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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
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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
36
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
60
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
83
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
268
Proposing a Diterpenoid Biosynthetic Pathway Based On Metabolites that 269
Accumulate in Tripterygium Adventitious Root Cultures 270
271
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
305
Identifying Genes with Roles in Generating Diterpenoid Structural Diversity 306
307
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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14
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
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421
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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
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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
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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
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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
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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
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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
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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
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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
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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
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684
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26
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Helmstädter A (2013) Tripterygium wilfordii Hook f - how a traditional Taiwanese medicinal plant 742 found its way to the West. Pharmazie 68: 643-646 743
Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from 744 protein sequences. Comp. Appl. Biosci 8: 275-282 745
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis 746 Version 7.0 for bigger datasets. Mol. Biol. Evol. 33: 1870-1874 747
Kutney JP, Han K (1996) Studies with plant-cell cultures of the Chinese herbal plant, 748 Tripterygium wilfordii Isolation and characterization of diterpenes. Rec Trav Chim Pays Bas 749 115: 77-93 750
Kutney JP, Hewitt GM, Kurihara T, Salisbury PJ, Sindelar RD, Stuart KL, Townsley PM, 751 Chalmers, WT, Jacoli GG (1981) Cyto-toxic diterpenes triptolide, tripdiolide, and cyto-toxic 752 triterpenes from tissue-cultures of Tripterygium wilfordii. Can J Chem 59: 2677-2683 753
Kutney JP, Hewitt GM, Lee G, Piotrowska K, Roberts M, Rettig SJ (1992) Studies with tissue-754 cultures of the Chinese herbal plant, Tripterygium-wilfordii - Isolation of metabolites of 755 interest in rheumatoid-arthritis, immunosuppression, and male contraceptive activity. Can J 756 Chem 70, 1455-1480 757
Kutney JP, Samija MD, Hewitt GM, Bugante EC, Gu H (1993) Antiinflammatory oleanane 758 triterpenes from Tripterygium-wilfordii cell-suspension cultures by fungal elicitation. Plant 759 Cell Rep 12: 356-359 760
Liu YF, Tu SH, Gao WN, Wang Y, Liu PL, Hu YH, Dong H (2013) Extracts of Tripterygium 761 wilfordii Hook F in the treatment of rheumatoid arthritis: A systemic review and meta-762 analysis of randomised controlled trials. Evid-Based Compl Alt Med, Article ID 410793 763
Meng CC, Zhu HC, Song HM, Wang ZM, Huang GH, Li DF, Ma J (2014) Targets and molecular 764 mechanisms of triptolide in cancer therapy. Chin J Cancer Res 26: 622-626 765
Miao GP, Zhu CS, Feng JT, Han J, Song XW, Zhang X (2013) Aggregate cell suspension 766 cultures of Tripterygium wilfordii Hook f for triptolide, wilforgine, and wilforine production. 767 Plant Cell Tiss Org Cult 112: 109-116 768
Miao GP, Zhu CS, Yang YQ, Feng MX, Ma ZQ, Feng JT, Zhang X (2014) Elicitation and in situ 769 adsorption enhanced secondary metabolites production of Tripterygium wilfordii Hook f 770 adventitious root fragment liquid cultures in shake flask and a modified bubble column 771 bioreactor. Bioproc Biosyst Eng 37: 641-650 772
Nakano K, Oose Y, Takaishi Y (1997) A novel epoxy-triterpene and nortriterpene from callus 773 cultures of Tripterygium wilfordii. Phytochemistry 46: 1179-1182 774
Nakano K, Yoshida C, Furukawa W, Takaishi Y, Shishido K (1998) Terpenoids in transformed 775 root culture of Tripterygium wilfordii. Phytochemistry 49: 1821-1824 776
Ro DK, Arimura GL, Lau SYW, Piers E, Bohlmann J (2005) Loblolly pine 777 abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate 778 cytochrome P450 monooxygenase. Proc Natl Acad Sci USA 102: 8060-8065 779
Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K et al (2010) De 780 novo assembly and analysis of RNA-seq data. Nat Methods 7: 909-U962 781
Srividya N, Davis E M, Croteau R B, Lange BM (2015) Functional Analysis of (4S)-limonene 782 synthase mutants reveals determinants of catalytic outcome in a model monoterpene 783 synthase. Proc Natl Acad Sci 112: 3332-3337 784
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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
Yamaguchi S, Sun T, Kawaide H, Kamiya Y (1998) The GA2 locus of Arabidopsis thaliana 788 encodes ent-kaurene synthase of gibberellin biosynthesis. Plant Physiol 116: 1271-1278 789
Zeng F, Wang W, Guan SH, Cheng CR, Yang M, Avula B, Khan IA, Guo DA (2013) 790 Simultaneous quantification of 18 bioactive constituents in Tripterygium wilfordii using liquid 791 chromatography-electrospray ionization-mass spectrometry. Planta Med 79: 797-805 792
Zerbe P, Hamberger B, Yuen MM, Chiang A, Sandhu HK, Madilao LL, Nguyen A Hamberger B, 793 Bach SS, Bohlmann J (2013) Gene discovery of modular diterpene metabolism in nonmodel 794 systems. Plant Physiol 162, 1073-1091 795
Zhou ZL, Yang YX, Ding J, Li YC, Miao ZH (2012) Triptolide: structural modifications, structure-796 activity relationships, bioactivities, clinical development and mechanisms. Nat Prod Rep 29: 797 457-475 798
Zhu B, Wang Y, Jardine M, Jun M, Lv JC, Cass A, Liyanage T et al (2013) Tripterygium 799 preparations for the treatment of CKD: A systematic review and meta-analysis. Am J Kidney 800 Dis 62: 515-530 801
Zhu CS, Miao GP, Guo J, Huo YB, Zhang X, Xie JH, Feng JT (2014) Establishment of 802 Tripterygium wilfordii Hook f hairy root culture and optimization of its culture conditions for 803 the production of triptolide and wilforine. J Microbiol Biotechnol 24: 823-834 804
Zi J, Peters R J (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid 805 biosynthesis in the Lamieaceae. Org Biomol Chem 11: 7650-7652 806
Zi JC, Mafu S, Peters RJ (2014) To gibberellins and beyond! Surveying the evolution of 807 (di)terpenoid metabolism. Annu Rev Plant Biol 65: 259-286 808
809
810
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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
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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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Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comp. Appl. Biosci8: 275-282
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis Version 7.0 for bigger datasets. Mol. Biol. Evol.33: 1870-1874
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nakano K, Oose Y, Takaishi Y (1997) A novel epoxy-triterpene and nortriterpene from callus cultures of Tripterygium wilfordii.Phytochemistry 46: 1179-1182
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nakano K, Yoshida C, Furukawa W, Takaishi Y, Shishido K (1998) Terpenoids in transformed root culture of Tripterygium wilfordii.Phytochemistry 49: 1821-1824
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yamaguchi S, Sun T, Kawaide H, Kamiya Y (1998) The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellinbiosynthesis. Plant Physiol 116: 1271-1278
Pubmed: Author and Title https://plantphysiol.orgDownloaded on December 16, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zi J, Peters R J (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamieaceae. Org Biomol Chem11: 7650-7652
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zi JC, Mafu S, Peters RJ (2014) To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism. Annu Rev Plant Biol65: 259-286
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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