. 630

Supplemental Table S2. List of dicot diterpene synthase protein sequences used in the 631

phylogenetic analysis. Physcomitrella patens CPS/KS serves as outgroup. 632

Supplemental Table S3. List of primers used in T. regelii terpene synthase gene 633

cloning. 634

Supplemental Methods and Data File S1. Purification and characterization of 635

diterpenoids from T. regelii adventitious root cultures. 636



23

637

638

Supplemental Fig. S1. Accumulation of abietane diterpenoids in the medium of T. 639

regelii adventitious root cultures. (A) HPLC-QTOF-MS chromatogram of organic extract 640

and corresponding integration table (B). Peaks for abietane diterpenoids are indicated 641

with arrows. (C) Detection of abietane hydrocarbon intermediates by GC-MS. 642

643

Supplemental Fig. S2. 1H-NMR spectra of abietane diterpenoids. (A) Triptophenolide, 644

(B) triptolide, (C) tripdiolide, (D) dehydroabietic acid. 645

646

Supplemental Fig. S3. Sequences of T. regelii diterpene synthases of the TPS-c 647

family. The aspartate-rich DXDD motif is indicated by a black square. 648

649

Supplemental Fig. S4. Sequences of T. regelii diterpene synthases of the TPS-e/f 650

family. The solid triangle indicates the predicted cleavage site of the plastidial targeting 651

sequence for TrTPS13 and TrTPS14. The hollow triangle indicates the same for 652

TrTPS15. The black square marks the aspartate-rich DDXXD motif. 653

Supplemental Fig. S5. Sequences of T. regelii diterpene synthases of the TPS-b 654

family. The solid triangle indicates the predicted cleavage site of the plastidial targeting 655

sequence for TrTPS8. The patterned and hollow triangles indicate the same for 656

TrTPS3/TrTPS4 and TrTPS7, respectively. The black square marks the aspartate-rich 657

DDXXD motif. 658

Supplemental Fig. S6. Modular in vivo combination of T. regelii class II (TrTPS2) and 659

class I (TrTPS13) diterpene synthases. (A) plasmid constructs, (B) chromatogram of 660

products, (C) mass spectra of peaks shown in (B), (D) mass spectra of authentic 661

standards. 662

663

Supplemental Fig. S7. Proposed mechanism for T. regelii monoterpene synthases. 664

OPP denotes the diphosphate moiety. 665

666



24

Supplemental Fig. S8. Comparison of expression levels of candidate diterpene 667

synthase genes in T. regelii adventitious root cultures (ARC) and roots based on RNA 668

Sequence by Expectation-Maximization analysis. 669

Supplemental Fig. S9. Alignment of diterpene synthase sequences included in the 670

phylogenetic analysis. 671

672

Supplemental Table S1. Changes in triptolide and celastrol concentrations in T. regelii 673

adventitious root cultures following various elicitor treatments. 674

675

Supplemental Table S2. List of dicot diterpene synthase protein sequences used in the 676

phylogenetic analysis. Physcomitrella patens CPS/KS serves as outgroup. 677

678

Supplemental Table S3. List of primers used in T. regelii terpene synthase gene 679

cloning. 680

681

Supplemental Methods and Data File S1. Purification and characterization of 682

diterpenoids from T. regelii adventitious root cultures. 683



25

684



26

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28

Tanaka N, Ooba N, Duan HQ, Takaishi Y, Nakanishi Y, Bastow K, Lee KH (2004) Kaurane and 785 abietane diterpenoids from Tripterygium doianum (Celastraceae). Phytochemistry 65: 2071-786 2076 787

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809

810



1

Table 1. Metabolites detected by HPLC-QTOF-MS in the medium of Tripterygium adventitious root cultures. MS/MS spectra were acquired with a

fragmentation voltage of 30 eV.

Metabolite RT Formula [M+H]

+ Error MS/MS signal patterns NMR- Auth. strd. Tentative

(Number in Fig. 1b) [min] (Hill notation) Calculated Observed [Δppm] (% relative abundance) confirmed confirmed ID

Tripdiolide (1) 1.8 C20H24O7 359.1489 359.1492 0.82 359 (100), 171 (45.1), 157 (60.3), 155 (59.7), 143 (54.3), 129 (44.8),

128 (51.9), 105 (50.0) X

Triptolide (2) 4.6 C20H24O6 361.1646 361.1647 0.31 361 (100), 157 (45.4), 145 (54.9), 143 (66.0), 142 (42.2), 131 (42.8), 119 (30.3), 105 (53.5)

X

11-O-β-D-Glucopyranosyl neotriptophenolide (3)

6.9 C27H36O9 505.2432 505.2433 0.13 343.2 (100), 301 (21.8), 297.2 (32.7), 255 (60.2), 205 (25.6), 179 (18.6), 163 (13.7), 127 (15.2)

X

Diterpenoid (unknown) (4) 9.4 C20H22O6 359.1489 359.1502 3.66 359 (100), 171 (14.5), 145 (19.2), 143 (27.8), 131 (15.8), 128 (13.0), 119 (13.5), 105 (26.2)

Diterpenoid (unknown) (5) 10.6 C21H26O3 327.1955 327.1938 5.16 203 (45.4), 161 (100), 147 (65.9), 135 (52.4), 133 (35.3), 131 (44.2), 111 (44.0), 105 (91.0)

Neotriptophenolide (6) 13.3 C21H26O4 343.1904 343.1902 0.48 283 (21.5), 237 (100), 223 (28.6), 222 (52.4), 209 (20.7), 197 (24.4), 185 (41.6), 111 (22.1)

X

Diterpenoid (unknown) (7) 14.9 C21H26O4 343.1904 343.1896 2.29 297 (24.5), 257 (22.7), 255 (100), 243 (22.9), 240 (42.1), 205 (62.8), 177 (28.6), 163 (57.5)

Triptophenolide (8) 15.2 C20H24O3 313.1798 313.1797 0.25 253 (18.7), 225 (100), 197 (19.1), 185 (23.5), 183 (47.8), 181 (25.8), 171 (25.8), 147 (22.2)

X X

Dehydroabietic acid (9) 18.1 C20H28O2 301.2162 301.2170 2.48 215 (90.4), 175 (61.3), 173 (71.2), 171 (60.2), 159 (68.7), 147 (94.9), 145 (51.7), 133 (100)

X X

Triptophenolide methyl ether (10)

18.8 C21H26O3 327.1982 327.1951 1.13 239 (100), 225 (24.3), 224 (28.9), 207 (24.9), 197 (43.8), 185 (32.4), 161 (26, 147 (40.7)

X

Triptinin A (11) 19.1 C21H28O3 329.2111 329.2116 1.35 255 (35.1), 237 (28.5), 189 (27.7), 175 (33.8), 171.8 (42.4), 149 (100), 147 (51.3), 133 (52.3)

X

Diterpenoid (unknown) (12)

21.9 C21H30O2 315.2324 315.2319 1.64 229.2 (56.6), 189 (43.2), 187 (65.1), 185 (64.0), 173 (46.4), 161 (100), 159 (41), 147 (73.3)

Celastrol (13) 23.0 C29H38O4 451.2843 451.2851 1.74 215 (10.3), 201 (100), 200 (5.2) X X



2

Table 2. Product distribution in in vitro assays with TrTPS3 and TrTPS4.

Monoterpene product

Retention time [min]

Geranyl diphosphate as substrate [% of total]

Linalyl diphosphate as substrate [% of total]

TrTPS3 TrTPS4 TrTPS3 TrTPS4

Myrcene 17.135 - 4.39

1.45 1.57

(-)-Limonene 20.567 7.77 - 2.05 2.06

(+)-Limonene 20.823 - - 1.87 1.85

Terpinolene 23.734 - - 2.18 2.22

(-)-Linalool 32.171 35.60 40.38 21.88 23.81

(+)-Linalool 32.327 44.77 46.04 28.32 29.93

(-)-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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Burke CC, Croteau RB (2002) Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificityof geranylgeranyl diphosphate synthase to produce geranyl diphosphate. J Biol Chem 277, 3141-3149


Brinker AM, Ma J, Lipsky PE, Raskin, I (2007) Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae)Phytochemistry 68: 1819-1819


Chen YZ, Gao Q, Zhao XZ, Chen XM, Zhang F, Chen J, Xu CG, Sun LL, Mei CL (2010) Meta-analysis of Tripterygium wilfordii Hook F inthe immunosuppressive treatment of IgA nephropathy. Internal Med 49: 2049-2055


Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes for specializedmetabolism that is highly diversified throughout the kingdom. Plant J 66: 212-229


Chugh R, Sangwan V, Patil SP, Dudeja V, Dawra RK, Banerjee S, Schumacher RJ et al (2012) A preclinical evaluation of minnelide as atherapeutic agent against pancreatic cancer. Sci Transl Med 4: 156ra139


Cuthbertson DJ, Johnson SR, Piljac-Zegarac J, Kappel J, Schäfer S, Wüst M, Ketchum et al (2013) Accurate mass-time tag library forLC/MS-based metabolite profiling of medicinal plants. Phytochemistry 91: 187-197


Cyr A, Wilderman PR, Determan M, Peters RJ (2007) A modular approach for facile biosynthesis of labdane-related diterpenes. J AmChem Soc 129: 6684-6685


Duan HQ, Takaishi Y, Momota H, Ohmoto Y, Taki T, Jia YF, Li D (1999) Immunosuppressive diterpenoids from Tripterygium wilfordii. JNat Prod 62: 1522-1525


Duan HQ, Takaishi Y, Momota H, Ohmoto Y, Taki T, Tori M, Takaoka S, Jia YF, Li D (2001) Immunosuppressive terpenoids from extractsof Tripterygium wilfordii. Tetrahedron 57: 8413-8424


Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Fischedick JT, Johnson SR, Ketchum REB, Croteau RB, Lange BM (2015) NMR spectroscopic search module for Spektraris, an onlineresource for plant natural product identification - Taxane diterpenoids from Taxus x media cell suspension cultures as a case study.Phytochemistry 113: 87-95



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http://www.ncbi.nlm.nih.gov/pubmed?term=Brinker AM%2C Ma J%2C Lipsky PE%2C Raskin%2C I %282007%29 Medicinal chemistry and pharmacology of genus Tripterygium %28Celastraceae%29 Phytochemistry 68%3A 1819%2D1819&dopt=abstract

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Gao W, Hillwig ML, Huang L, Cui G, Wang X, Kong J, Yang B, Peters RJ (2009) A functional genomics approach to tanshinonebiosynthesis provides stereochemical insights. Org Lett 11: 5170-5173


Geisler K, Jensen NB, Yuen MM, Madilao L, Bohlmann J (2016) Modularity of conifer diterpene resin acid biosynthesis: P450 enzymesof different CYP720B clades use alternative substrates and converge on the same products. Plant Physiol 171: 152-164


Gonzalez MA, Perez-Guaita D, Correa-Royero J, Zapata B, Agudelo L, Mesa-Arango A, Betancur-Galvis L (2010) Synthesis andbiological evaluation of dehydroabietic acid derivatives. Eur J Med Chem 45: 811-816


Guo L, Duan L, Liu K, Liu EH, Li P (2014) Chemical comparison of Tripterygium wilfordii and Tripterygium hypoglaucum based onquantitative analysis and chemometrics methods. J Pharm Biomed 95: 220-228


Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB et al (2013) De novo transcript sequencereconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protocols 8: 1494-1512


Hamberger B, Ohnishi T, Hamberger B, Seguin A, Bohlmann J (2011) Evolution of diterpene metabolism: Sitka spruce CYP720B4catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects Plant Physiol 157: 1677-1695


Hansen NL, Heskes AM, Hamberger B, Olsen CE, Hallström BM, Andersen-Ranberg J, Hamberger B (2017) The terpene synthase genefamily in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily. Plant J89: 429-441


Helmstädter A (2013) Tripterygium wilfordii Hook f - how a traditional Taiwanese medicinal plant found its way to the West. Pharmazie68: 643-646


Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comp. Appl. Biosci8: 275-282


Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis Version 7.0 for bigger datasets. Mol. Biol. Evol.33: 1870-1874


Kutney JP, Han K (1996) Studies with plant-cell cultures of the Chinese herbal plant, Tripterygium wilfordii Isolation andcharacterization of diterpenes. Rec Trav Chim Pays Bas 115: 77-93


Kutney JP, Hewitt GM, Kurihara T, Salisbury PJ, Sindelar RD, Stuart KL, Townsley PM, Chalmers, WT, Jacoli GG (1981) Cyto-toxicditerpenes triptolide, tripdiolide, and cyto-toxic triterpenes from tissue-cultures of Tripterygium wilfordii. Can J Chem 59: 2677-2683


Kutney JP, Hewitt GM, Lee G, Piotrowska K, Roberts M, Rettig SJ (1992) Studies with tissue-cultures of the Chinese herbal plant,Tripterygium-wilfordii - Isolation of metabolites of interest in rheumatoid-arthritis, immunosuppression, and male contraceptive activity.https://plantphysiol.orgDownloaded on December 16, 2020. - Published by

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

http://www.ncbi.nlm.nih.gov/pubmed?term=Gao W%2C Hillwig ML%2C Huang L%2C Cui G%2C Wang X%2C Kong J%2C Yang B%2C Peters RJ %282009%29 A functional genomics approach to tanshinone biosynthesis provides stereochemical insights%2E Org Lett 11%3A 5170%2D5173&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gao W, Hillwig ML, Huang L, Cui G, Wang X, Kong J, Yang B, Peters RJ (2009) A functional genomics approach to tanshinone biosynthesis provides stereochemical insights. Org Lett 11: 5170-5173

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gao&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=A functional genomics approach to tanshinone biosynthesis provides stereochemical insights&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gao&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Geisler K%2C Jensen NB%2C Yuen MM%2C Madilao L%2C Bohlmann J %282016%29 Modularity of conifer diterpene resin acid biosynthesis%3A P450 enzymes of different CYP720B clades use alternative substrates and converge on the same products%2E Plant Physiol 171%3A 152%2D164&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Geisler K, Jensen NB, Yuen MM, Madilao L, Bohlmann J (2016) Modularity of conifer diterpene resin acid biosynthesis: P450 enzymes of different CYP720B clades use alternative substrates and converge on the same products. Plant Physiol 171: 152-164

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http://scholar.google.com/scholar?as_q=Modularity of conifer diterpene resin acid biosynthesis: P450 enzymes of different CYP720B clades use alternative substrates and converge on the same products&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Gonzalez MA%2C Perez%2DGuaita D%2C Correa%2DRoyero J%2C Zapata B%2C Agudelo L%2C Mesa%2DArango A%2C Betancur%2DGalvis L %282010%29 Synthesis and biological evaluation of dehydroabietic acid derivatives%2E Eur J Med Chem 45%3A 811%2D816&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Gonzalez MA, Perez-Guaita D, Correa-Royero J, Zapata B, Agudelo L, Mesa-Arango A, Betancur-Galvis L (2010) Synthesis and biological evaluation of dehydroabietic acid derivatives. Eur J Med Chem 45: 811-816

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gonzalez&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Synthesis and biological evaluation of dehydroabietic acid derivatives&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Synthesis and biological evaluation of dehydroabietic acid derivatives&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gonzalez&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Guo L%2C Duan L%2C Liu K%2C Liu EH%2C Li P %282014%29 Chemical comparison of Tripterygium wilfordii and Tripterygium hypoglaucum based on quantitative analysis and chemometrics methods%2E J Pharm Biomed 95%3A 220%2D228&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Guo L, Duan L, Liu K, Liu EH, Li P (2014) Chemical comparison of Tripterygium wilfordii and Tripterygium hypoglaucum based on quantitative analysis and chemometrics methods. J Pharm Biomed 95: 220-228

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Guo&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Chemical comparison of Tripterygium wilfordii and Tripterygium hypoglaucum based on quantitative analysis and chemometrics methods&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Chemical comparison of Tripterygium wilfordii and Tripterygium hypoglaucum based on quantitative analysis and chemometrics methods&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Guo&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Haas BJ%2C Papanicolaou A%2C Yassour M%2C Grabherr M%2C Blood PD%2C Bowden J%2C Couger MB et al %282013%29 De novo transcript sequence reconstruction from RNA%2Dseq using the Trinity platform for reference generation and analysis%2E Nat Protocols 8%3A 1494%2D1512&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB et al (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protocols 8: 1494-1512

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http://scholar.google.com/scholar?as_q=De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Haas&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hamberger B%2C Ohnishi T%2C Hamberger B%2C Seguin A%2C Bohlmann J %282011%29 Evolution of diterpene metabolism%3A Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects Plant Physiol 157%3A 1677%2D1695&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hamberger B, Ohnishi T, Hamberger B, Seguin A, Bohlmann J (2011) Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects Plant Physiol 157: 1677-1695

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http://www.ncbi.nlm.nih.gov/pubmed?term=Hansen NL%2C Heskes AM%2C Hamberger B%2C Olsen CE%2C Hallstr�m BM%2C Andersen%2DRanberg J%2C Hamberger B %282017%29 The terpene synthase gene family in Tripterygium wilfordii harbors a labdane%2Dtype diterpene synthase among the monoterpene synthase TPS%2Db subfamily%2E Plant J 89%3A 429%2D441&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hansen NL, Heskes AM, Hamberger B, Olsen CE, Hallstr�m BM, Andersen-Ranberg J, Hamberger B (2017) The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily. Plant J 89: 429-441

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http://www.ncbi.nlm.nih.gov/pubmed?term=Helmst�dter A %282013%29 Tripterygium wilfordii Hook f %2D how a traditional Taiwanese medicinal plant found its way to the West%2E Pharmazie 68%3A 643%2D646&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Helmst�dter A (2013) Tripterygium wilfordii Hook f - how a traditional Taiwanese medicinal plant found its way to the West. Pharmazie 68: 643-646

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Helmst�dter&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Jones DT%2C Taylor WR%2C Thornton JM %281992%29 The rapid generation of mutation data matrices from protein sequences%2E Comp%2E Appl%2E Biosci 8%3A 275%2D282&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comp. Appl. Biosci 8: 275-282

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http://www.ncbi.nlm.nih.gov/pubmed?term=Kumar S%2C Stecher G%2C Tamura K %282016%29 MEGA7%3A Molecular evolutionary genetics analysis Version 7%2E0 for bigger datasets%2E Mol%2E Biol%2E Evol%2E 33%3A 1870%2D1874&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis Version 7.0 for bigger datasets. Mol. Biol. Evol. 33: 1870-1874

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http://www.ncbi.nlm.nih.gov/pubmed?term=Kutney JP%2C Han K %281996%29 Studies with plant%2Dcell cultures of the Chinese herbal plant%2C Tripterygium wilfordii Isolation and characterization of diterpenes%2E Rec Trav Chim Pays Bas 115%3A 77%2D93&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kutney JP, Han K (1996) Studies with plant-cell cultures of the Chinese herbal plant, Tripterygium wilfordii Isolation and characterization of diterpenes. Rec Trav Chim Pays Bas 115: 77-93

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http://scholar.google.com/scholar?as_q=Studies with plant-cell cultures of the Chinese herbal plant, Tripterygium wilfordii Isolation and characterization of diterpenes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Studies with plant-cell cultures of the Chinese herbal plant, Tripterygium wilfordii Isolation and characterization of diterpenes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kutney&as_ylo=1996&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kutney JP%2C Hewitt GM%2C Kurihara T%2C Salisbury PJ%2C Sindelar RD%2C Stuart KL%2C Townsley PM%2C Chalmers%2C WT%2C Jacoli GG %281981%29 Cyto%2Dtoxic diterpenes triptolide%2C tripdiolide%2C and cyto%2Dtoxic triterpenes from tissue%2Dcultures of Tripterygium wilfordii%2E Can J Chem 59%3A 2677%2D2683&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kutney JP, Hewitt GM, Kurihara T, Salisbury PJ, Sindelar RD, Stuart KL, Townsley PM, Chalmers, WT, Jacoli GG (1981) Cyto-toxic diterpenes triptolide, tripdiolide, and cyto-toxic triterpenes from tissue-cultures of Tripterygium wilfordii. Can J Chem 59: 2677-2683


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http://scholar.google.com/scholar?as_q=Cyto-toxic diterpenes triptolide, tripdiolide, and cyto-toxic triterpenes from tissue-cultures of Tripterygium wilfordii&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kutney&as_ylo=1981&as_allsubj=all&hl=en&c2coff=1


Can J Chem 70, 1455-1480Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kutney JP, Samija MD, Hewitt GM, Bugante EC, Gu H (1993) Antiinflammatory oleanane triterpenes from Tripterygium-wilfordii cell-suspension cultures by fungal elicitation. Plant Cell Rep 12: 356-359


Liu YF, Tu SH, Gao WN, Wang Y, Liu PL, Hu YH, Dong H (2013) Extracts of Tripterygium wilfordii Hook F in the treatment of rheumatoidarthritis: A systemic review and meta-analysis of randomised controlled trials. Evid-Based Compl Alt Med, Article ID 410793


Meng CC, Zhu HC, Song HM, Wang ZM, Huang GH, Li DF, Ma J (2014) Targets and molecular mechanisms of triptolide in cancertherapy. Chin J Cancer Res 26: 622-626


Miao GP, Zhu CS, Feng JT, Han J, Song XW, Zhang X (2013) Aggregate cell suspension cultures of Tripterygium wilfordii Hook f fortriptolide, wilforgine, and wilforine production. Plant Cell Tiss Org Cult 112: 109-116


Miao GP, Zhu CS, Yang YQ, Feng MX, Ma ZQ, Feng JT, Zhang X (2014) Elicitation and in situ adsorption enhanced secondarymetabolites production of Tripterygium wilfordii Hook f adventitious root fragment liquid cultures in shake flask and a modified bubblecolumn bioreactor. Bioproc Biosyst Eng 37: 641-650


Nakano K, Oose Y, Takaishi Y (1997) A novel epoxy-triterpene and nortriterpene from callus cultures of Tripterygium wilfordii.Phytochemistry 46: 1179-1182


Nakano K, Yoshida C, Furukawa W, Takaishi Y, Shishido K (1998) Terpenoids in transformed root culture of Tripterygium wilfordii.Phytochemistry 49: 1821-1824


Ro DK, Arimura GL, Lau SYW, Piers E, Bohlmann J (2005) Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is amultifunctional, multisubstrate cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102: 8060-8065


Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K et al (2010) De novo assembly and analysis of RNA-seqdata. Nat Methods 7: 909-U962


Srividya N, Davis E M, Croteau R B, Lange BM (2015) Functional Analysis of (4S)-limonene synthase mutants reveals determinants ofcatalytic outcome in a model monoterpene synthase. Proc Natl Acad Sci 112: 3332-3337


Tanaka N, Ooba N, Duan HQ, Takaishi Y, Nakanishi Y, Bastow K, Lee KH (2004) Kaurane and abietane diterpenoids from Tripterygiumdoianum (Celastraceae). Phytochemistry 65: 2071-2076


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Pubmed: Author and Title https://plantphysiol.orgDownloaded on December 16, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

http://www.ncbi.nlm.nih.gov/pubmed?term=Kutney JP%2C Hewitt GM%2C Lee G%2C Piotrowska K%2C Roberts M%2C Rettig SJ %281992%29 Studies with tissue%2Dcultures of the Chinese herbal plant%2C Tripterygium%2Dwilfordii %2D Isolation of metabolites of interest in rheumatoid%2Darthritis%2C immunosuppression%2C and male contraceptive activity%2E Can J Chem 70%2C 1455%2D1480&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kutney JP, Hewitt GM, Lee G, Piotrowska K, Roberts M, Rettig SJ (1992) Studies with tissue-cultures of the Chinese herbal plant, Tripterygium-wilfordii - Isolation of metabolites of interest in rheumatoid-arthritis, immunosuppression, and male contraceptive activity. Can J Chem 70, 1455-1480


http://scholar.google.com/scholar?as_q=Studies with tissue-cultures of the Chinese herbal plant, Tripterygium-wilfordii - Isolation of metabolites of interest in rheumatoid-arthritis, immunosuppression, and male contraceptive activity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Studies with tissue-cultures of the Chinese herbal plant, Tripterygium-wilfordii - Isolation of metabolites of interest in rheumatoid-arthritis, immunosuppression, and male contraceptive activity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kutney&as_ylo=1992&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kutney JP%2C Samija MD%2C Hewitt GM%2C Bugante EC%2C Gu H %281993%29 Antiinflammatory oleanane triterpenes from Tripterygium%2Dwilfordii cell%2Dsuspension cultures by fungal elicitation%2E Plant Cell Rep 12%3A 356%2D359&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kutney JP, Samija MD, Hewitt GM, Bugante EC, Gu H (1993) Antiinflammatory oleanane triterpenes from Tripterygium-wilfordii cell-suspension cultures by fungal elicitation. Plant Cell Rep 12: 356-359


http://scholar.google.com/scholar?as_q=Antiinflammatory oleanane triterpenes from Tripterygium-wilfordii cell-suspension cultures by fungal elicitation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Antiinflammatory oleanane triterpenes from Tripterygium-wilfordii cell-suspension cultures by fungal elicitation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kutney&as_ylo=1993&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Liu YF%2C Tu SH%2C Gao WN%2C Wang Y%2C Liu PL%2C Hu YH%2C Dong H %282013%29 Extracts of Tripterygium wilfordii Hook F in the treatment of rheumatoid arthritis%3A A systemic review and meta%2Danalysis of randomised controlled trials%2E Evid%2DBased Compl Alt Med%2C Article ID 410793&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Liu YF, Tu SH, Gao WN, Wang Y, Liu PL, Hu YH, Dong H (2013) Extracts of Tripterygium wilfordii Hook F in the treatment of rheumatoid arthritis: A systemic review and meta-analysis of randomised controlled trials. Evid-Based Compl Alt Med, Article ID 410793

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CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zeng F, Wang W, Guan SH, Cheng CR, Yang M, Avula B, Khan IA, Guo DA (2013) Simultaneous quantification of 18 bioactiveconstituents in Tripterygium wilfordii using liquid chromatography-electrospray ionization-mass spectrometry. Planta Med 79: 797-805


Zerbe P, Hamberger B, Yuen MM, Chiang A, Sandhu HK, Madilao LL, Nguyen A Hamberger B, Bach SS, Bohlmann J (2013) Genediscovery of modular diterpene metabolism in nonmodel systems. Plant Physiol 162, 1073-1091


Zhou ZL, Yang YX, Ding J, Li YC, Miao ZH (2012) Triptolide: structural modifications, structure-activity relationships, bioactivities,clinical development and mechanisms. Nat Prod Rep 29: 457-475


Zhu B, Wang Y, Jardine M, Jun M, Lv JC, Cass A, Liyanage T et al (2013) Tripterygium preparations for the treatment of CKD: Asystematic review and meta-analysis. Am J Kidney Dis 62: 515-530


Zhu CS, Miao GP, Guo J, Huo YB, Zhang X, Xie JH, Feng JT (2014) Establishment of Tripterygium wilfordii Hook f hairy root culture andoptimization of its culture conditions for the production of triptolide and wilforine. J Microbiol Biotechnol 24: 823-834


Zi J, Peters R J (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamieaceae. Org Biomol Chem11: 7650-7652


Zi JC, Mafu S, Peters RJ (2014) To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism. Annu Rev Plant Biol65: 259-286



http://search.crossref.org/?page=1&rows=2&q=Yamaguchi S, Sun T, Kawaide H, Kamiya Y (1998) The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis. Plant Physiol 116: 1271-1278

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http://www.ncbi.nlm.nih.gov/pubmed?term=Zeng F%2C Wang W%2C Guan SH%2C Cheng CR%2C Yang M%2C Avula B%2C Khan IA%2C Guo DA %282013%29 Simultaneous quantification of 18 bioactive constituents in Tripterygium wilfordii using liquid chromatography%2Delectrospray ionization%2Dmass spectrometry%2E Planta Med 79%3A 797%2D805&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zeng&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Simultaneous quantification of 18 bioactive constituents in Tripterygium wilfordii using liquid chromatography-electrospray ionization-mass spectrometry&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Simultaneous quantification of 18 bioactive constituents in Tripterygium wilfordii using liquid chromatography-electrospray ionization-mass spectrometry&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zeng&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zerbe P%2C Hamberger B%2C Yuen MM%2C Chiang A%2C Sandhu HK%2C Madilao LL%2C Nguyen A Hamberger B%2C Bach SS%2C Bohlmann J %282013%29 Gene discovery of modular diterpene metabolism in nonmodel systems%2E Plant Physiol 162%2C 1073%2D1091&dopt=abstract

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zerbe&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Gene discovery of modular diterpene metabolism in nonmodel systems&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Gene discovery of modular diterpene metabolism in nonmodel systems&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zerbe&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhou ZL%2C Yang YX%2C Ding J%2C Li YC%2C Miao ZH %282012%29 Triptolide%3A structural modifications%2C structure%2Dactivity relationships%2C bioactivities%2C clinical development and mechanisms%2E Nat Prod Rep 29%3A 457%2D475&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhou ZL, Yang YX, Ding J, Li YC, Miao ZH (2012) Triptolide: structural modifications, structure-activity relationships, bioactivities, clinical development and mechanisms. Nat Prod Rep 29: 457-475

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhou&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Triptolide: structural modifications, structure-activity relationships, bioactivities, clinical development and mechanisms&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Triptolide: structural modifications, structure-activity relationships, bioactivities, clinical development and mechanisms&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhu B%2C Wang Y%2C Jardine M%2C Jun M%2C Lv JC%2C Cass A%2C Liyanage T et al %282013%29 Tripterygium preparations for the treatment of CKD%3A A systematic review and meta%2Danalysis%2E Am J Kidney Dis 62%3A 515%2D530&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhu B, Wang Y, Jardine M, Jun M, Lv JC, Cass A, Liyanage T et al (2013) Tripterygium preparations for the treatment of CKD: A systematic review and meta-analysis. Am J Kidney Dis 62: 515-530

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhu&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Tripterygium preparations for the treatment of CKD: A systematic review and meta-analysis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Tripterygium preparations for the treatment of CKD: A systematic review and meta-analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhu&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhu CS%2C Miao GP%2C Guo J%2C Huo YB%2C Zhang X%2C Xie JH%2C Feng JT %282014%29 Establishment of Tripterygium wilfordii Hook f hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine%2E J Microbiol Biotechnol 24%3A 823%2D834&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhu CS, Miao GP, Guo J, Huo YB, Zhang X, Xie JH, Feng JT (2014) Establishment of Tripterygium wilfordii Hook f hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine. J Microbiol Biotechnol 24: 823-834

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhu&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Establishment of Tripterygium wilfordii Hook f hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Establishment of Tripterygium wilfordii Hook f hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhu&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zi J%2C Peters R J %282013%29 Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamieaceae%2E Org Biomol Chem 11%3A 7650%2D7652&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zi J, Peters R J (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamieaceae. Org Biomol Chem 11: 7650-7652

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http://scholar.google.com/scholar?as_q=Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamieaceae&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamieaceae&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zi&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zi JC%2C Mafu S%2C Peters RJ %282014%29 To gibberellins and beyond%21 Surveying the evolution of %28di%29terpenoid metabolism%2E Annu Rev Plant Biol 65%3A 259%2D286&dopt=abstract

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http://scholar.google.com/scholar?as_q=To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zi&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1


biosynthesis of diterpenoids in tripterygium …...2017/07/27 · 97 triptolide, a diterpenoid...

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