Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a

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Biosynthesis of antroquinonol and 4-acetylantroquinonol B via polyketide pathway using orsellinic acid as a ring precursor in Antrodia cinnamomea Kevin Chi-Chung Chou, Shang-Han Yang, Hsiang-Lin Wu, Pei-Yin Lin, TsuLiang Chang, Fuu Sheu, Kai-Hsien Chen, and BEEN-HUANG CHIANG J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04346 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016

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Journal of Agricultural and Food Chemistry

Biosynthesis of antroquinonol and 4-acetylantroquinonol B via polyketide pathway using orsellinic acid as a ring precursor in Antrodia cinnamomea

Kevin Chi-Chung Choua,b, Shang-Han Yangc, Hsiang-Lin Wua, Pei-Yin Linb, Tsu-Liang Changa, Fuu Sheua, Kai-Hsien Chena* and Been-Huang Chiangc* a

Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei, Taiwan, ROC.

b

Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan, ROC

c

Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, ROC.

*Corresponding author Kai-Hsien Chen

E-Mail: [email protected]

Tel: +886-2-33664120

Fax: +886-2-23620849

Been-Huang Chiang

E-Mail: [email protected]

Tel: +886-2-33664120

Fax: +886-2-23620849

-1-

ACS Paragon Plus Environment


Abstract

1 2

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the

3

mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ).

4

Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts

5

from the polyketide pathway, we cultivated this fungus in a culture medium containing

6

[U-13C]oleic acid, then analyzed the crude extracts of the mycelium using UHPLC-MS. We

7

found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2]

8

fragments on the ring backbone were detected in mass spectrum for [13C2]AQ, [13C2]4-AAQB

9

and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA

10

and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring

11

precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated

12

intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A.

13

cinnamomea.

14 15 16

Key words: Antrodia cinnamomea, antroquinonol, 4-acetylantroquinonol B, orsellinic acid,

17

polyketide pathway

18

-2-


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1. Introduction Fungi produce a wide variety of bioactive secondary metabolites, particularly

21

meroterpenoids,1 which are natural products of mixed biosynthetic origin and partially

22

derived from terpenoids.2 Although diverse in structure, meroterpenoids can be grouped into

23

two major classes based on their biosynthetic origins: non-polyketide terpenoids and

24

polyketide terpenoids.1 The non-polyketide group of meroterpenoids is often formed from

25

compounds which arise from the shikimate pathway. Meroterpenoids in which a quinone ring

26

is formed through the shikimate pathway, like coenzyme Q (from 4-hydroxybenzoic acid),

27

plastoquinones (from 4-hydroxyphenylpyruvate) and menaquinones (from isochorismate), are

28

widespread in living organisms and are essential for several life processes.3-5

29

The meroterpene 4-acetylantroquinonol B (4-AAQB, Figure 1(a)) was isolated only

30

from the mycelium cells but not from the fruiting bodies of Antrodia cinnamomea,6 a

31

Taiwanese medicinal mushroom. 4-AAQB has attracted attention in recent years due to its

32

potent cytotoxic activities and anti-hepatocellular carcinoma functions.7-17 Antroquinonol B

33

(AQB, Figure 1(b)) and 4-AAQB were isolated from A. cinnamomea in 2008.6 Antroquinonol

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(AQ, Figure 1(c)), identified in 2007,18 induces crosstalk between apoptosis, autophagy and

35

senescence in human pancreatic carcinoma cells.19 Antroquinonol D

36

(5-demethoxy-antroquinonol, AQD, Figure 1(d)), isolated in 2014, which induces DNA

37

demethylation, reverses the silencing of multiple tumor suppressor genes and induces cancer

38

cell death and inhibits cell migration.20 These AQ-like compounds, 4-AAQB, AQB, AQ and

39

AQD, have similar chemical backbones to that of coenzyme Q (CoQ).6, 18, 20

40

Several researchers have tried to find the precursor via the shikimate pathway in order

41

to increase the yields of AQ-like compounds from the mycelium of A. cinnamomea.

42

4-hydroxybenzoic acid and coenzyme Q0 (CoQ0, Figure 1(e)) were chosen as the potential

43

precursors to provide the possible building blocks for the benzoquinone ring backbone of AQ -3-



44

in submerged fermentation.21 Tyrosine and phenylalanine were suggested to be the potential

45

benzoquinone precursor of AQ.22

46

(TMBA) to the culture medium resulted in a significantly higher yield of 4-AAQB compared

47

with the control group, suggesting that TMBA, a volatile constituent produced via the

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shikimate pathway during submerged fermentation of A. cinnamomea, may be a building

49

block of 4-AAQB.7 Due to the structural similarity, the biosynthetic routes of AQ and

50

4-AAQB are thought to be closely related to the biosynthesis of CoQ via the shikimate

51

pathway in A. cinnamomea.7, 21, 22

52

An addition of 0.2% of 2,4,5-trimethoxybenzaldehyde

The genome of A. cinnamomea isolates was recently deciphered, and eleven putative

53

Coq proteins have been identified based on the query sequences of yeast Coq1∼Coq9.23

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Except for Coq1, all the other Coq protein candidates have a much higher expression in the

55

mycelium than in the fruiting body,23 consistent with the much higher content of AQ in the

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mycelium.24 Therefore, AQ biosynthesis is assumed to be highly related to the CoQ

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biosynthesis genes.23 Current knowledge about the CoQ biosynthetic pathway is mostly

58

derived from the characterization of accumulated intermediates in CoQ-deficient mutant

59

strains of Saccharomyces cerevisiae.3 In yeast, Coq1 synthesizes the hexaprenyl diphosphate

60

tail, and Coq2 adds the hexaprenyl tail to 4-hydroxybenzoic acid, forming

61

3-hexaprenyl-4-hydroxybenzoic acid (HHB). Coq6 adds the first hydroxyl group to the C-5

62

position of the aromatic ring, forming 3-hexaprenyl-4,5- dihydroxybenzoic acid, and further

63

performing O-methylation to form 3-hexaprenyl-5-methoxy-4- hydroxybenzoic acid by Coq3.

64

An undetermined enzyme catalyzes the decarboxylation and hydroxylation steps at C-1,

65

forming 2-demethyl-6-demethoxy CoQ6. Coq5 catalyzes the C-methylation at the C-2

66

position of the aromatic ring, producing 6-demethoxy-CoQ6 (6-DMQ6). Coq7 adds the second

67

hydroxyl to the C-6 position, generating 6-demethyl-CoQ6, followed by the second

68

O-methylation catalyzed by Coq3 to synthesize CoQ6. Coq4, Coq9, Coq10, and Coq11 are -4-


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required for efficient CoQ6 biosynthesis, but their function is yet to be determined.3, 25-30

70

Previous research about the biosynthesis of CoQ had showed that the 4-hydroxybenzoic acid,

71

4-aminobenzoic acid, resveratrol and 4-coumaric acid serve as the benzoquinonol ring

72

precursor of CoQ.29,30 This suggests that CoQ may be formed from different precursors.

73

The quinone ring of meroterpenoids can be formed through the polyketide pathway.

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Albatrellin, isolated from Albatrellus flettii, is a dimeric meroterpenoid with a

75

furylbenzoquinone chromophore responsible for the beautiful velvet-blue color shown by the

76

fruit bodies. The partial structure of albatrellin, via tetraketide product orsellinic acid (OA,

77

Figure 1(f)), is similar to the backbone of CoQ.31 In addition, grifolinone C, formed from OA

78

with similar partial structure to the backbone of CoQ, was obtained from the fruiting bodies of

79

Albatrellus confluens.32 These findings suggest that products of polyketide pathway may have

80

similar chemical backbones to CoQ.

81

A recent study has identified OA and CoQ0 in A. cinnamomea mycelium and proposed

82

that the pks63787, a polyketide synthase gene responsible for the biosynthesis of benzenoids

83

in A. cinnamomea, may also participate in the biosynthesis of 4-AAQB.33 Based on this and

84

other related studies, we hypothesized that A. cinnamomea generates benzoquinone ring

85

precursors for 4-AAQB and AQ biosynthesis via the polyketide pathway. The objective of this

86

study was to confirm this hypothesis. In addition, we also wanted to be certain that OA,

87

formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, is a precursor of AQ

88

and 4-AAQB. The overall objective of this study was to reveal possible biosynthesis

89

pathways of AQ and 4-AAQB.

-5-



90

2. Materials and Methods

91

2.1. Microorganism and reagents

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A. cinnamomea BCRC35716 was obtained from the Bioresource Collection and

93

Research Center of the Food Industry Research and Development Institute (FIRDI, Hsinchu,

94

Taiwan). Potato dextrose agar, malt extract, and peptone were obtained from Difco (Sparks,

95

MD, USA). All the potential aromatic ring precursor compounds, oleic acid, OA, CoQ0 and

96

stable isotope-labeling compounds [U-13C]oleic acid, were obtained from Sigma-Aldrich (St.

97

Louis, MO, USA).

98

2.2. Shake-flask fermentation of A. cinnamomea

99

The seed culture of A. cinnamomea was maintained on potato dextrose agar (39 g/L) at

100

25°C and transferred to a fresh potato dextrose agar plate every 28 days. Normal medium

101

(2.0% glucose, 2.0% malt extract, 0.1% peptone) was prepared as described.7-9 The pH of the

102

medium was adjusted to 5 by adding 0.1 N NaOH or 0.1 N HCl. The prepared medium was

103

sterilized at 121°C for 20 minutes before use. A. cinnamomea colonies from seed culture

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potato dextrose agar were first inoculated into 500 mL flasks containing 200 mL liquid

105

medium and incubated at 25°C for 7 days for mycelium growth. An inoculum of 20 mL was

106

transferred to a 500 mL flask containing 200 mL sterilized medium. Normal and [U-13C]oleic

107

acid of 0.01% were added into medium and the mixture was incubated at 25°C for 7, 14, 21,

108

and 28 days in a rotary shaker at 100 rpm.

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2.3. Sample preparation

110

The mycelium in the fermentation broth was collected through a Whatman No. 1 filter

111

paper and then washed twice with distilled water. After lyophilization, the freeze-dried

112

mycelium (0.1 g) was extracted by 2 mL of 95% ethanol along with sonication for 1 hour. The

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ethanol crude extract was centrifuged at 25°C for 1 hour at 10,000 rpm. The supernatant was

114

moved to a new vial, another 2 mL of 95% ethanol was added to the remaining mycelium and -6-


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same procedure of extraction was repeated two more times. The collected supernatant was

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filtered through 0.45 µm membrane and evaporated to 2 mL using pure nitrogen gas. Samples

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were routinely analyzed by LC-MS after extraction.

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2.4. LC-MS data acquisition and analysis

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The crude extracts of A. cinnamomea mycelium were analyzed by using

120

ultrahigh-performance liquid chromatography coupled to a photo-diode array detector

121

(UltiMate 3000, Thermo Fisher Scientific) and a quadrupole orbital trap mass spectrometer

122

(Q-Exactive, Thermo Fisher Scientific) equipped with an electrospray ionization (ESI)

123

interface. The LC separation was carried out on a Zorbax SB-Aq C18 column (100 mm x 2.1

124

mm i.d., 1.8 µm particle size, Agilent Technologies). The elution was carried out using DI

125

water containing 0.01% formic acid as an eluent (A) and acetonitrile containing 0.01% formic

126

acid as an eluent (B). The elution gradient began at 20% eluent (B) and we ramped the eluent

127

(B) linearly up to 100% over 30 min and it was held at 100% for 10 min. The column was

128

equilibrated for 5 min in the initial condition and was ready for next sample separation round.

129

The flow rate of elution was 0.4 mL/min, and the column temperature was set at 40°C. The

130

injection volume was 5 µL. The photo-diode array detector was used (210~600 nm) and the

131

absorption at wavelength of 254 nm was monitored to detect aromatic ring structures. The

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quadrupole orbital mass spectrometer equipped with a heated electrospray ionization (HESI)

133

probe was operated in the electrospray positive-ion (ESI+) mode. Nitrogen gas generated from

134

a nitrogen generator (Genius 1022, Peak Scientific) was used for sheath, auxiliary and sweep

135

gases. The parameters of the HESI probe was set as follows: sheath gas flow rate, 50 L/min;

136

aux gas flow rate, 13 L/min; sweep gas flow rate, 1 L/min; spray voltage, 3.5 kV; capillary

137

temperature, 320°C; S-lens RF level, 55.0 kV; aux gas heater temperature, 425°C. In full-scan

138

mode, the mass resolution was set at 70,000, and the scan ranges were set from m/z 154 to

139

212 (for OA and Q0) and from m/z 328 to 495 (for the farnesylated intermediates of AQ and -7-



140

4-AAQB). In data-dependent MS/MS (DDMS2) mode, the mass isolation window of

141

quadrupole was set at 0.4 m/z to select target ions as precursors and high-energy collision

142

dissociation (HCD) was performed in the collision cell using normalized collision energies

143

(NCE) to generate product ions. The mass resolution setting was 70,000 in DDMS2 mode.

144

Data processing was performed using the Xcalibur Qual Browser (ver. 4.0.27.10, Thermo

145

Fisher Scientific). The qualitative mass spectrum data collected in this study were from

146

triplicate samples (n=3), independently extracted with duplicate or triplicate instrumental

147

analysis.

-8-


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3. Results and Discussion

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3.1. Prediction of biosynthetic intermediates via the polyketide pathway in A.

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cinnamomea

151

For the consistency of the carbon’s position on the ring backbone in this study, we used

152

the same numbering to describe the carbon position on the ring of all the related compounds.

153

The carbon position of the aromatic farnesylation is assigned at C-3. Rather than the name of

154

2,4-dihydroxy-6-methyl-benzoic acid, the name 2-methyl-4,6- dihydroxybenzoic acid is used

155

to describe the functional group position on the aromatic ring of OA in this report. We

156

assumed that the benzoquinone ring formation is via the polyketide pathway and follows

157

similar biosynthetic sequences to that of CoQ. Therefore, the first farnesyl intermediate of AQ

158

should be the 2-methyl-3-farnesyl-4,6-dihydroxybenzoic acid or 3-farnesyl-orsellinic acid

159

(FOA, Figure 1(g)). After C-3 farnesylation, FOA undergoes the O-methylation at C-6, a

160

decarboxylation following a hydroxylation at C-1 to form 5-demethoxy-CoQ3 (5-DMQ3,

161

Figure 1(h)). Finally, a further hydroxylation and a methylation at C-5 occurs to form CoQ3

162

(Figure 1(i)), and then AQ.

163

There are two types of gamma-lactone groups on the prenyl tail of meroterpenoid,

164

formed by condensation of the terminal carboxyl group with the hydroxyl group on terpene

165

backbones, or formed by the cyclization of oxidized terpene.1 4-AAQB, which has the

166

traditional farnesyl backbone with a terminal 5-carbon lactone ring modification, is more

167

likely to be formed by condensation of the terminal carboxyl group with the hydroxyl group

168

on the terpene backbone. If the terminal gamma-lactone modification proceeds after

169

farnesylation of the aromatic precursors, intermediates similar to erythrolic acid E should be

170

detected. Erythrolic acid E is an unusual meroterpenoid isolated from the bacterium

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Erythrobacter sp. derived from a marine sediment sample collected in Galveston, TX.

172

Erythrolic acid E has a terminal carboxyl group and a hydroxyl group on a farnesyl backbone. -9-



173

The unusual nature of the terpene side chain involves an oxidation of a terminal methyl group

174

to a carboxylic acid and subsequent Claisen condensation with acetyl-CoA.34 If the terminal

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gamma-lactone modification proceeds before the prenylation of the aromatic precursors, the

176

first prenylated intermediate for OA should be FOAB (Figure 1(j)), an FOA with a

177

gamma-lactone modification on the farnesyl tail terminal.

178

The similar suffix “B” of the intermediate FOAB as the suffix of 4-AAQB, is used to

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describe intermediates with a gamma-lactone modification on the farnesyl tail terminal. The

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farnesyl with a terminal gamma-lactone modification is called “farnesyl B”, and farnesyl

181

diphosphate (FPP) with terminal gamma-lactone modification is called “FPPB” in this report.

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A parallel route with similar biosynthetic sequences for OA to form 4-AAQB can be expected.

183

After the formation of FOAB, the intermediate follows similar sequences of ring modification

184

to form 5-DMQ3B (Figure 1(k)), CoQ3B (Figure 1(l)) and 4-AAQB.

185 186

3.2. Detection of the predicted intermediates of AQ and 4-AAQB biosynthesis

187

in A. cinnamomea

188

The related information of molecular formula, retention time (RT) of HPLC elution,

189

exact mass of the precursor ion and predominant product ions used for the detection of the

190

target compounds in this study, are listed in Table 1. The crude extract prepared from A.

191

cinnamomea mycelium was analyzed by reverse-phase UHPLC-PDA coupled with a

192

high-resolution quadrupole orbital trap mass spectrometer. To identify the predicted

193

intermediates, the high mass resolution of mass spectrometer is needed to distinguish m/z

194

differences smaller than 0.001 at the same retention time due to the complicated co-elute

195

composition in the crude extract. The identification of these farnesylated intermediates was

196

based on the presence of an HPLC peak with a high-resolution mass spectrum with the mass

197

accuracy of 5 ppm for both precursor and product ions. The product ion information of these -10-


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intermediates was based on the mass spectrum data of CoQ6, 6-DMQ6 and HHB reported in

199

previous yeast CoQ6 biosynthesis studies.25-30

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Most of the prenylated intermediates of yeast CoQ6 are unstable and difficult to detect

201

by GC-MS without derivatization.25 To accumulate HHB in the yeast cell, a yeast coq3 mutant

202

was cultured.30 However, there is no available A. cinnamomea mutant similar to the yeast

203

coq3 mutant. The amount of the predicted intermediates, FOA and FOAB, in the crude extract

204

was below the limit of detection and we were not able to obtain the DDMS2 product ion

205

spectrum of these unstable intermediates in the crude extract of A. cinnamomea mycelium cell

206

in this study.

207

6-DMQ6 is a relatively stable intermediate present in lipid extracts of wild-type

208

yeast.26,28 In the crude extract of A. cinnamomea mycelium, we also detected the 5-DMQ3

209

(RT=19.97 min). The precursor ion [M+H]+ of 357.24242, and predominant tropylium m/z

210

167.07027 product ions of 5-DMQ3 (Figure 2(a)) were consistent with the mass spectrum

211

obtained from 6-DMQ6 of yeast in a previous study.25 The tropylium-like product ion is a

212

transition ion generated from prenylated aromatic and benzoquinone rings which is formed

213

under dissociation conditions by incorporation of a methylene remnant (produced by

214

fragmentation after the first carbon of prenyl tail) to form a 7-carbon membered ring.25 A

215

similar product ion had been reported in HHB, 6-DMQ6 and CoQ6 analysis of yeast CoQ6

216

biosynthesis.25-30 This is an important characteristic collision dissociation behavior of the

217

compounds with the structure of prenylated aromatic and benzoquinone rings.35 CoQ3 was

218

also detected in the crude extract. The precursor ion [M+H]+ of 387.25299, and the

219

predominant tropylium m/z 197.08084 product ions were found at RT=20.41 min (Figure

220

2(e)). The mass spectrum of CoQ3 was consistent with that of the methoxyl group added

221

5-DMQ3.

222

5-DMQ3B and CoQ3B, the predicted intermediates of 4-AAQB with the terminal -11-



223

gamma-lactone modification of 5-DMQ3 and CoQ3, were also detected in the crude extract. A

224

similar fragmentation pattern in mass spectrometry as those of 5-DMQ3 and CoQ3 were also

225

found. The precursor ion [M+H]+ of 387.21660 and predominant tropylium m/z 167.07027

226

product ions of 5-DMQ3B at RT=15.56 min (Figure 3(a)), were consistent with the collision

227

induced dissociation patterns present in 5-DMQ3. The precursor ion [M+H]+ of 417.22717,

228

and predominant tropylium m/z 197.08084 product ions of CoQ3B at RT=16.02 min (Figure

229

3(c)), were consistent with the product ion spectrum present in CoQ3.

230

The chromenylium-like ion was found in the product ion spectrum of 5-DMQ3

231

(C12H15O3+; exact mass, 207.10157, Figure 2(a)), CoQ3 (C13H17O4+; exact mass, 237.11214,

232

Figure 2(e)), 5-DMQ3B (C12H15O3+; exact mass, 207.10157, Figure 3(a)) and CoQ3B

233

(C13H17O4+; exact mass, 237.11214, Figure 3(c)). The chromenylium-like ion is larger than

234

the tropylium ion in mass by m/z 40.03130 (C3H4) under the electrospray ionization and is

235

derived by fragmentation and cyclization to include the first four prenyl tail carbons.30,35

236

The precursor ion [M+H]+ of 391.28429, m/z 181.08592 (tropylium ion) and m/z

237

221.11722 (chromenylium-like ion) of AQ were detected at RT=22.26 min (Figure 2(g)). The

238

precursor ion [M+H]+ of 421.25847, m/z 181.08592 (tropylium ion) and m/z 221.11722

239

(chromenylium-like ion) of AQB were also detected at RT=13.10 min (Figure 3(e)). The

240

precursor ion [M+H]+ of 463.26903, m/z 181.08592 (tropylium ion) and m/z 221.11722

241

(chromenylium-like ion) of 4-AAQB were detected at RT=15.94 min (Figure 3(g)). Similar

242

collision dissociation behaviors of 4-AAQB, AQB and AQ were found in the mass spectrum,

243

and the product ion spectrum of AQ, AQB and 4-AAQB were consistent with spectrum of

244

these compounds purified in a previous study.7

245

We also found OA and CoQ0 in the crude extract from mycelium cells cultured in

246

normal broth. The precursor ion [M+H]+ of 169.04954, and m/z 151.03897 product ions of

247

OA at RT=2.45 min (Figure 4(b)) were consistent with those of OA commercial standard -12-


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248

(Figure 4(a)). The precursor ion [M+H]+ of 183.06519, m/z 168.04171 and m/z 137.05971

249

product ions of CoQ0 at RT= 3.43 min (Figure 4(e)) were also consistent with those of CoQ0

250

commercial standard (Figure 4(d)). An HPLC peak eluting at RT=3.58 min with a mass

251

spectrum in accordance with [M+H]+=C8H11O3+ was found (Figure 4(g)). The precursor ion

252

[M+H]+ of 155.07027, m/z 109.02841 and m/z 127.03897 product ions were consistent with

253

the predicted intermediate of 5-demethoxy-CoQ0 (5-DMQ0, Figure 1(m)).

254 255

3.3. Comparison of normal and 13C-labeled form of predicted intermediates Metabolic labeling studies with stable isotopes provide a definitive result, because the

256 257

labeled carbons can be detected in both precursor and product ions by mass spectrometry.

258

After identifying predicted intermediates, we wished to generate both the normal and

259

13

260

commercial [13C6-ring]OA or [13C6-ring]CoQ0 available, [U-13C]oleic acid was added as an

261

alternative in the fermentation culture medium to provide [13C2]acetyl-CoA, the

262

beta-oxidation product of oleic acid and the precursor in the polyketide pathway. The

263

theoretical carbon isotope natural distribution [13C2]/[U-12C] ratio of OA is 0.8019% and that

264

of CoQ0 is 0.8046%. We expected to see a much higher [13C2]/[U-12C] ratio with significant

265

differences in the ring backbone of target compounds from the [U-13C]oleic acid cultured

266

mycelium compared with those from normal oleic acid mycelium if OA was formed via the

267

polyketide pathway. Both the normal isotopic form and the [13C2] form of OA and CoQ0 were

268

detected in crude extracts from the [U-13C]oleic acid cultured mycelium. The [13C2]/[U-12C]

269

ratio of OA was about 10.2% and the [13C2]/[U-12C] ratio of CoQ0 was about 10.6% in the

270

crude extract from [U-13C] oleic acid cultured mycelium. The [13C2]/[U-12C] ratio of OA was

271

about 0.9% and the [13C2]/[U-12C] ratio of CoQ0 was about 0.9% in the crude extract from

272

normal oleic acid cultured mycelium. A much higher amount of [13C2]OA and [13C2]CoQ0

C-labeled form of predicted intermediates for the purpose of comparison. Since there is no

-13-



273

were found from the [U-13C] oleic acid cultured mycelium as compared with normal oleic

274

acid cultured mycelium. This result suggests that most of the [13C2]OA and [13C2]CoQ0

275

detected in the crude extract from [U-13C]oleic acid cultured mycelium are formed through

276

[13C2]acetyl-CoA. Additionally, the obvious [13C2] pattern on the ring fragments were found in

277

the [13C2]OA, [13C2]5-DMQ0 and [13C2]CoQ0 product ion spectrum (Figure 4(c), 4(h) and

278

4(f)). These results also suggest that the both OA and CoQ0 are formed via the polyketide

279

pathway. The modification of OA and CoQ0 to form 4-AAQB in A. cinnamomea should not

280

be rate-limiting due to the high yields of 4-AAQB and extremely low content of OA and

281

CoQ0 that were found in the crude extract of mycelium.

282

Two HPLC peaks with very similar pattern of precursor ion [C23H32O3+H]+ (DMQ3)

283

and tropylium ion m/z 167.07027 in the product ion spectrum were found at RT=14.84 min

284

and RT=19.97 min. A much higher amount of [13C2]DMQ3 at RT=19.97 min was found from

285

the [U-13C] oleic acid cultured mycelium as compared with that from normal oleic acid

286

cultured mycelium. The [13C2]/[U-12C] ratio of DMQ3 eluted at RT=19.97 min was about

287

3.2% in the crude extract from normal oleic acid cultured mycelium. The [13C2] /[U-12C] ratio

288

of DMQ3 eluted at RT=19.97 min was about 22.1% in the crude extract from [U-13C]oleic

289

acid cultured mycelium. Both the normal isotopic form and the [13C2] form of DMQ3 were

290

observed in the crude extract from [U-13C]oleic acid cultured mycelium at RT=19.97 min. As

291

shown in Figure 2(a) and 2(b), the tropylium product ion of [13C2] DMQ3 shows the obvious

292

[13C2] fragments, which means that the 13C-label is on the benzoquinone ring via

293

[13C2]acetyl-CoA. The [13C2]/[U-12C] ratio of DMQ3 eluted at RT=14.84 min from

294

[U-13C]oleic acid cultured mycelium is identical with that from normal oleic acid cultured

295

mycelium, and no obvious [13C2] fragments pattern on the ring fragments in the spectrum

296

were found (Figure 2(c) and 2(d)). The result suggests that the [13C2] from acetyl-CoA is

297

labeled on the compound eluted at RT=19.97 min, but not on that eluted at RT=14.84 min. In -14-


Page 14 of 37

Page 15 of 37


298

other words, the DMQ3 eluted at RT=19.97 min was formed via the polyketide pathway.

299

Referring to the polyketide pathway product OA, the first methoxyl modification of OA

300

should be at the C-6 hydroxyl methylation of the benzoquinone ring. The compound eluted at

301

RT=19.97 min should be 5-DMQ3. The compound eluted at RT=14.84 min could be 6-DMQ3

302

(Figure 1(n)) via the shikimate pathway, and further confirmation for this compound is

303

needed.

304

The [13C2]CoQ3 (Figure 2(f)),and [13C2]AQ (Figure 2(h)) detected in the crude extract

305

from the [U-13C]oleic acid cultured mycelium also show the obvious [13C2] fragments on the

306

ring backbone in mass spectrum as compared with normal isotopic forms of these compounds,

307

and the much higher [13C2]/[U-12C] ratio of CoQ3 and AQ were found (23.5% and 22.7%,

308

respectively). These mass spectrum evidences suggest that the ring precursor of AQ is

309

synthesized via the polyketide pathway.

310

An HPLC peak of precursor ion [C23H30O5+H]+ (DMQ3B) in the ion chromatogram at

311

RT=15.56 min was found. A much higher amount of [13C2]DMQ3B in the extract from the

312

[U-13C] oleic acid cultured mycelium was found as compared with that from normal oleic acid

313

cultured mycelium ([13C2] /[U-12C] ratio =26.4%). In the product ion spectrum of

314

[13C2]DMQ3B, the tropylium product ion showed the obvious [13C2] fragments. In reference to

315

the polyketide pathway product OA, the compound that eluted at RT=15.56 min should be

316

5-DMQ3B.

317

As shown in Figure 3, both the normal isotopic form and [13C2] form of 5-DMQ3B

318

(Figure 3(a) and 3(b)), CoQ3B (Figure 3(c) and 3(d)), AQB (Figure 3(e) and 3(f)) and

319

4-AAQB (Figure 3(g) and 3(h)) were detected in the crude extract from [U-13C]oleic acid

320

cultured mycelium cells. A much higher [13C2]/[U-12C] ratio (24.1%, 22.9% and 23.1%,

321

respectively) and similar pattern of obvious [13C2] fragments as that of [13C2]5-DMQ3 were

322

also found in CoQ3B, AQB and 4-AAQB. This result suggests that the benzoquinone rings of -15-



323

4-AAQB is also derived from the polyketide pathway.

324 325

3.4. Postulated biosynthetic routes of AQ and 4-AAQB in A. cinnamomea via

326

the polyketide pathway

327

Based on our findings in this study, the postulated biosynthetic routes of AQ and

328

4-AAQB via the polyketide pathway in A. cinnamonea are shown in Figure 5. AQ

329

biosynthesis through the polyketide pathway starts from the formation of OA via the

330

condensation of one unit of acetyl-CoA and three units of malonyl-CoA. Two parallel routes

331

are possible to form AQ after the formation of OA. One route follows the similar biosynthetic

332

sequences as that of yeast CoQ6, starts from the farnesylaton of OA to form FOA by Coq2,

333

and undergoes C-6 O-methylation by Coq3. A decarboxylation with a hydroxylation at C-1

334

forms 5-DMQ3, and a C-5 hydroxylation with a methylation forms CoQ3 by Coq6 and Coq3,

335

and then through an unknown process, forms AQ. Alternatively, farnesylation of 5-DMQ0 or

336

CoQ0, the ring modification product of OA, can proceed to form 5-DMQ3 or CoQ3, and then

337

through an unknown process, forms AQ. Although Wang et al. isolated AQD,

338

5-demethoxy-antroquinonol, from A. cinnamomea, we were not able to identify this

339

compound in the mass spectrum of the crude extract of mycelium in this study. However, if

340

AQD does exists, judging by its structure, this compound may be directly formed from

341

5-DMQ3, bypassing the methoxylation modification by Coq6 and Coq3. The lack of a

342

methoxyl at C-5 of the AQD structure suggests that AQD is also formed via the polyketide

343

pathway.

344

In this study, we did not find any mass spectrum evidences to support the existence of

345

farnesyl terminal hydroxylated or carboxylated intermediates in crude extracts. These results

346

suggest that the farnesyl B is formed before the aromatic farnesylation in 4-AAQB

347

biosynthesis. It is assumed that the bonding of farnesyl B to OA, 5-DMQ0 or CoQ3 is the -16-


Page 16 of 37

Page 17 of 37


348

decision-making step of AQ and 4-AAQB biosynthesis via the polyketide pathway. Similar to

349

the parallel branches in AQ biosynthesis, one possible route of 4-AAQB biosynthesis is from

350

the addition of farnesyl B to OA, further modification to form 5-DMQ3B, CoQ3B, AQB and

351

then 4-AAQB. Other alternatives are adding farnesyl B to the ring modification product of

352

OA, 5-DMQ0 or CoQ0, to form 5-DMQ3B or CoQ3B, and further modifications to form AQD,

353

AQB and 4-AAQB.

354

In summary, our analyses deduced that orsellinic acid synthesized via the polyketide

355

pathway is a novel benzoquinone ring precursor for biosynthesis of AQ and 4-AAQB in A.

356

cinnamomea. Findings in this study suggest that AQ and 4-AAQB follow the similar

357

biosynthetic sequences as that of CoQ, and the two biosynthetic routes for AQ and 4-AAQB

358

branch at the decision-making aromatic farnesylation step.

-17-



Reference

359 360

(1)

Geris, R.; Simpson, T. J., Meroterpenoids produced by fungi. Nat. Prod. Rep. 2009, 26, 1063-94.

361 362

(2)

Cornforth, J. W., Terpenoid biosynthesis. Chem. Brit. 1968, 4, 102-6.

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(3)

Tran, U. C.; Clarke, C. F., Endogenous synthesis of coenzyme Q in eukaryotes. Mitochondrion. 2007, 7 Suppl, S62-71.

364 365

(4)

Norris, S. R.; Barrette, T. R.; DellaPenna, D., Genetic dissection of carotenoid synthesis

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in Arabidopsis defines plastoquinone as an essential component of phytoene

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desaturation. Plant Cell. 1995, 7, 2139-49.

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BBA-Bioenergetics. 2010, 1797, 1587-605.

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Nowicka, B.; Kruk, J., Occurrence, biosynthesis and function of isoprenoid quinones.

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Yang, S. S.; Wang, G. J.; Wang, S. Y.; Lin, Y. Y.; Kuo, Y. H.; Lee, T. H., New

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Constituents with iNOS inhibitory activity from mycelium of Antrodia camphorata.

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Planta Med. 2009, 75, 512-16.

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Chiang, C. C.; Huang, T. N.; Lin, Y. W.; Chen, K. H.; Chiang, B. H., Enhancement of

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4-acetylantroquinonol B production by supplementation of its precursor during

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submerged fermentation of Antrodia cinnamomea. J. Agric. Food Chem. 2013, 61,

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9160-5.

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Lin, Y. W.; Pan, J. H.; Liu, R. H.; Kuo, Y. H.; Sheen, L. Y.; Chiang, B. H., The

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4-acetylantroquinonol B isolated from mycelium of Antrodia cinnamomea inhibits

379

proliferation of hepatoma cells. J. Sci. Food Agric. 2010, 90, 1739-44.

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Lin, Y. W.; Chiang, B. H., 4-Acetylantroquinonol B isolated from Antrodia cinnamomea

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arrests oroliferation of human hepatocellular carcinoma HepG2 cell by affecting p53,

382

p21 and p27 levels. J. Agric. Food Chem. 2011, 59, 8625-31.

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(10) Hsu, Y. L.; Kuo, Y. C.; Kuo, P. L.; Ng, L. T.; Kuo, Y. H.; Lin, C. C., Apoptotic effects of -18-


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extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma

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cell lines. Cancer Lett. 2005, 221, 77-89.

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(11) Song, T. Y.; Hsu, S. L.; Yen, G. C., Induction of apoptosis in human hepatoma cells by

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mycelia of Antrodia camphorata in submerged culture. J. Ethnopharmacol. 2005, 100,

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158-67.

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(12) Song, T. Y.; Hsu, S. L.; Yeh, C. T.; Yen, G. C., Mycelia from Antrodia camphorata in

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submerged culture induce apoptosis of human hepatoma HepG2 cells possibly through

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regulation of Fas pathway. J. Agric. Food Chem. 2005, 53, 5559-64.

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(13) Kuo, P. L.; Hsu, Y. L.; Cho, C. Y.; Ng, L. T.; Kuo, Y. H.; Lin, C. C., Apoptotic effects of

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Antrodia cinnamomea fruiting bodies extract are mediated through calcium and

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calpain-dependent pathways in Hep 3B cells. Food Chem. Toxicol. 2006, 44, 1316-26.

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(14) Hsu, Y. L.; Kuo, P. L.; Cho, C. Y.; Ni, W. C.; Tzeng, T. F.; Ng, L. T.; Kuo, Y. H.; Lin, C.

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C., Antrodia cinnamomea fruiting bodies extract suppresses the invasive potential of

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human liver cancer cell line PLC/PRF/5 through inhibition of nuclear factor κB pathway.

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Food Chem. Toxicol. 2007, 45, 1249-57.

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(15) Chang, C. Y.; Huang, Z. N.; Yu, H. H.; Chang, L. H.; Li, S. L.; Chen, Y. P.; Lee, K. Y.;

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Chuu, J. J., The adjuvant effects of Antrodia camphorata extracts combined with

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anti-tumor agents on multidrug resistant human hepatoma cells. J. Ethnopharmacol.

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2008, 118, 387-95.

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(16) Chiang, P. C.; Lin, S. C.; Pan, S. L.; Kuo, C. H.; Tsai, I. L.; Kuo, M. T.; Wen, W. C.;

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Chen, P.; Guh, J. H., Antroquinonol displays anticancer potential against human

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hepatocellular carcinoma cells: a crucial role of AMPK and mTOR pathways. Biochem.

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Pharmacol. 2010, 79, 162-71.

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(17) Hsieh, Y. C.; Rao, Y. K.; Whang-Peng, J.; Huang, C. Y. F.; Shyue, S. K.; Hsu, S. L.; Tzeng, Y. M., Antcin B and its ester derivative from Antrodia camphorata induce -19-



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apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress

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coincident with activation of intrinsic and extrinsic apoptotic pathway. J. Agric. Food

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Chem. 2011, 59, 10943-54.

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(18) Lee, T. H.; Lee, C. K.; Tsou, W. L.; Liu, S. Y.; Kuo, M. T.; Wen, W. C., A new cytotoxic

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agent from solid-state fermented mycelium of Antrodia camphorata. Planta Med. 2007,

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73, 1412-5.

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(19) Yu, C. C.; Chiang, P. C.; Lu, P. H.; Kuo, M. T.; Wen, W. C.; Chen, P. N.; Guh, J. H.,

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Antroquinonol, a natural ubiquinone derivative, induces a cross talk between apoptosis,

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autophagy and senescence in human pancreatic carcinoma cells. J. Nutr. Biochem. 2012,

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23, 900-7.

419

(20) Wang, S. C.; Lee, T. H.; Hsu, C. H.; Chang, Y. J.; Chang, M. S.; Wang, Y. C.; Ho, Y. S.;

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Wen, W. C.; Lin, R. K., Antroquinonol D, isolated from Antrodia camphorata, with

421

DNA demethylation and anticancer potential. J. Agric. Food Chem. 2014, 62, 5625-35.

422

(21) Hu, Y. D.; Zhang, B. B.; Xu, G. R.; Liao, X. R.; Cheung, P. C.K., A mechanistic study

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on the biosynthetic regulation of bioactive metabolite antroquinonol from edible and

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medicinal mushroom Antrodia camphorata. J. Funct. Foods. 2016, 25, 70-9.

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(22) Hu, Y. D.; Zhang, H.; Lu, R. Q.; Liao, X. R.; Zhang, B. B.; Xu, G. R., Enabling the

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biosynthesis of antroquinonol in submerged fermentation of Antrodia camphorata.

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Biochem. Eng. J. 2014, 91, 157-62.

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(23) Lu, M. Y.; Fan, W. L.; Wang, W. F.; Chen, T. C.; Tang, Y. C.; Chu, F. H.; Chang, T. T.;

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Wang, S. Y.; Li, M. Y.; Chen, Y. H.; Lin, Z. S.; Yang, K. J.; Chen, S. M.; Teng, Y. C.; Lin,

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Y. L.; Shaw, J. F.; Wang, T. F.; Li, W. H., Genomic and transcriptomic analyses of the

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medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual

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development. P. Natl. Acad. Sci. U.S.A. 2014, 111, E4743-52.

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(24) Kumar, K. J. S.; Chu, F. H.; Hsieh, H. W.; Liao, J. W.; Li, W. H.; Lin, J. C. C.; Shaw, J. -20-


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F.; Wang, S. Y., Antroquinonol from ethanolic extract of mycelium of Antrodia

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cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2

436

activation. J. Ethnopharmacol. 2011, 136, 168-77.

437

(25) Poon, W. W.; Marbois, B. N.; Faull, K. F.; Clarke, C. F., 3-Hexaprenyl-4-

438

hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis

439

in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 1995, 320, 305-14.

440

(26) Padilla, S.; Jonassen, T.; Jimenez-Hidalgo, M. A.; Fernandez-Ayala, D. J. M.;

441

Lopez-Lluch, G.; Marbois, B.; Navas, P.; Clarke, C. F.; Santos-Ocana, C.,

442

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration

443

in Saccharomyces cerevisiae and lacks antioxidant activity. J. Biol. Chem. 2004, 279,

444

25995-6004.

445

(27) Marbois, B.; Gin, P.; Faull, K. F.; Poon, W. W.; Lee, P. T.; Strahan, J.; Shepherd, J. N.;

446

Clarke, C. F., Coq3 and Coq4 define a polypeptide complex in yeast mitochondria for

447

the biosynthesis of coenzyme Q. J. Biol. Chem. 2005, 280, 20231-8.

448

(28) Tran, U. C.; Marbois, B.; Gin, P.; Gulmezian, M.; Jonassen, T.; Clarke, C. F.,

449

Complementation of Saccharomyces cerevisiae coq7 mutants by mitochondrial

450

targeting of the Escherichia coli UbiF polypeptide - Two functions of yeast Coq7

451

polypeptide in coenzyme Q biosynthesis. J. Biol. Chem. 2006, 281, 16401-9.

452

(29) Marbois, B.; Xie, L. X.; Choi, S.; Hirano, K.; Hyman, K.; Clarke, C. F.,

453

para-Aminobenzoic acid is a precursor in coenzyme Q6 biosynthesis in Saccharomyces

454

cerevisiae. J. Biol. Chem. 2010, 285, 27827-38.

455

(30) Xie, L. X.; Williams, K. J.; He, C. H.; Weng, E.; Khong, S.; Rose, T. E.; Kwon, O.;

456

Bensinger, S. J.; Marbois, B. N.; Clarke, C. F., Resveratrol and para-coumarate serve as

457

ring precursors for coenzyme Q biosynthesis. J. Lipid Res. 2015, 56, 909-19.

458

(31) Koch, B.; Steglich, W., Meroterpenoid pigments from Albatrellus flettii -21-



459

(Basidiomycetes). Eur. J. Org. Chem. 2007, 1631-5.

460

(32) Yang, X. L.; Qin, C.; Wang, F.; Dong, Z. J.; Liu, J. K., A new meroterpenoid pigment

461

from the basidiomycete Albatrellus confluens. Chem. Biodivers. 2008, 5, 484-9.

462

(33) Yu, P. W.; Chang, Y. C.; Liou, R. F.; Lee, T. H.; Tzean, S. S., pks63787, a polyketide

463

synthase gene responsible for the biosynthesis of benzenoids in the medicinal

464

mushroom Antrodia cinnamomea. J. Nat. Prod. 2016, 79, 1485-91.

465

(34) Hu, Y. C.; Legako, A. G.; Espindola, A. P. D. M.; MacMillan, J. B., Erythrolic acids A-E,

466

meroterpenoids from a marine-derived Erythrobacter sp. J. Org. Chem. 2012, 77,

467

3401-7.

468 469

(35) Elliot, W. H.; Waller, G. R., In Biochemical applications of mass spectrometry, Waller, G. R., Ed. Wiley-Interscience: New York, N.Y., 1972; pp 499-536.

-22-


Page 22 of 37

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Figure captions

470 471 472

Figure 1. Structures of the predicted intermediates of AQ and 4-AAQB biosynthesis in A. cinnamomea.

473

(a) 4-acetylantroquinonol B (4-AAQB); (b) antroquinonol B (AQB); (c) antroquinonol (AQ);

474

(d) antroquinonol D (AQD); (e) conenzyme Q0 (CoQ0) (f) orsellinic acid

475

(2-methyl-4,6-dihydroxybenzoic acid, OA); (g) 3-farnesyl-orsellinic acid (FOA); (h)

476

5-demethoxy-coenzyme Q3 (5-DMQ3); (i) Coenzyme Q3 (CoQ3); (j) 3-farnesyl-orsellinic

477

acid B (FOAB); (k) 5-demethoxy-coenzyme Q3 B (5-DMQ3B); (l) coenzyme Q3 B

478

(CoQ3B); (m) 5-demethoxy-coenzyme Q0 (5-DMQ0); (n) 6-demethoxy-coenzyme Q3

479

(6-DMQ3).

480 481 482

Figure 2. Detection of 5-DMQ3, 6-DMQ3, CoQ3, and AQ in crude extracts of A. cinnamomea cultured in the absence or presence of [U-13C]oleic acid.

483

(a)-(h) show the DDMS2 product ion spectrum:

484

(a) 5-DMQ3 [M+H]+ precursor ion (C23H33O3+; exact mass 357.24242), the 5-DMQ3

485

tropylium product ion [m]+ (C9H11O3+; exact mass, 167.07027) and the

486

chromenylium-like product ion [m]+ (C12H15O3+; exact mass, 207.10157);

487

(b) [13C2]5-DMQ3 [M+H]+ precursor ion (13C2C21H33O3+; exact mass 359.24913), the

488

[13C2]5-DMQ3 tropylium product ion [m]+ (13C2C7H11O3+; exact mass, 169.07698) and

489

the chromenylium-like product ion [m]+ (13C2C10H15O3+; exact mass, 209.10828);

490

(c) 6-DMQ3 [M+H]+ precursor ion (C23H33O3+; exact mass 357.24242), the 6-DMQ3

491

tropylium product ion [m]+ (C9H11O3+; exact mass, 167.07027) and the

492


493 494

(d) [13C2]6-DMQ3 [M+H]+ precursor ion (13C2C21H33O3+; exact mass 359.24913), no obvious [13C2] fragments of tropylium and chromenylium-like product ion were found in -23-



495 496

this mass spectrum; (e) CoQ3 [M+H]+ precursor ion (C24H35O4+; exact mass 387.25299), the CoQ3 tropylium

497

product ion [m]+ (C10H13O4+; exact mass, 197.08084) and the chromenylium-like product

498

ion [m]+ (C13H17O4+; exact mass, 237.11214);

499

(f) [13C2]CoQ3 [M+H]+ precursor ion (13C2C22H35O4+; exact mass 389.25970), the

500

[13C2]CoQ3 tropylium product ion [m]+ (13C2C8H13O4+; exact mass, 199.08755) and the

501

chromenylium-like product ion [m]+ (13C2C11H17O4+; exact mass, 239.11885);

502

(g) AQ [M+H]+ precursor ion (C24H39O4+; exact mass 391.28429), the AQ tropylium product

503

ion [m]+ (C10H13O3+; exact mass, 181.08592) and AQ chromenylium-like product ion

504

[m]+ (C13H17O3+; exact mass, 221.11722);

505

(h) [13C2]AQ [M+H]+ precursor ion (13C2C22H39O4+; exact mass 393.29100), the [13C2]AQ

506

tropylium product ion [m]+ (13C2C8H13O3+; exact mass, 183.09263) and AQ

507

chromenylium-like product ion [m]+ (13C2C11H17O3+; exact mass, 223.12393).

508 509 510

Figure 3. Detection of 5-DMQ3B, CoQ3B, AQB and 4-AAQB in crude extracts of A. cinnamomea cultured in the absence or presence of [U-13C]oleic acid.

511

(a)-(h) show the DDMS2 product ion spectrum:

512

(a) 5-DMQ3B [M+H]+ precursor ion (C23H31O5+; exact mass 387.21660), the 5-DMQ3B

513

tropylium product ion [m]+ (C9H11O3+; exact mass, 167.07027), and chromenylium-like

514

product ion [m]+ (C12H15O3+; exact mass, 207.10157);

515

(b) [13C2]5-DMQ3B [M+H]+ precursor ion (13C2C21H31O5+; exact mass 389.22331), the

516

[13C2]5-DMQ3B tropylium product ion [m]+ (13C2C7H11O3+; exact mass, 169.07698) and

517


518

(c) CoQ3B [M+H]+ precursor ion (C24H33O6+; exact mass 417.22717), the CoQ3B tropylium

519

product ion [m]+ (C10H13O4+; exact mass, 197.08084) and chromenylium-like product -24-


Page 24 of 37

Page 25 of 37

520 521


ion [m]+ (C13H17O4+; exact mass, 237.11214); (d) [13C2]CoQ3B [M+H]+ precursor ion (13C2C22H33O6+; exact mass 419.23388), the

522

[13C2]CoQ3B tropylium product ion [m]+ (13C2C8H13O4+; exact mass, 199.08755) and

523


524

(e) AQB [M+H]+ precursor ion (C24H37O6+; exact mass 421.25847), the AQB tropylium

525

product ion [m]+ (C10H13O3+; exact mass, 181.08592) and AQB chromenylium-like

526

product ion [m]+ (C13H17O3+; exact mass, 221.11722);

527

(f) [13C2]AQB [M+H]+ precursor ion (13C2C22H37O6+; exact mass 423.26518), the

528

[13C2]AQB tropylium product ion [m]+ (13C2C8H13O3+; exact mass, 183.09263) and

529

[13C2]AQB chromenylium-like product ion [m]+ (13C2C11H17O3+; exact mass,

530

223.12393);

531

(g) 4-AAQB [M+H]+ precursor ion (C26H39O7+; exact mass 463.26903) and the 4-AAQB

532

tropylium product ion [m]+ (C10H13O3+; exact mass, 181.08592) and 4-AAQB

533


534

(h) [13C2] 4-AAQB [M+H]+ precursor ion (13C2C24H39O7+; exact mass 465.27574), the

535

[13C2]4-AAQB tropylium product ion [m]+ (13C2C8H13O3+; exact mass, 183.09263) and

536

[13C2]4-AAQB chromenylium-like product ion [m]+ (13C2C11H17O3+; exact mass,

537

223.12393).

538 539 540

Figure 4. Detection of OA, CoQ0 and 5-DMQ0 in crude extracts of A. cinnamomea cultured in the absence or presence of [U-13C]oleic acid.

541

(a)-(f) show the DDMS2 product ion spectrum:

542

(a) OA standard

543

(b) OA [M+H]+ precursor ion (C8H9O4+; exact mass 169.04954) and the OA product ion

544

[m]+ (C8H7O3+; exact mass, 151.03897); -25-



545 546

(c) [13C2]OA [M+H]+ precursor ion (13C2C6H9O4+; exact mass 171.05625) and the [13C2]OA product ion [m]+ (13C2C6H7O3 +; exact mass, 153.04568);

547

(d) CoQ0 standard

548

(e) CoQ0 [M+H]+ precursor ion (C9H11O4+; exact mass 183.06519) and the CoQ0 product

549 550

ions [m]+ (C8H8O4+; exact mass, 168.04171; C8H9O2+; exact mass, 137.05971); (f) [13C2]CoQ0 [M+H]+ precursor ion (13C2C7H11O4+; exact mass 185.07190) and the

551

[13C2]CoQ0 product ions [m]+ (13C2C6H8O4+; exact mass, 170.04842; 13C2C6H9O2+; exact

552

mass, 139.06642);

553 554 555

(g) 5-DMQ0 [M+H]+ precursor ion (C8H10O3+; exact mass 155.07027) and the 5-DMQ0 product ions [m]+ (C6H5O2+; exact mass, 109.02841; C6H7O3+; exact mass, 127.03897); (h) [13C2]5-DMQ0 [M+H]+ precursor ion (13C2C6H10O3+; exact mass 157.07698) and the

556

[13C2] 5-DMQ0 product ions [m]+ (13C2C4H5O2+; exact mass, 111.03512; 13C2C4H7O3+;

557

exact mass, 129.04568).

558 559 560

Figure 5. Postulated biosynthetic routes of AQ-like compounds in A. cinnamomea mycelium cells via polyketide pathway

561

Parallel routes are possible to form AQ-like compounds. One route follows the similar

562

biosynthetic sequences as that of yeast CoQ6, starts from the farnesylaton of OA to form

563

FOA, further ring modification to form 5-DMQ3, CoQ3, and then AQ. Alternatively, adding

564

farnesyl group to the ring modification product of OA, 5-DMQ0 or CoQ0, to form 5-DMQ0

565

or CoQ3, and then AQ are possible. AQD may be formed directly from 5-DMQ3 through an

566

unknown processes. Similar to the parallel routes of AQ biosynthesis, one possible route of

567

4-AAQB biosynthesis is from an addition of farnesyl B to OA, further modification to form

568

5-DMQ3B, CoQ3B, AQB and then 4-AAQB. Other alternatives are adding farnesyl B to the

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569

ring modification product of OA, 5-DMQ0 or CoQ0, to form 5-DMQ3B or CoQ3B, followed

570

by a further modification to AQB then 4-AAQB.

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Table 1. Related information of predicted compounds detection in this study. Compound 5-DMQ3 6-DMQ3 CoQ3 AQ 5-DMQ3B CoQ3B AQB 4-AAQB OA 5-DMQ0 CoQ0

RT (min) 19.97 14.84 20.41 22.26 15.56 16.02 13.10 15.94 2.45 3.58 3.43

Molecular formula C23H32O3 C23H32O3 C24H34O4 C24H38O4 C23H30O5 C24H32O6 C24H36O6 C26H38O7 C8H8O4 C8H10O3 C9H10O4

[M+H]+ formula + C23H33O3 + C23H33O3 + C24H35O4 + C24H39O4 + C23H31O5 + C24H33O6 + C24H37O6 + C26H39O7 + C8H9O4 + C8H11O3 + C9H11O4

[M+H]+ exact mass 357.24242 357.24242 387.25299 391.28429 387.21660 417.22717 421.25847 463.26903 169.04954 155.07027 183.06519

13

C2 [M+H]+ exact mass 359.24913 359.24913 389.25970 393.29100 389.22331 419.23388 423.26518 465.27574 171.05625 157.07698 185.07190

PI #1 formula + C9H11O3 + C9H11O3 + C10H13O4 + C10H13O3 + C9H11O3 + C10H13O4 + C10H13O3 + C10H13O3 + C8H7O3 + C6H5O2 + C8H8O4

PI #1 exact mass 167.07027 167.07027 197.08084 181.08592 167.07027 197.08084 181.08592 181.08592 151.03897 109.02841 168.04171

13

C2 PI #1 exact mass 169.07698 199.08755 183.09263 169.07698 199.08755 183.09263 183.09263 153.04568 111.03512 170.04842

PI #2 formula + C12H15O3 + C12H15O3 + C13H17O4 + C13H17O3 + C12H15O3 + C13H17O4 + C13H17O3 + C13H17O3 + C6H7O3 + C8H9O2

PI #2 exact mass 207.10157 207.10157 237.11214 221.11722 207.10157 237.11214 221.11722 221.11722 127.03897 137.05971

13

C2 PI #2 exact mass 209.10828 239.11885 223.12393 209.10828 239.11885 223.12393 223.12393 129.04568 139.06642

PI: product ion; 5-DMQ3: 5-demethoxy-coenzyme Q3; 6-DMQ3: 6-demethoxy-coenzyme Q3; CoQ3: coenzyme Q3; AQ: antroquinonol; 5-DMQ3B: 5-demethoxy-coenzyme Q3 B; CoQ3B: coenzyme Q3 B; AQB: antroquinonol B; 4-AAQB: 4-acetylantroquinonol B; OA: orsellinic acid; 5-DMQ0: 5-demethoxy-coenzyme Q0; CoQ0: coenzyme Q0.

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Figure 1. O

O O

O

O

O O

O

O

O

O

1(a)

OH O

1(b)

O O

O

OH O

O

1(c) O

OH

O

O

O O

1(e)

OH

1(d)

1(f) HO

OH

OH

O

O O

1(g) HO

OH

O

1(h)

O

OH O

O O

1(i)

HO

1(j)

OH

O

O

O

O

O

O

O

O

O

O

O

O

1(k)

O O

1(l) O O

O O

1(m)

O

O

1(n)

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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TOC Graphics

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Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a

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