CYP76 Oxidation Network of Abietane Diterpenes in Lamiaceae

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Biotechnology and Biological Transformations

A CYP76 oxidation network of abietane diterpenes in Lamiaceae reconstituted in yeast Ulschan Bathe, Andrej Frolov, Andrea Porzel, and Alain Tissier J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00714 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 21, 2019

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Journal of Agricultural and Food Chemistry

1

Title: A CYP76 oxidation network of abietane diterpenes in

2

Lamiaceae reconstituted in yeast

3 4

Ulschan Bathea, Andrej Frolovb, Andrea Porzelb, and Alain Tissier*a

5 6

Affiliation:

7

aDepartment

8

3, 06120 Halle, Germany.

9

bDepartment

of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Weinberg

of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3,

10

06120 Halle, Germany.

11

*Corresponding author: [email protected]

12 13

Abstract

14

Rosemary and sage species from the Lamiaceae contain high amounts of structurally related

15

but diverse abietane diterpenes. A number of substances from this compound family have

16

potential pharmacological activities, and are used in the food and cosmetic industry. This has

17

raised interest in their biosynthesis. Investigations in Rosmarinus officinalis and some sage

18

species have uncovered two main groups of cytochrome P450 oxygenases that are involved

19

in the oxidation of the precursor abietatriene. CYP76AHs produce ferruginol and 11-hydroxy

20

ferruginol while CYP76AKs catalyze oxidations at the C20 position. Using a modular Golden

21

Gate-compatible assembly system for yeast expression, these enzymes were systematically

22

tested either alone or in combination. A total of 14 abietane diterpenes could be detected, 8

23

of which have not been reported so far. We demonstrate here that yeast is a valid system for

24

engineering and reconstituting the abietane diterpene network, allowing the discovery of

25

novel compounds with potential bioactivity.

26 27

Keywords 1

ACS Paragon Plus Environment


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CYP76AH, CYP76AK, abietane diterpene, Lamiaceae, yeast expression, CYP plasticity

29 30

Introduction

31

Plants all over the world have been exploited for centuries by Humans for various uses,

32

including as medicine, aromas or fragrances. The Lamiaceae constitute a plant family of

33

special interest that includes various Mediterranean species such as rosemary (Rosmarinus

34

officinalis) and sages (Salvia sp.). They are utilized to treat hypertension and inflammation

35

(rosemary) or to alleviate menopausal symptoms and dental caries (common sage)

36

Moreover, Chinese sage (Danshen; Salvia miltiorrhiza), known from traditional Chinese

37

medicine, has anti-cancer properties and could be used to prevent heart attack 4. Among the

38

many secondary metabolites from the Lamiaceae that are or could be of potential relevance

39

for therapeutic or health benefit usage, abietane diterpenes (ADs) have recently attracted

40

attention. Among them, carnosic acid and its derivative carnosol, which are already approved

41

as preservative agents in the food and cosmetic industry 5, have been shown to protect

42

neurons against oxidative damage and have been proposed as preventive treatment for

43

neurodegenerative diseases 6. Additional advantageous effects of ADs but also of the

44

structurally related tanshinones from danshen and totarol include anti-bacterial, anti-cancer

45

and anti-oxidative properties 7-10.

46

ADs found in the Lamiaceae carry oxidations at several positions, including C12, C11, C20

47

and C7 (Fig. 1). In rosemary and sage species investigated so far, the biosynthesis of ADs

48

starts with the cyclization of geranylgeranyl diphosphate (GGPP) to miltiradiene, which is the

49

product of two successive diterpene synthase reactions, namely a copalyl diphosphate

50

synthase (CPS) and the miltiradiene synthase (MiS) (Fig. 1)

51

miltiradiene to the aromatic abietatriene is assumed to occur spontaneously or by exposure

52

to UV-irradiation, but which olefin, i.e. either miltiradiene or abietatriene, is used for

53

downstream modification has not been fully clarified yet

54

(CYPs) carry out all oxidations downstream of abietatriene reported so far. Most of the CYPs

13-14.

11-12.

1-3.

The conversion of

Cytochrome P450 oxygenases

2


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55

involved in Lamiaceae AD biosynthesis belong to the CYP76 family from the CYP71 clan.

56

CYP76AH1

57

miltiradiene/abietatriene to ferruginol

58

(CYP76AH4, CYP76AH22, and CYP76AH23) and Greek sage

(Salvia fruticosa)

59

(CYP76AH24) with the same activity followed soon after

However, further

60

characterization of these enzymes and of their orthologues from a related sage species

61

(Salvia pomifera) showed that all of them, except SmCYP76AH1, do a second hydroxylation

62

to produce 11-hydroxy ferruginol

63

contains a ferruginol synthase (FS, CYP76AH1) and 11-hydroxy ferruginol synthases (HFS,

64

CYP76AH4, and CYP76AH22-24). More recently, CYP76AH3 from S. miltiorrhiza was shown

65

to oxidize ferruginol at positions C11 and C7 18, indicating that members of this group of CYP

66

enzymes can oxidize ADs at positions other than C11 and C12.

67

The CYP76AK subfamily constitutes a second group of enzymes that catalyze oxygenation

68

at position C20 of ADs, and here are referred to as C20-oxidases (C20ox) 16-18. C20ox accept

69

a variety of substrates, including miltiradiene/abietatriene, ferruginol and 11-hydoxy

70

ferruginol, but the latter appears to be the preferred one

71

species (S. fruticosa, S. pomifera) these CYP76AK enzymes catalyze successive oxidations

72

at the same position to generate the carboxylic acid. The co-expression of enzymes of the

73

CYP76AH and CYP76AK in yeast resulted in the production of carnosic acid

74

substrate flexibility is not specific to the biosynthesis of ADs in Lamiaceae and has been

75

observed in the biosynthesis of other labdane-related diterpenoids, such as rice phytoalexins

76

and diterpene resin acids from conifers

77

with substrates that are known to occur in the species from which they originate. Their

78

already established promiscuity offers the possibility to test them with a yet broader set of

79

substrates. This can be done by combining CYPs from different species together in the same

80

expression host. Such combinatorial biosynthesis was successfully reported for labdanoid

81

diterpene synthases resulting in a set of 50 diterpene labdane scaffolds, 40 of which appear

from

Salvia

miltiorrhiza

16-17

13.

was

the

first

enzyme

reported

to

oxidize

The identification of related genes from rosemary

14-15.

(Fig. 1). The CYP76AH group of enzymes therefore

19-22.

16, 18.

In rosemary and several sage

16-17.

Such

In most cases, these CYPs have been tested

3



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82

to be “new-to-nature”

83

system is required. Although Nicotiana benthamiana has proved useful for the production of

84

olefinic diterpenes, the endogenous non-specific enzymatic activities such as glycosyl-

85

transferases, acyl- or methyl-transferases interfere with the introduced pathway, making the

86

identification of novel products difficult. We and others have been successful in using yeast

87

(Saccharomyces cerevisiae) as an engineering platform for plant diterpenoids

88

Advantages of yeast are the paucity of specialized metabolic pathways that may interfere

89

with engineered pathways and the amenability to high-throughput. In addition, the Golden

90

Gate compatible cloning system we have established for yeast expression allows the rapid

91

and efficient assembly of multiple combinations of genes

92

advantages to explore the expanded CYP oxidation network of ADs from Lamiaceae.

93

Through a deeper analysis of already characterized CYPs, we detected novel products which

94

had been overlooked so far. Furthermore, we show that by combining CYPs from different

95

Lamiaceae species, novel products can be generated.

23-24.

To be able to do this with CYPs, an appropriate expression

16.

16-17.

Here, we capitalize on these

96 97

Material and methods

98

Plant material

99

R. officinalis variety Majorca Pink and S. miltiorrhiza were grown in the greenhouse under

100

long day conditions (16 h light/8 h darkness) with temperatures of 25 °C during the day and

101

of 20 °C at night, with 53 % humidity. Plant material for chromatographic analysis was

102

collected in the vegetative phase.

103 104

Isolation and cloning of genes

105

RNA from 130 mg homogenized S. miltiorrhiza roots was prepared using Spectrum™ Plant

106

Total RNA Kit (Sigma-Aldrich) with additional ethanol washing steps. Total RNA with

107

remaining gDNA was digested using TURBO DNA-free™ Kit (Thermo Fisher Scientific). The

108

obtained RNA solution was purified again using Spectrum™ Plant Total RNA Kit (Sigma4


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109

Aldrich) with the following adaptions: 500 µL binding Solution were mixed with the RNA,

110

loaded to a Binding Column and subsequently processed according to manufacturers’

111

instructions. The cDNA was generated from 1 mg total RNA using ProtoScript® II First

112

Strand cDNA Synthesis Kit (NEB) with random primers and oligo-dT primers. Full-length

113

coding sequences for SmCYP76AH3 and SmCYP76AK1 were PCR amplified with KOD Hot

114

Start DNA Polymerase (Merck) in two separate fragments to remove internal BpiI and BsaI

115

restriction sites by introducing single point silent mutations. The gene specific primers

116

contained

117

TTTGAAGACAAAATGGATTCTTTCTCTCTTCTGGCTG

118

TTTGAAGACCATGCTCTGGTGCGAGAACATC;

119

TTTGAAGACAGAGCATGGAGGACAGCCAG

120

TTTGAAGACAAAAGCTCATGCCTTATACGGAACGATCCTG; SmCYP76AK1 Fragment 1:

121

TTTGAAGACAAAATGCAAGTTTTAATAGTTGCATCCCTAG

122

TTTGAAGACGAGCACGGATACCTCGCCG;

123

TTTGAAGACCCGTGCTCATGCTGCCG

124

TTTGAAGACAAAAGCCTAAACCTTGACGGGAATAGCTTTG). The amplified fragments

125

were purified using QIAquick PCR Purification Kit (Qiagen). Plasmids containing promoters,

126

genes of interest and terminators for yeast expression were produced according to prior

127

established protocols 16.

overhangs

for

further

cloning

(SmCYP76AH3

Fragment

1: and

SmCYP76AH3

Fragment

2: and

and

SmCYP76AK1

Fragment

2: and

128 129

Phylogenetic analysis

130

For the sake of clarity, the name of the CYP proteins is preceded by the letters of the species

131

from which they are coming. For example, RoCYP76AH22 is from Rosemary (Rosmarinus

132

officinalis). Amino acid sequences (GenBank accessions: SmCYP76H1, JX422213;

133

SmCYP76AH3, KR140168; RoCYP76AH4

134

KP091844;

SfCYP76AH24,

KP091842;

SpCYP76AH24,

KT157044;

SmCYP76AK1,

135

KR140169;

SmCYP76AK2,

KP337688;

SmCYP76AK3,

KP337689;

RoCYP76AK7,

16;

RoCYP76AH22, KP091843; RoCYP76AH23,

5



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136

KX431219; RoCYP76AK8, KX431220; SfCYP76AK6, KX431218; SpCYP76AK6, KT157045;

137

SmKO,

138

(http://mafft.cbrc.jp/alignment/server/). The phylogenetic tree was computed using Mega6

139

with the maximum likelihood method based on the JTT/+G model with five categories

140

a bootstrap with 1,000 replicates.

KJ606394)

were

aligned

using

MAFFT

version

7

with

default

settings

26

25

and

141 142

Yeast microsome isolation and in vitro CYP assays

143

Plasmids containing the CYPs (SmCYP76AH1, SmCYP76AH3, RoCYP76AH22, and/or

144

SmCYP76AK1) and ATR1 (Tab. S1) were transformed into S. cerevisiae strain INVSc1

145

(genotype: MATα his 3D1 leu2 trp1-289 ura3-52; Invitrogen) and plated onto uracil-free

146

selection medium (1 g/L Yeast Synthetic Drop-out Medium Supplements without uracil

147

(Sigma-Aldrich), 6.7 g/L Yeast Nitrogen Base With Amino Acids (Sigma-Aldrich) and 20 g/L

148

Micro Agar (Duchefa Biochemie)). Preparation of yeast cultures and isolation of yeast

149

microsomes were performed as previously described

150

carried out as stated with 40 µL microsomes and the substrates miltiradiene/abietatriene and

151

ferruginol. When multiple enzymes were required in the assays, the volume of 40 µL

152

microsomes was distributed equally among them.

16.

The in vitro CYP assays were

153 154

Production of diterpenes in yeast

155

Yeast expression vectors (Tab. S1) were transformed into the yeast strain INVSc1

156

(Invitrogen) and plated out onto uracil-free selection medium. Positively transformed colonies

157

were inoculated into 5 mL YPD medium (20 g/L tryptone and 10 g/L yeast extract) containing

158

2 % of glucose and grown for 24 h with shaking at 30 °C. Protein expression was induced by

159

resuspending the cell pellet in fresh YPD medium containing 2 % galactose. After another

160

24 h of cultivation, whole cultures were extracted using 2 mL n-hexane.

161 162

Chromatographic analysis 6


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Plant surface extracts of R. officinalis leaves were prepared by shaking three young leaves in

164

1 mL n-hexane for 2 min at room temperature. S. miltiorrhiza roots were homogenized and

165

extracted using 1 mL n-hexane. ADs produced in yeast were extracted from 5 mL culture

166

with 2 mL n-hexane. The extracts were evaporated to complete dryness under a nitrogen

167

stream and resuspended in 200 µL n-hexane for GC-MS analysis. Using a Trace GC Ultra

168

gas chromatograph (Thermo Scientific) coupled to ATAS Optic 3 injector and an ISQ single

169

quadrupole mass spectrometer (Thermo Scientific) with electron impact ionization, the plant

170

and yeast extracts were separated on a ZB-5ms capillary column (30 m x 0.32 mm,

171

Phenomenex) using splitless injection and an injection volume of 1 µl. An injection

172

temperature gradient from 60 °C to 250 °C with 10 °C/s was used and the flow rate of helium

173

was 1 ml/min. The GC oven temperature gradient was as follows: 50 °C for 1 min, 50 to

174

300 °C with 7 °C/min, 300 to 330 °C with 20 °C/min and 330 °C for 5 min. Mass spectrometry

175

was performed at 70 eV, in a full scan mode with m/z from 50 to 450. Data analysis was

176

done with the device specific software Xcalibur (Thermo Scientific) 16.

177

For LC-MS measurement, the dried plant and yeast extracts were resuspended in 150 µL

178

methanol and subjected to reversed phase-ultraperformance LC-(ESI)-MS/MS analysis in

179

which metabolites were separated using a Nucleoshell RP18 column (2 x 150 mm, particle

180

size 2.7 µm, Macherey-Nagel) and a ACQUITY UPLC System (Waters), including an

181

ACQUITY Binary Solvent Manager and an ACQUITY Sample Manager (10 mL sample loop,

182

partial loop injection mode, 5 mL injection volume). For elution, solvents A (aqueous

183

0.3 mmol/L NH4HCOO (adjusted to pH 3.5 with formic acid)) and B (acetonitrile) were used.

184

The elution conditions were as follows: isocratic from 0 to 2 min at 5% eluent B, from 2 to

185

19 min linear from 5 to 95 %, from 19 to 22 min isocratically at 95 %, from 22 to 22.01 min

186

linear from 95 to 5 % and from 22.01 to 24 min isocratically at 5 % eluent B. The flow rate

187

was 400 µl/min and the column temperature was set to 40 °C. To detect the metabolites, a

188

TripleToF® 5600 mass spectrometer (AB Sciex) was used, which was equipped with an ESI-

189

Duo-TurboIon-Spray interface. It operated in the negative ion mode and was controlled by 7



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190

Analyst® 1.6 TF software (AB Sciex). The LC-ESI source parameters were as follows: ion

191

spray voltage: -4,500 V, nebulizing gas: 60 p.s.i., source temperature: 600 °C, drying gas:

192

70 p.s.i., curtain gas: 35 p.s.i. Data acquisition was performed in the MS1-ToF mode

193

scanned from 250 to 500 Da with an accumulation time of 50 ms, and the MS2-SWATH

194

mode, divided into 5 Da segments of 20 ms accumulation time. 56 separate scan

195

experiments were carried out covering the mass range from 65 to 500 Da. In this process, a

196

declustering potential of -35 V was applied and collision energies were set to 55 V with a

197

collision energy spread of -45 V. Data independent acquisition of MS2 spectra using SWATH-

198

MS/MS allowed for cycle times of 1 s to access MS1 and MS2 data

199

acquisition frequency is high, it was possible to manually purify all spectra by superimposing

200

the chromatographic peak shape of individual fragment ions with the MS1 precursor ion.

27.

27,

Since SWATH

201 202

Liquid chromatography and MSn experiments

203

The methanol extracts of yeast cultures were prepared as described in section

204

“Chromatographic analysis”. 3 µL of each sample were loaded on a EC 150/2 Nucleoshell

205

RP18 column (encaped C18 phase, ID 2 mm, length 150 mm, particle size 2.7 μm, Macherey

206

Nagel, Düren, Germany) using a Dionex Ultimate 3000 UHPLC, equipped with a 3400RS

207

pump, 3000TRS autosampler and DAD3000 photodiode array (PDA) detector (Thermo-

208

Fisher Scientific, Bremen, Germany). The eluents A and B were 0.3 mmol/L ammonium

209

formate (adjusted to pH 3.5 with formic acid) and acetonitrile, respectively. After a 2-min

210

isocratic step (5 % eluent B), analytes were eluted at a flow rate of 400 µL/min at 25 °C in a

211

17-min linear gradient to 45 % eluent B. The column effluents were introduced on-line in an

212

Orbitrap Elite mass spectrometer operating in negative ion mode, equipped with a heated

213

electrospray ion source (HESI), and controlled by Xcalibur (Thermo-Fisher Scientific,

214

Bremen, Germany). The source and transfer capillary temperatures were set to 300 and

215

325 °C, respectively. The spray voltage was -3.5 kV, while sheath, auxiliary and sweep

216

gases were set to 5 pounds per square inch gauge (psig) each. Analytes were annotated in 8


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217

linear ion trap-orbital trap (LIT-Orbitrap) scans (m/z 100-1500, mass resolution 30000) by

218

their tR, m/z and isotopic patterns. The structures of the annotated analytes were suggested

219

by tandem mass spectrometric (MSn) analysis. Thereby, collision induced fragmentation

220

(CID) was performed in LIT by resonance activation in presence of helium as a

221

collision/cooling gas. The corresponding quasi-molecular ions were isolated with the width of

222

2 m/z, activation time and relative activation frequency were 10 ms and 0.250, respectively.

223

Normalized collision energy was experiment-specific and varied in the range of 20-45 %.

224 225

Structure confirmation by NMR analysis

226

For isolation of pisiferol, two single yeast colonies expressing GGPPS, CPS, MiS, ATR1,

227

SmCYP76AH1 and SmCYP76AK1 were inoculated into each 5 mL YPD medium with 2 %

228

glucose and grown at 30 °C with shaking. After 24 h, the suspensions were transferred into

229

200 mL culture medium for shake-flask expression and grown for another 24 h. The

230

expression was induced by resuspending the cell pellet in 200 mL YPD containing 2 %

231

galactose and the culture was further incubated for another 24 h. The diterpenes were

232

extracted from the yeast culture by adding 200 mL n-hexane and shaking thoroughly. The

233

completely dried extract was dissolved in 0.75 mL C6D6 with a final concentration of ca.

234

60 µmol/L. NMR spectra were recorded on a Varian/Agilent VNMRS 600 NMR spectrometer

235

operating at a proton NMR frequency of 599.829 MHz, using a 5 mm inverse detection

236

cryoprobe. 1H NMR spectra were recorded with a digital resolution of 0.37 Hz/point, a pulse

237

width (pw) of 2.2 μs (30°), a relaxation delay of 0.27 s, an acquisition time of 2.73 s; and

238

number of transients of 40. 2D NMR spectra were recorded using standard CHEMPACK 7.1

239

pulse sequences (gDQCOSY, gHSQCAD, gHMBCAD) implemented in Varian VNMRJ 4.2A

240

spectrometer software. The HSQC experiment was optimized for 1JCH = 146 Hz with DEPT-

241

like editing and 13C-decoupling during acquisition time. The HMBC experiment was optimized

242

for a long-range coupling of 8 Hz; a 2-step 1JCH filter was used (130-165 Hz). 1H as well as

243

13C

chemical shifts are referenced to internal TMS (0 ppm). 9



Page 10 of 37

244 245

Structural suggestions of ADs produced in yeast

246

Structures of ADs produced in yeast were suggested by the help of either comparative

247

analysis of mass spectral data with the literature, by NMR analysis (see section “Structure

248

confirmation by NMR analysis”) or by liquid chromatography coupled to MSn experiments

249

(see section “Liquid chromatography and MSn experiments”).

250 251

Results

252

Reliable reconstruction of AD biosynthesis in yeast

253

Ferruginol synthase (FS) and 11-hydroxy ferruginol synthase (HFS) of the CYP76AH sub-

254

family

255

miltiradiene/abietatriene and they do so at multiple defined positions (Fig. 1)

256

CYP76AH members have been described with FS and/or HFS activity. Based on reports

257

published to date, SmCYP76AH1 oxidizes at C12 only, SmCYP76AH3 at C7 and C11,

258

RoCYP76AH4, RoCYP76AH22-23 and SfCYP76AH24 (or SpCYP76AH24) oxidize at

259

positions C11 and C12 13-18. Using the Golden Gate modular cloning system for expression in

260

yeast

261

diphosphate synthase (GGPS), a copalyl diphosphate synthase (CPS), a miltiradiene

262

synthase (MiS) and a cytochrome P450 reductase from Arabidopsis thaliana (ATR1). The

263

latter four enzymes constitute the core module (CM) providing sufficient supply of

264

miltiradiene/abietatriene as substrates for downstream CYP activity. Diterpenes produced by

265

engineered yeast strains were extracted with hexane, and the dried extracts were measured

266

by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-

267

MS) in appropriate solvents (see the methods section). In agreement with previous reports 13,

268

co-expression of the CM with SmCYP76AH1 led to the production of ferruginol (3) along with

269

the precursors miltiradiene (1) and abietatriene (2) and co-expression of RoCYP76AH22 with

270

the CM yielded additionally 11-hydroxy ferruginol (4), which spontaneously oxidized to the

from

16,

rosemary

and

sage

species

are

the

first

enzymes

to

oxidize

16, 18.

Several

we co-expressed SmCYP76AH1 or RoCYP76AH22 with a geranylgeranyl

10


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271

corresponding quinone, here named abietaquinone (11)

272

enzymes (regardless whether RoCYP76AH4, RoCYP76AH22-23 or SfCYP76AH24 was

273

used) produced 11-hydroxy sugiol (6) (Fig. S1). The structures of 6 and other ADs were not

274

only suggested by comparative analysis of mass spectral data from the literature

275

by liquid chromatography coupled to multiple tandem mass spectrometry (LC-MSn). For that

276

purpose, measurements were run in a targeted mode by surveying the [m-H]- from MS1

277

experiments and the first daughter ion from MS2 experiments which we obtained from LC-MS

278

measurements (Fig. S2). For example, extracts that were assumed to contain 6, were

279

analyzed for the m/z ratios in the negative ion mode of 315.196 ([m-H]-) and 300.173 (first

280

daughter ion). In case [m-H]- and the daughter ion were found at the same retention time, the

281

fragmentation patterns from up to MS4 experiments were interpreted to propose an AD

282

structure (Fig. S3 and Fig. S4). Using this method, we obtained high quality data with mass

283

accuracies between -0.5 and 2.8 ppm (Tab. 1). The interpretation of fragmentation pattern

284

from MSn experiments allowed us to assign measured fragment ions to specific structural

285

features (Tab. 1 and Fig. S4). The interpreted characteristic fragmentation pattern of 6

286

further served as a template for other AD structures by LC-MSn. A representative example of

287

the AD fragmentation pattern is given in Fig. 2.

288

Although we had not detected 6 in our previous study, the results here demonstrate that all

289

HFS enzymes (SmCYP76AH3, RoCYP76AH4, RoCYP76AH22-23 and SfCYP76AH24)

290

exhibit oxidative activity at position C7. Previously, we demonstrated the importance of

291

amino acids at positions 301, 303 and 479 for CYP76AH enzymes in determining whether

292

the enzyme has 11-hydroxy ferruginol synthase activity as in RoCYP76AH22 or is a strict FS

293

like SmCYP76AH1

294

301, 303 and 479 with those of SmCYP76AH1 prevented the formation of not only 11-

295

hydroxy ferruginol (4), but also of 6. Results presented in Fig. S1 show that this is the case.

296

This indicates that the sequence of oxidation is first C12 (ferruginol), C11 and then C7.

16.

16, 18.

In addition we found that HFS

18,

but also

We tested if exchanging amino acids of RoCYP76AH22 at positions

297 11



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298

SmCYP76AH3 has ferruginol synthase activity

299

SmCYP76AH3 was previously reported to display exclusively HFS activity on ferruginol

300

Therefore, we did not expect to detect oxidized diterpenes beyond miltiradiene and

301

abietatriene when co-expressing the CM and SmCYP76AH3. Surprisingly, we detected

302

activity of SmCYP76AH3 on miltiradiene/abietatriene, which resulted in the formation of

303

ferruginol (3), 11-hydroxy ferruginol (4) and 11-hydroxy sugiol (6) as with RoCYP76AH22

304

(Fig. 3A-B, Fig. S1 and Fig. S5). To confirm this, we carried out in vitro enzyme assays with

305

a microsome fraction from yeast strains expressing ATR1 and SmCYP76AH3 using

306

miltiradiene/abietatriene and ferruginol as substrates (Fig. S6-S8). Our results show that the

307

activity of SmCYP76AH3 is similar to that of other HFS from rosemary and Greek sage (e.g.

308

RoCYP76AH22). In agreement with these observations, the protein sequence of

309

SmCYP76AH3 is highly similar to all other HFS with 80-90 % identity (Fig. 4A). Accordingly,

310

SmCYP76AH3 contains amino acids E301, S303 and F479 like most other HFS, and in a

311

phylogenetic analysis forms a conserved HFS cluster together with RoCYP76AH4,

312

RoCYP76AH22-23 and Sp/SfCYP76AH24 but distinct from SmCYP76AH1 (Fig. 4A-B).

18.

313 314

Novel ADs produced in yeast by activity of CYP76AH enzymes

315

Next to ferruginol (3), 11-hydroxy ferruginol (4) and 11-hydroxy sugiol (6), two new main

316

products, namely 5 and 15, could be detected by LC-MS measurements of engineered yeast

317

strains expressing the CM and HFS (Fig. 3B/D and Fig. S1). Compound 5 eluted shortly

318

before 6 and had a m/z ratio in the negative ion mode of 317.211. The mass difference of +2

319

with 11-hydroxy sugiol (compound 6, m/z 315.196) suggests that 5 is a precursor of 6 with a

320

hydroxyl group instead of a ketone at position C7. Product 15 had a m/z ratio of 331.227.

321

Here, the mass difference of +14 suggests the addition of a methyl group to 6. Notably, both

322

compounds were also found in extracts of rosemary leaf surface and total S. miltiorrhiza

323

roots (Fig. S9 and Fig. S10). Due to their relatively low abundance, it was not possible to

324

isolate these new compounds from yeast cultures in appropriate quality and quantity for an 12


Page 13 of 37


325

unambiguous structural elucidation by NMR analysis. To gain insights into the structure of

326

these compounds anyhow, we performed LC-MSn. Interpretation of the fragmentation pattern

327

suggested that compound 5 is 7,11-dihydroxy ferruginol and compound 15 7-methoxy-11-

328

hydroxy ferruginol (Tab. 1, Fig. S3, Fig. S4A and I).

329

We then tested if 7-methoxy-11-hydroxy ferruginol (15) is a product of HFS activity, because

330

methylation is not a common CYP reaction. For this, we performed in vitro enzyme assays

331

with microsomes containing ATR1 and HFS. No formation of 15 could be detected, neither

332

when miltiradiene/abietatriene nor when ferruginol was used as substrate (Fig. S8).

333

Therefore, we conclude that 15 is the product of a non-specific methyl transferase activity

334

from yeast on 7,11-dihydroxy ferruginol (5).

335

In addition to the major products of HFS activity, we found one additional minor product (12)

336

with much lower abundance than 5 and 15 in engineered yeast strains (Fig. 3C/D). A

337

compound with the same retention time and mass spectrum also occurs in S. miltiorrhiza root

338

extracts, suggesting that 12 is a true product of HFS and not an artefact due to expression in

339

yeast (Fig. S2 and Fig. S9). Compound 12 had a m/z ratio in LC-MS analysis of 313.180.

340

Since the catechol function on the C-ring of ADs is prone to spontaneous oxidation to the

341

corresponding quinone (Fig. 1), as shown for 11-hydroxy ferruginol and carnosic acid

342

12 was hypothesized to be the quinone derivative of 11-hydroxy sugiol (6). Analysis of LC-

343

MSn data supported this and 12 is therefore proposed to be 7-keto abietaquinone (Tab. 1,

344

Fig. S3 and Fig. S4F).

345

Taking all major and minor products into account, FS/HFS activity overall resulted in the

346

formation of six ADs (3-6, 11 and 12) and in an additional product due to an unspecific

347

methyl transferase activity from yeast (15) (Tab. 2). To our knowledge, three of them (5, 12

348

and 15) have never been described before, although we could detect them in rosemary and

349

sage. This set of products is the result of the capacity of CYP76AH enzymes to oxidize the

350

miltiradiene/abietatriene scaffold at three distinct positions, respectively C7, C11 and C12.

16, 28-29,

13



Page 14 of 37

351

Depending on the specific enzyme and species, different combinations of these oxidations

352

are possible (Tab. 2).

353 354

SmCYP76AK1 promiscuity further expands the network of ADs in Lamiaceae

355

In the downstream biosynthesis of ADs from Lamiaceae, SmCYP76AK1, Sf/SpCYP76AK6

356

and RoCYP76AK7-8 are C20ox that respectively generate either an alcohol or a carboxyl

357

group at position C20

358

RoCYP76AK7-8) highly favor 11-hydroxy ferruginol as substrate and produce primarily

359

carnosic acid

360

sought to explore the oxidation landscape when CYP76AK1 is combined with the CYP76AH

361

group of enzymes. Co-expression of the CM, SmCYP76AH1 and SmCYP76AK1 led to the

362

formation of miltiradiene (1), abietatriene (2), ferruginol (3) and a higher oxidized AD (7) (Fig.

363

3A). The relatively low abundance of 7 suggests that ferruginol is not the favored substrate of

364

SmCYP76AK1. However, this compound could also be detected in extracts of S. miltiorrhiza

365

total roots (Fig. S5) supporting the natural occurrence of this reaction in planta. In vitro

366

enzyme assays with microsomal fractions of yeast strains expressing ATR1, SmCYP76AH1

367

and SmCYP76AK1, or ATR1 and SmCYP76AK1 confirmed production of 7 when incubated

368

with miltiradiene/abietatriene (Fig. S6 and Fig. S10) and ferruginol (Fig. S7 and Fig. S10),

369

respectively. Finally, we identified 7 as pisiferol by comparison of mass spectral data with the

370

literature

371

and Tab. S2).

372

Guo et al. (2016) reported that SmCYP76AK1 uses 11-hydroxy ferruginol (4) and 11-hydroxy

373

sugiol (6) as substrates to produce 11,20-dihydroxy ferruginol (8) and 11,20-dihydroxy sugiol

374

(10) respectively, the former spontaneously oxidizing to 20-hydroxy abietaquinone (13).

375

When co-expressing the CM, HFS and SmCYP76AK1 in yeast, we could confirm these data

376

(Tab. 1, Fig. S3, Fig. S4C, E, G and Fig. S6), but detected three additional peaks (9, 14 and

377

16) (Fig. 3C-D). We could also detect them in extracts of rosemary leaf surfaces and total S.

30

16.

16-18.

C20ox that introduce the carboxyl group (Sf/SpCYP76AK6 and

In contrast, SmCYP76AK1 oxidizes only once at position C20

18.

We then

and by NMR analysis of the compound isolated from yeast cultures (Fig. S11-13

14


Page 15 of 37


378

miltiorrhiza roots (Fig. S2 and Fig. S9). This suggests that the enzymatic reactions observed

379

in yeast are not artefacts, but accurately reflect the network of reactions occurring in

380

Lamiaceae. Based on LC-MSn data, we propose 7,11,20-trihydroxy ferruginol for 9, 7,20-

381

dihydroxy abietaquinone for compound 14 and 7-methoxy-11,20-dihydroxy ferruginol for

382

compound 16 (Tab. 1, Fig. S3 and Fig. S4D, H, J). The latter was most likely due to the

383

action of a yeast methyl transferase on the 7-hydroxyl group of 7,11,20-trihydroxy ferruginol

384

(9), as in the case of 7-methoxy-11-hydroxy ferruginol (15). In summary, the activity of

385

SmCYP76AK1 in combination with FS and HFS enzymes resulted in the production of seven

386

ADs (7-10, 13, 14 and 16), 16 being the result of an unspecific methyl transferase activity on

387

9. Notably, three of them (9, 14 and 16) had not been reported before.

388 389

Discussion

390

In recent years, great progress was made in the biosynthesis of ADs from Lamiaceae. Using

391

a Golden Gate cloning system adapted for yeast expression, we recently described the

392

reconstitution of the pathway for carnosic acid from rosemary and S. fruticosa in this host

393

Here, we exploited the modularity of this system to rapidly combine CYP enzymes from a

394

number of related Lamiaceae species (rosemary and S. miltiorrhiza), where the pathway for

395

ADs is well represented. We have chosen yeast rather than Nicotiana benthamiana as an

396

expression platform because of the activity of modifying enzymes from N. benthamiana that

397

interfere with the introduced pathway and make the analysis difficult. This has been shown

398

for example for sesquiterpenes

399

pathway in N. benthamiana gives very poor yields (data not shown). In contrast, the yeast S.

400

cerevisiae has no known terpenoid specialized metabolite and, to our knowledge, there are

401

no reports of interfering reactions when plant terpenoid pathways are expressed in this host.

402

Interestingly, we found two compounds (15 and 16) that were methylated, a reaction that the

403

CYP used was not able to carry out in vitro. A shown by Loussouarn et al. (2017)35, such a

32-34,

31.

and in our hands reconstitution of the carnosic acid

15



Page 16 of 37

404

methylation can happen spontaneously in oxidizing conditions in vitro. Alternatively this could

405

be the result of a promiscuous methyltransferase activity from yeast.

406

By

407

miltiradiene/abietatriene) with either FS or HFS and/or the C20ox that oxidizes only once

408

(SmCYP76AK1), we were able to produce and detect in total 16 diterpenes, 14 of them

409

resulting from the oxidative activity of the expressed CYPs and subsequent spontaneous

410

conversions (3-16). Although some of the detected products confirm previous studies

411

16, 18,

412

compounds framed with green dashed lines). We could show that SmCYP76AH3 can oxidize

413

miltiradiene/abietatriene, which had not been shown previously

414

is comparable to other characterized HFS enzymes, such as RoCYP76AH22. Furthermore,

415

we detected new ADs when FS or HFS are combined with SmCYP76AK1. Due to the

416

relatively low abundance of these compounds we were able to unambiguously assign a

417

structure to only one of them, pisiferol (7). Other compounds (5, 9, 12 and 14, see Fig. 5)

418

were putatively identified using fragmentation patterns from LC-MSn data. None of these

419

have been reported by previous studies, but we could detect them in planta (either in

420

rosemary or sage species), confirming that they are authentic products of the AD metabolic

421

network in these species. Some compounds reported by Guo et al. (2016), namely sugiol as

422

well as its expected precursor 7-hydroxy ferruginol and potentially derived derivatives upon

423

oxidation by SmCYP76AK1, could not be clearly identified in our expression system (see

424

Tab. 2, compounds framed with grey dashed lines). The reason for this is not clear, but we

425

also failed to detect these compounds in rosemary or S. miltiorrhiza extracts, indirectly

426

supporting our pathway reconstitution in yeast.

427

However, SmCYP76AK1, which possesses exclusively one hydroxylation activity on position

428

C20, is unique to tanshinone producing S. miltiorrhiza and accepts C7-oxidized ADs as

429

substrates, resulting in the production of compounds 9, 10, 14 and 16. In contrast, C20ox

430

that introduce a carboxyl group (CYP76AK6-8) can only be found in rosemary and Greek

co-expression

of

the

core

module

(to

produce

the

diterpene

precursors

11, 13, 15-

to the best of our knowledge, eight of them have not been reported before (Tab. 2,

18,

and has an activity which

16


Page 17 of 37


431

sage and their main products are carnosic acid and its derivatives. We could not find any

432

activity on C7-oxidized intermediates when CYP76AK8 is co-expressed with HFS (data not

433

shown). This confirms the observation that 11-hydroxy ferruginol (oxidation at C11 and C12)

434

is the favored substrate en route to carnosic acid 16 and suggests that the HFS cannot accept

435

substrates that carry a carboxyl group at C20. Despite this, products with a hydroxyl or a keto

436

group at position C7 can be found in rosemary and sage extracts. This is the case for

437

example for 11-hydroxy sugiol (6), 7,11-dihydroxy ferruginol (5) and its methylated derivative

438

(15). Because RoCYP76AK8 is dominant over the C7-oxidation activity of CYP76AHs, this

439

could indicate that in rosemary either the expression of CYP76AHs and CYP76AKs is not

440

exactly overlapping (in space and/or time) or that CYP76AHs are more strongly expressed

441

than CYP76AK8. By revisiting our transcriptome data

442

RoCYP76AH22 is 2.5 fold higher than that of RoCYP76AK8 (Fig. S14). Thus, it is possible

443

that this relative expression in favor of HFS level allows the production of C7-oxidized

444

diterpenes in planta.

445

Although misinterpretations resulting from heterologous expression cannot be entirely

446

excluded, the yeast expression system to elucidate biosynthetic pathways and to reconstitute

447

the complex network of related compounds that is typically associated with plant secondary

448

metabolism is of high reliability as all compounds reported in this study could be detected in

449

rosemary and/or sage extracts. In that regard, the Golden Gate cloning system adapted for

450

yeast expression

451

Lamiaceae AD network (Fig. 5). This system can be extended to further explore the chemical

452

space of ADs, for example by expressing CYPs that have been shown to oxidize at different

453

positions on the labdanoid backbone

454

generated with different backbones by combining diterpene synthases and CYPs from

455

various plants or organisms for large scale diterpene combinatorial biosynthesis. This would

456

allow to access rare or also “new-to-nature” compounds. The high-throughput cloning

457

capacity of Golden Gate combined with the high-throughput screening potential of yeast

16

11, 16,

we found that the expression of

provides a powerful tool to reconstruct and better understand the

36.

Beyond this, other diterpene libraries could be

17



Page 18 of 37

458

holds much promise for the creation of libraries of diterpenoids, which can then be screened

459

for novel biological activities of interest.

460

In conclusion, the complex and diverse abietane diterpene network in Lamiaceae is based on

461

the substrate flexibility of CYP76AH and CYP76AK enzymes, but also of yet unknown

462

enzymes, for example in the biosynthesis of the tanshinones. Within this study we were able

463

to produce 14 ADs in yeast by combining the expression of FS, HFS and C20ox. Our results

464

thus expand the network of naturally occurring ADs beyond carnosaldehyde, carnosic acid,

465

carnosol, carnosic acid quinone, pisiferal and pisiferic acid to a total of 20 compounds in

466

rosemary and sage species. Although the full extent of metabolites and biosynthetic CYPs

467

remains to be discovered, the Golden Gate cloning tool combined with yeast expression

468

have proven highly efficient and provide a powerful tool to enlighten the unidentified parts of

469

the AD network in Lamiaceae.

470 471

Acknowledgements

472

We are very grateful to Jürgen Schmidt for guidance in the analysis of MSn experiments.

473 474

Funding

475

The work was supported by the Leibniz Institute of Plant Biochemistry in Halle, Germany.

476 477

Author contributions

478

A.T. conceived the project. A.T. and U.B. conceived and designed the experiments. U.B.,

479

A.F., and A.P. performed the experiments. U.B., A.T., A.F., and A.P. analyzed the data and

480

wrote the paper.

481 482

Supporting Information statement

483

The Supporting Information is available free of charge on the ACS Publications website.

18


Page 19 of 37


484

Supporting information include GC-MS and LC-MS data of all tested yeast strains, plant

485

extracts and in vitro assays as well as tandem mass spectral data of all reported oxidized

486

diterpenes, NMR data of pisiferol, transcriptome data, and a list of analyzed constructs and

487

corresponding yeast strains.

19



Page 20 of 37

488

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605

diversity. Phytochemistry 2019, 161, 149-162.

Liu, Q.; Manzano, D.; Tanic, N.; Pesic, M.; Bankovic, J.; Pateraki, I.; Ricard, L.;

Loussouarn, M.; Krieger-Liszkay, A.; Svilar, L.; Bily, A.; Birtic, S.; Havaux, M.,

Bathe, U.; Tissier, A., Cytochrome P450 enzymes: A driving force of plant diterpene

606 607

Figure captions

608

Figure 1: Simplified scheme of the CYP oxidation network in Lamiaceae. The upstream

609

reactions are catalyzed by diterpene synthases copalyl diphosphate synthase (CPS) and

610

miltiradiene synthase (MiS) followed by CYPs from the subfamilies CYP76AH (HFS) and

611

CYP76AK (C20ox). The affected oxidized positions (C7, C11, C12 and C20) are labeled with

612

blue or red arrows. Blue arrows and red arrows designate positions oxidized by CYP76AKs

613

or CYP76AHs respectively. The number of arrows indicates the number of successive

614

oxidations that can occur at a single position, i.e. to an alcolhol, an aldehyde or a carboxylic

615

acid. Due to the incorporation of hydroxy-, keto-, aldehyde and carboxylic groups in different

616

combinations a wide range of derived structures is formed. Additionally, the hydroxyl groups

617

at positions 11 and 12 can spontaneously oxidize to form a quinone.

618 619

Figure 2: Fragmentation pattern of 7-methoxy-11,20-dihydroxy ferruginol (16) as

620

representative example of all other ADs described in this study. A, 7-methoxy-11,2024


Page 25 of 37


621

dihydroxy ferruginol (16) with a m/z ratio in the negative ion mode of 347.222 looses its

622

methoxy group after MS2, yielding a fragment with m/z 315.1966 (a). The MS3 fragmentation

623

then gives m/z 285.186 (b) with additional smaller fragments (c-g). B, the experiments from

624

MS2 to MS4 are shown with the assignment of fragment ions to the corresponding peaks.

625 626

Figure 3: Chromatographic analysis of extracts from yeast strains expressing

627

enzymes of the AD biosynthesis. GC-MS (A; selected m/z signals: 270, 272, 286, 300 and

628

302) and LC-MS (B; selected m/z signals in the negative ion mode: 301.2017, 315.196,

629

317.211 and 331.227) results of extracts of yeast strain expressing the CM (control) and the

630

given CYPs. C, Presented are the traces of the given m/z signals of extracts from yeast

631

strains expressing GGPPS + RoCPS + RoMiS + ATR1 and the indicated CYPs. D, ESI mass

632

spectra of novel ADs from the CYP oxidation network. Miltiradiene (1), abietatriene (2),

633

ferruginol (3), 11-hydroxy ferruginol (4), 7,11-dihydroxy ferruginol (5), 11-hydroxy sugiol (6),

634

pisiferol (7), 7,11,20-trihydroxy ferruginol (9), 11,20-dihydroxy sugiol (10), abietaquinone

635

(11), 7-keto abietaquinone (12), 7,20-dihydroxy abietaquinone (14), 7-methoxy-11-hydroxy

636

ferruginol (15) and 7-methoxy-11,20-dihydroxy ferruginol (16).

637 638

Figure 4: Comparison of the amino acid sequences of CYP76AH and CYP76AK

639

enzymes. A, part of the amino acid sequence alignment of HFS enzymes. The conserved

640

motifs, I-helix, K-helix, PERF and heme-binding domains are indicated. Amino acids which

641

determine the activity as FS or HFS are indicated with arrows. SmCYP76AH3 shows high

642

sequence similarity to the other CYP76AHs confirming its function as HFS. B, phylogenetic

643

analysis of CYPs from the AD biosynthesis in Lamiaceae. The included enzymes from

644

CYP76AHs and CYP76AKs cluster in subgroups which match their function as FS, HFS or

645

C20ox. S. miltiorrhiza kaurene oxidase (SmKO) serves as outgroup.

646

25



Page 26 of 37

647

Figure 5: The biosynthetic network of ADs in Lamiaceae. Ferruginol synthase

648

(CYP76AH1), 11-hydroxy ferruginol synthases (CYP76AH3-4, CYP76AH22-24) and C20

649

oxidases (CYP76AK1, CYP76AK6-8) catalyze successive oxidations at positions C12, C11,

650

C7 and C20, respectively. In addition, unspecific yeast methyl transferases act on 7,11-

651

dihydroxy ferruginol (5) and 7,11,20-trihydroxy ferruginol (9). The color code for the boxes is

652

as follows: orange is for reactions and compounds involving a ferruginol synthase

653

(CYP76AH1) and C20 oxidases; blue is for reactions and compounds involving 11-hydroxy

654

ferruginol synthases alone; grey for reactions involving 11-hydroxy ferruginol synthases and

655

an alcohol producing C20 oxidase (CYP76AK1); green is for reactions and compounds

656

involving 11-hydroxy ferruginol synthases and a carboxylic acid producing C20 oxidase

657

(CYP76AK8).

658

26


Page 27 of 37


Table 1: Mass spectrometric information obtained in MS and MSn experiments performed with extracts of engineered yeast strains which expressed the CM and the given CYPs. Fragmentation patternsc ADa

CYP

tR

[M-H]-

Elemental

Error

(min)

(m/z)

compositionb

(ppm)

a

b

c1

c2

c3

d

e

f

g

h

-MeOH

-CH2O

-CH3

-2CH3

-3CH3

-C3H7

-C4H8-10

-C5H8-11

-C6H11-12

-H2O/-CO

284.1782

255.1395

241.1234

227.1078

214.1003

-

-

SmCYP 5

15.0

317.2130

76AH3

C20H30O3

2.6

/0.16*

/1.68**

299.2027 -

/-0.21**

/0.22**

/1.58**

/3.59

217.0870 SmCYP 6

300.1728 15.8

315.1964

76AH3

C20H28O3

-0.5

-

285.1479

270.1249

-

229.0869

/0.23*

/-0.65*

244.1099

/-1.01

/0.14

/-4.46*

-

-

/-2.33 RoCYP 76AH22 8

287.2018 18.3

317.2122

SmCYP

C20H30O3

-0.1

272.1784

257.1543

-

244.1472

229.1237

/0.66

/0.83*

/-1.58*

203.1080 -

/1.32*

/1.44*

/1.15*

76AK1 RoCYP

303.1968

76AH22 9

/0.65 14.0

333.2073

SmCYP

C20H30O4

0.4

RoCYP

288.1729

-

260.1418 -

273.1859

76AK1 10

315.1965 231.1387

-

/-0.84*

203.1074

/-0.22

/-1.93*

285.1858

/0.20*

/-1.32*

/-0.64*

/-0.33* 15.0

331.1915

C20H28O4

0.1

-

301.1813

286.1574

-

-

258.1262

-

230.0948

-

273.1860

27



76AH22

/1.29

Page 28 of 37

/-0.26*

/0.05*

/-0.04*

/-0.03*

SmCYP 76AK1 SmCYP 12

298.1574 15.3

313.1812

76AH3

C20H26O3

1.0

C20H28O3

-0.2

C20H28O4

2.8

C21H32O3

0.4

C21H32O4

-0.5

-

283.1340

-

/-0.21

257.1184

244.1105

230.0949

/0.23

/-0.02

/0.09

214.1001

201.0923

/0.65*

/0.75*

-

-

-

/0.12

-

RoCYP 76AH22 13

285.1860 16.0

315.1965

SmCYP

270.1627

-

/0.43

-

-

-

/0.60*

-

76AK1 RoCYP 76AH22 14

301.1811 17.4

331.1924

SmCYP

-

258.1262 -

-

-

/0.68

283.1704 -

/0.10*

/0.04*

76AK1 SmCYP 15

299.2018 18.1

331.2280

76AH3

284.1781

268.1469

255.1393 -

/0.43

/-0.17*

/-0.04**

315.1966

285.1864

270.1625

227.1079

214.1000

/0.68**

/0.22**

242.1300

229.1234

214.0999

201.0921

/-0.33**

/0.04**

/-0.06**

/-0.01**

-

/0.81**

-

-

RoCYP 76AH22 16

16.4 SmCYP

347.2226

/-0.02

/1.22*

/-0.08**

-

-

76AK1

The measurements were done with a linear ion trap-orbital trap (LIT-Orbitrap) hybrid mass spectrometer operating in negative ion mode and equipped with a heated electrospray ionization (HESI) ion source. The structural information was derived from MS, MS2, MS3 and MS4 experiments acquired in FT-MS mode (LITOrbitrap scans) with the resolution 15,000 – 30,000 and mass accuracy better than 5 ppm. a The ADs are labeled according to Tab. 2; b all values represented the

28


Page 29 of 37


best hits within the mass tolerance of 5 ppm; c the fragmentation patterns and corresponding mass spectra are presented in Fig. S3-4, and the nomenclature of fragments is explained in Fig. 2. The fragments obtained in MS3 and MS4 spectra are marked with one and two asterisks, respectively.

29



Page 30 of 37

Table 2: CYP oxidation network of ADs. The given enzymes use the indicated substrates to form the products in vertical orientation. Green labeled: new from this study. Gray labeled: expected intermediate, but not detected. Miltiradiene (1), abietatriene (2), ferruginol (3), 11-hydroxy ferruginol (4), 7,11-dihydroxy ferruginol (5), 11-hydroxy sugiol (6), pisiferol (7), 11,20-dihydroxy ferruginol (8), 7,11,20-trihydroxy

ferruginol

(9),

11,20-dihydroxy

sugiol

(10),

abietaquinone

(11),

7-keto

abietaquinone (12), 20-hydroxy abietaquinone (13), 7,20-dihydroxy abietaquinone (14), 7-methoxy-11hydroxy ferruginol (15) and 7-methoxy-11,20-dihydroxy ferruginol (16). Reaction Line

Substrates

Products

catalyzed by

2

1 A

RoMiS

spontaneous

CPP

oxidation

3 B

SmCYP76AH1

Line A

2

SmCYP76AH3

C

RoCYP76AH22

4

3

RoCYP76AH4 Line A and B

RoCYP76AH23 6

5

SfCYP76AH24 SmCYP76AH3 RoCYP76AH4 D

RoCYP76AH22

Line C

RoCYP76AH23 8

SfCYP76AH24 7 E

Line C SmCYP76AK1 9

30 ACS Paragon Plus Environment

Page 31 of 37


10 F

SmCYP76AK1

Line D

Pisiferal

Carnosaldehyde

Pisiferic acid

Carnosic acid

SfCYP76AK6 G

RoCYP76AK7

Line C

RoCYP76AK8

11

Spontaneous I

Line C

-

oxidation

12 Spontaneous

Line D

J

-

-

oxidation

Spontaneous

13

Line E

K

O HO

O

oxidation 14 Spontaneous

L

Line F

-

-

oxidation

Spontaneous M

oxidation Line G

Carnosic acid quinone



Page 32 of 37

Spontaneous N

Line G

-

oxidation Carnosol

15 O

unspecific

Line D

-

-

-

16 P

unspecific

Line F

-

-

-


Page 33 of 37


Figures 16 12

13

11 1

GGPP

CPS

CPP

MiS

9 8

19

20

spontaneous

7

5 6

18

12

14

10 4

11 17

20

2

3

15

Miltiradiene

7

Abietatriene

Figure 1

Figure 2



Page 34 of 37

Figure 3


Page 35 of 37


Figure 4



Page 36 of 37

16 12 20 1 2

11 9

15 17

14

10 4

13

8 5

7

6

19

18

Abietatriene (2) CYP76AH1 CYP76AH3-4 CYP76AH22-24

OH

CYP76AK1 CYP76AK8

CYP76AH3-4 CYP76AH22-24

Ferruginol (3)

OH

OH HO

HO

CYP76AK8

Pisiferol (7)

CYP76AK1 CYP76AK8

11-Hydroxy ferruginol (4)

CYP76AH3-4 CYP76AH22-24

spontaneous

OH

OH

OH

O

HO HO

O

HO

O

OH 11,20-Dihydroxy ferruginol (8)

Pisiferal

Abietaquinone (11) 7,11-Dihydroxy ferruginol (5) Methylation CYP76AH3-4 CYP76AK1 spontaneous CYP76AH22-24

CYP76AK8

CYP76AK8

OH

OH HO O

HOOC

OH

O HO

HO HO

O

OH HO

OH Pisiferic acid

Carnosaldehyde CYP76AK8

OH O

HO C

spontaneous

OH

O

HO HO

HO

O 11-Hydroxy sugiol (6) CYP76AK1

O 7-Methoxy-11-hydroxy ferruginol (15) spontaneous

O

OH O

HO HO

O

spontaneous

O Carnosol

7,11,20-Trihydroxy ferruginol (9)

Methylation

OH HO HOOC

O

20-Hydroxy abietaquinone (13)

OH HO

Carnosic acid

7-Methoxy-11,20dihydroxy ferruginol (16)

O

OH 7,20-Dihydroxy bietaquinone (14)

11,20-Dihydroxy sugiol (10)

O 7-Keto abietaquinone (12)

Figure 5


Page 37 of 37


TOC graphic


CYP76 Oxidation Network of Abietane Diterpenes in Lamiaceae

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