Phosphorylation of Isoflavones by Bacillus subtilis BCRC 80517 May

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Phosphorylation of Isoflavone by Bacillus subtilis BCRC 80517 May Represent Xenobiotic Metabolism Chen Hsu, Bo-Yuan Wu, Yu-Chuan Chang, Chi-Fon Chang, Tai-Ying Chiou, and Nan-Wei Su J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04647 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Journal of Agricultural and Food Chemistry

Phosphorylation of Isoflavone by Bacillus subtilis BCRC 80517 May Represent Xenobiotic Metabolism Chen Hsu1, Bo-Yuan Wu1, Yu-Chuan Chang1, Chi-Fon Chang2, Tai-Ying Chiou3, Nan-Wei Su1*

1

Laboratory of Food chemistry, Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan 2

3

Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan

Laboratory of Food Science and Technology, Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, Kitami, Japan

* Corresponding author. Tel.: +886-2-33664806；Fax: +886-2-23632714 E-mail address: [email protected] Postal address: Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan

1

ACS Paragon Plus Environment


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1

Abstract

2

Soy isoflavones, daidzein (DAI) and genistein (GEN), have beneficial effects on

3

human health. However, their oral bioavailability is hampered by low aqueous

4

solubility. Our previous study revealed two water-soluble phosphorylated isoflavones

5

conjugates of isoflavones, daidzein 7-O-phosphate and genistein 7-O-phosphate,

6

generated by biotransformation of Bacillus subtilis BCRC80517 cultivated with

7

isoflavones. In this study, two novel derivatives of isoflavones, daidzein

8

4’-O-phosphate and genistein 4’-O-phosphate, were identified by HPLC-ESI–MS/MS

9

and 1H, 13C and 31P NMR and the biotransformation road map was proposed.

10

Isoflavone glucosides were primarily deglycosylated, then phosphorylated into

11

7-O-phosphate conjugates predominantly and trace of 4’-O-phosphate conjugates.

12

Inevitably, trace quantities of glucosides were converted into 6’’-O-succinyl

13

glucosides. GEN was more efficiently phosphorylated than DAI. Nevertheless, GEN

14

prolonged the time into the exponential phase of cell growth, whereas other

15

isoflavones showed little effect on cell growth. Our findings provide new insights into

16

the novel microbial phosphorylation of isoflavones involved in xenobiotic

17

metabolism.

18 19

Key words: Isoflavone; Biotransformation; Phosphorylation; Bacillus subtilis;

20

Xenobiotic metabolism

21

2


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22

Introduction

23

The relationship between soy isoflavones and Bacillus subtilis var. natto has been

24

extensively studied in the past two decades. Natto, cheonggukjang and doenjang,

25

made from soybeans fermented with B. subtilis, are popular traditional foods that have

26

been consumed in East Asia for thousands of years 1. Soybean and soy-based food

27

products contain high amounts of isoflavones, including the aglycones daidzein (DAI)

28

and genistein (GEN); the glycosides daidzin (DAI-G) and genistin (GEN-G); and the

29

malonyl glycosides malonyl daidzin (DAI-GM) and malonyl genistin (GEN-GM) 2-3.

30

Much evidence has indicated that soy isoflavones possess phytoestrogenic

31

activity and physiological activities, including lowering the risk of breast cancer and

32

prostate cancer 4-5, decreasing cholesterol levels and preventing cardiovascular

33

diseases 6, increasing bone mass density to prevent osteoporosis and reducing

34

menopause symptoms 7-9, preventing obesity and diabetes 10-11, and improving

35

cognitive function 12, all with high efficacy, low toxicity, and minimal side effects.

36

However, according to the Merck Index, both DAI and GEN are practically

37

water-insoluble and belong to the Biopharmaceutical Classification System (BCS)

38

class IV, so they are not easily physiologically absorbed because of their low aqueous

39

solubility, low gastrointestinal permeability and, therefore, low bioavailability 13-14 .

40

In the past decade, enzyme or microbial biocatalysis has been promoted as a new

41

strategy and powerful tool to modify the chemical structure of isoflavones 15. Several

42

recent studies have used a microbial biotransformation method focused on

43

hydroxylation and glycosylation to generate new water-soluble isoflavones. Many

44

studies have cloned and expressed genes encoding cytochrome P450 (CYP)16-18 or

45

glycosyltransferase 19-20 to biosynthesize regio- and stereo-specific hydroxylated or

46

glycosylated isoflavones, which showed higher antioxidant activities and solubility 3



47

than their parent form. Despite many studies of the biotransformation of isoflavones

48

by diverse enzymes or microbial factors, to the best of our knowledge, the biological

49

phosphorylation of isoflavones has rarely been explored 21.

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In our previous study, we generated two highly water-soluble isoflavones,

51

daidzein 7-O-phosphate (DAI-7P) and genistein 7-O-phosphate (GEN-7P), by

52

cultivating B. subtilis BCRC 80517 with aglycones and glucosides of soy isoflavones.

53

This bacterial strain could also convert isoflavone glucosides via aglycones into the

54

corresponding 7-O-phosphate conjugates 22. Furthermore, we found that GEN-7P

55

improved water solubility and enhanced intestinal permeability in vitro and in situ as

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well as greatly increased plasma exposure to GEN after oral administration in rats 23.

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The major metabolites of isoflavones, namely 7-O-phosphate conjugates,

58

produced by B. subtilis BCRC 80517 have been investigated, but some minor

59

metabolites and the related metabolism remain unclear and uncharacterized. In this

60

study, we isolated and identified new metabolites of isoflavones, daidzein

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4’-O-phosphate (DAI-4’P) and genistein 4’-O-phosphate (GEN-4’P), from culture

62

with B. subtilis BCRC 80517, then characterized each metabolite during the

63

biotransformation. We further examined the bioconversion rate of individual

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isoflavones with B. subtilis BCRC 80517 culture and their inhibition of cell growth.

65

We proposed that phosphorylation modification of isoflavones may play a role in

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xenobiotic metabolism in B. subtilis.

67 68

Materials and methods

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Chemicals and biomaterials

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Isoflavone standards DAI, GEN, DAI-G, GEN-G were obtained from Shanghai

71

Aladdin Bio-Chem Technology Co. (Shanghai, China). High-performance liquid 4


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chromatography (HPLC)-grade acetonitrile and methanol were from Merck

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(Darmstadt, Germany). Analytical-grade acetic acid, benzoic acid, ethanol and formic

74

acid were from Sigma-Aldrich (St. Louis, MO, USA). B. subtilis BCRC 80517 was

75

from the Bioresource Collection and Research Centre (Hsinchu, Taiwan). The strain

76

was maintained on nutrient agar, stored at 4 oC and subcultured periodically. Nutrient

77

broth (NB) and agar were from Difco (Detroit, MI, USA).

78 79

Culture conditions of B. subtilis BCRC 80517 with individual isoflavone

80

A colony of B. subtilis BCRC 80517 from an agar plate was placed in a 30-mL glass

81

tube containing 5 mL sterilized NB medium (pH 7.0) and cultivated at 37 oC, 150 rpm

82

until OD600 = 1.0 (108 CFU/mL); the culture broth was used as the seed culture for

83

further incubation. For microbial biotransformation, a 500-mL Hinton’s flask

84

containing 85 mL medium, 10 mL individual isoflavones at 15 mg/mL and 5 mL

85

inoculum from seed culture was incubated at 37 oC, 150 rpm for 48 h. Cell growth

86

was determined by colony-forming units (CFUs) in an NB agar plate. Experimental

87

blank and control were incubated under the same culture conditions without the

88

addition of isoflavones and inoculum, respectively.

89 90

Analysis of isoflavones and their derivatives by HPLC-DAD

91

An amount of 500 µL culture broth was mixed with 500 µL methanol containing

92

10,000 mg/L benzoic acid as an internal standard (I.S.) at various intervals, then the

93

mixture was centrifuged to remove insoluble matter, and the supernatant underwent

94

HPLC. An analytical Shimadzu LC-20 AD HPLC system equipped with a YMC-Pack

95

ODS-AM C18 column (250×4.6 mm, 5 µm) and a Thermo Scientific UV6000

96

photodiode array detector (DAD) were used. The mobile phase was involving use of 5



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0.1% acetic acid in H2O (solvent A) and 0.1% acetic acid in acetonitrile (solvent B)

98

under a linear gradient of combination. After the injection of 20 µL sample, solvent B

99

was increased from 15% to 20% over 20 min, to 24% within the next 10 min,

100

remained isocratic at 24% for next 6 min, and then was increased to 35% within the

101

next 8 min, remained isocratic at 35% over 6 min, and then decreased to 15% within 5

102

min and held there for the next 15 min. The flow rate was set at 1 mL/min. The eluted

103

components were detected at 254 nm. The bioconversion rate was calculated by

104

dividing the generation of isoflavone 7-O-phosphate in culture broth by isoflavone in

105

initial culture broth and expressed as a percentage of molar ratios.

106 107

Isolation and identification of DAI-4’P and GEN-4’P

108

The purification of isoflavone derivatives was previously described in detail 22.

109

Briefly, the harvested broth was centrifuged to remove cells, then the supernatant was

110

adjusted to pH 1.0 with concentrated HCl and extracted three times with an equal

111

volume of ethyl acetate. The organic layers were combined and then evaporated under

112

reduced pressure to remove solvent. The residue was suspended in a small amount of

113

distilled water, placed on a prepacked DIAION HP-20 resin column, and eluted with

114

distilled water. The non-adsorbed fraction was collected and lyophilized. Further

115

separation of DAI-4’P and GEN-4’P involved a semipreparative HPLC system

116

equipped with a Hypersil ODS C18 HPLC column (250×10 mm, 10 µm). The mobile

117

phase consisted of 0.2% formic acid in H2O (solvent A) and 0.2% formic acid in

118

methanol (solvent B) with a gradient elution. After the injection of 100 µL sample,

119

solvent B was increased from 20% to 35% over 20 min, then to 80% within the next 5

120

min, then decreased to 20% within 5 min and held there for the next 10 min. The flow

121

rate was 5.0 mL/min. The eluate was collected and monitored at 254 nm. Fractions 6


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containing the desired compounds were further concentrated and resuspended with

123

hot alcohol to obtain a pale amorphous crystal for identifying the chemical structure.

124

DAI-4’P and GEN-4’P were identified according to LC-MS spectra data and NMR

125

analyses. LC-ESI-MS/MS involved the Thermo Finnigan LXQ Mass Spectrometer

126

System (San Jose, CA, USA) equipped with an ESI source. Experiments were carried

127

out in the positive ion mode. The source parameters were spray voltage, 3.73 kV;

128

cone voltage, 33.0 V; collision voltage, 35 V; sheath gas, 20.0 arbitrary units;

129

auxiliary gas, 20.0 arbitrary units; sweep gas, 2.0 arbitrary units; capillary temperature,

130

200.5 °C. Nitrogen was used as a nebulizing gas at 50 psi pressure and 350.0 °C.

131

Separation involved use of a Finnigan Surveyor LC Pump (San Jose, CA, USA) and a

132

flow rate of 1.0 mL/min with a post-column split volume ratio of 1/5 to the mass

133

detector at room temperature. The same column and chromatographic conditions as

134

for the HPLC-DAD analyses were as mentioned previously. Full scan spectra were

135

acquired from 100-1000 m/z range. Helium was used as the damping gas. NMR

136

spectra were recorded on a Bruker Avanve-500 spectrometer. Chemical shifts (δ) and

137

coupling constants (J) were expressed in ppm and Hz, respectively. 1H and 13C

138

chemical shifts were calibrated with DMSO-d6 used as the internal standard at 2.58

139

and 39.9, respectively. For phosphorus-31 NMR spectra, triphenylphosphate (TPP) in

140

acetone-d6 was used as an external standard with -17.5 ppm for chemical shift

141

calibration of 31P resonance.

142 143

Effect of isoflavones and the corresponding 7-O-phosphate conjugates on the growth

144

of B. subtilis BCRC 80517

145

An amount of 1 mL seed culture broth (OD600 = 1.0, 108 CFU/mL) was inoculated into

146

a 500-mL Hinton’s flask containing 100 mL NB medium, then the individual 7



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isoflavones pre-dissolved in DMSO was added to the culture broth to give a final

148

substrate concentration of 100 µM, and the bacterium was incubated at 37 oC, 150

149

rpm on a rotary shaker for 24 h. DMSO was added without the isoflavone substrate as

150

a control. Aliquots of 1 mL culture broth were withdrawn at intervals of 2 h to

151

monitor the cell growth by measuring absorbance at 600 nm.

152 153

Data analysis

154

The isoflavone profiles from HPLC were analyzed by using a SISC Chromatography

155

Data Station v3.2 (SISC Inc., Taipei, Taiwan). LC-ESI-MS/MS data were analyzed by

156

using Xcalibur v2.0 (ThermoFinnigan, San Jose, CA, USA). All NMR spectra were

157

analyzed by using TopSpin 3.5 (Bruker BioSpin, Billerica, MA, USA), and chemical

158

shifts were referenced to those of the solvent signals. Data are mean ± SD calculated

159

by using Microsoft Excel 2013. All experiments were performed in triplicate.

160 161

Results

162

HPLC profiles of isoflavones and their derivatives during biotransformation with B.

163

subtilis BCRC 80517

164

To investigate the change in composition of isoflavones during biotransformation, we

165

used HPLC to analyze the isoflavone profiles at different times of cultivation (Fig. 1).

166

As described previously, two major peaks, representing DAI-7P and GEN-7P (Fig. 1b,

167

peaks 1 and 3, respectively), were predominant products of biotransformation. In

168

addition, we observed two new minor peaks (peaks 2 and 4) eluted after DAI-7P and

169

GEN-7P. The next section describes the identification of these two novel compounds.

170

Moreover, during the biotransformation process, peaks 5 and 6 showed slight

171

accumulation after 24 h and were identified as 6''-O-succinyl daidzin (DAI-GS) (peak 8


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5, RT: 27.3 min, [M+H] + m/z 517.4, MS2 m/z 255.2) (Fig. S1) and 6''-O-succinyl

173

genistin (GEN-GS) (peak 6, RT: 37.4 min, [M+H] + m/z 533.1, MS2 m/z 271.2) (Fig.

174

S2), which agrees with the literature for UV absorbance and ESI-MS/MS spectra 24.

175

With the polar functional group, the above minor isoflavone derivatives were

176

considered more water-soluble than their parent aglycones. Characterization of all

177

newly generated peaks elucidated by HPLC-ESI-MS/MS is in Table 1.

178 179

Identification of novel derivatives of isoflavones: daidzein 4’-O-phosphate (DAI-4’P)

180

and genistein 4’-O-phosphate (GEN-4’P)

181

Peaks 2 and 4 showed the same spectra of UV absorption and peak tailing profile as

182

for isoflavone 7-O-phosphate (Fig. 1b). The tailing peaks of ionic compounds are

183

often caused by a silanol effect, so these two peaks should be ionic compounds. The

184

ESI-MS/MS spectra for peak 2 exhibited [M+H]+ at m/z 335.2 and MS2 peak at m/z

185

255.2 [M-H2PO3+H]+ (Fig. 2a-b). The fragment ion m/z 80.0 might suggest the

186

presence of a phosphate moiety. MS3 analysis of the m/z 255.2 ion brought out the

187

DAI spectrum (Fig. 2c). The mass fragmentation profiles of peak 2 were the same as

188

for DAI-7P, so the two compounds are structural isomers. The NMR spectral data for

189

peak 2 were 1H NMR (DMSO-d6, 500 MHz) δ: 6.93 (d, J=2.0 Hz, H-8), 7.00 (dd,

190

J=8.8, 2.0 Hz, H-6), 7.27 (2H, d, J=8.3 Hz, H-3’, 5’), 7.48 (2H, d, J=8.3 Hz, H-2’, 6’),

191

8.00 (d, J=8.8 Hz, H-5), 8.35 (s, H-2); 13C NMR (DMSO-d6, 125 MHz) δ: 102.5 (C-8),

192

115.7 (C-6), 120.0 (C-3’, 5’), 123.7 (C-3), 125.8 (C-1’), 127.5 (C-5), 129.8 (C-2’, 6’),

193

153.5 (C-2, 4’), 163.4 (C-7), 175.0 (C-4); and 31P NMR (TPP in acetone-d6, 202 MHz)

194

δ: -5.46 (s) (Fig. S3). The 2D HMQC 1H-31P NMR spectra show the presence of

195

correlation cross-peaks between the main phosphorus resonance at δ -5.46 with the

196

hydrogens of peak 2 at δ 7.27 (H-3’, 5’) and δ 7.48 (H-2’, 6’) (Fig. 2d). Therefore, the 9



197

monophosphate was located at the 4’-O position. From all this evidence, peak 2 was

198

characterized as daidzein 4’-O-phosphate (DAI-4’P) (Fig. 2e).

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The ESI-MS/MS spectra for peak 4 showed [M+H] + at m/z 351.2 and MS2 peak

199 200

at m/z 271.1 [M-H2PO3+H] + (Fig. 3a-b). MS3 analysis of the m/z 271.1 ion revealed

201

the GEN spectrum (Fig. 3c). The NMR spectral data for peak 4 were 1H NMR

202

(DMSO-d6, 500 MHz) δ: 6.28 (d, J=1.8 Hz, H-6), 6.44 (d, J=1.8 Hz, H-8), 7.26 (2H, d,

203

J=8.5 Hz, H-3’, 5’), 7.45 (2H, d, J=8.5 Hz, H-2’, 6’), 8.37 (s, H-2); 13C NMR

204

(DMSO-d6, 125 MHz) δ: 94.2 (C-8), 99.6 (C-6), 120.0 (C-3’, 5’), 122.6 (C-4), 123.9

205

(C-1’), 129.7 (C-2’, 6’), 154.5 (C-2, 4’), 162.3 (C-5), 165.4 (C-7), 180.5 (C-4); and

206

31

207

4’-O position of GEN but not the 5-O position was established by measuring the 2D

208

HMQC 1H-31P NMR spectra, which showed correlations of phosphorus at δ -5.45

209

with protons of peak 4 at δ 7.26 (H-3’, 5’) and δ 7.45 (H-2’, 6’) (Fig. 3d). These data

210

together confirmed that the structure of peak 4 was genistein 4’-O-phosphate

211

(GEN-4’P) (Fig. 3e).

P NMR (TPP in acetone-d6, 202 MHz) δ: -5.45 (s) (Fig. S4). The phosphate at the

212 213

Biotransformation of individual DAI and GEN by B. subtilis BCRC 80517

214

Time-course study was used to investigate the biotransformation of individual DAI

215

and GEN on culture with B. subtilis BCRC 80517 (Fig. 4 and 5, respectively). At the

216

beginning of the biotransformation, both DAI and GEN contents were decreased

217

slowly in the lag phase of growth. After 12 h of cultivation, both contents were

218

decreased rapidly during the exponential phase. GEN in culture broth was almost

219

consumed after 48 h of incubation, whereas DAI showed approximately 22% of the

220

original content. DAI-7P and GEN-7P started to accumulate at 6 h, then GEN-7P

221

content peaked at 5.4 mM in the culture broth at 48 h (Fig. 5a). In contrast, DAI-7P 10


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222

content was relatively low, around 4.3 mM (Fig. 4a). At the end of 48-h incubation,

223

the bioconversion rate of GEN-7P was 96% but only 78% for DAI-7P. Although GEN

224

could be phosphorylated more efficiently than DAI by B. subtilis BCRC 80517, it

225

retarded the growth of bacteria and prolonged the time into the exponential phase of

226

cell growth, whereas DAI had only minor effects (Fig. 4b and 5b). Moreover,

227

according to the bioconversion rate and cell growth results, the generation of

228

phosphate derivatives seems to be highly related to the cell growth. Moreover, two

229

new minor peaks were generated and accumulated in the culture broth separately with

230

increasing biotransformation time; these peaks were further identified as DAI-4’P and

231

GEN-4’P. Furthermore, a small amount of DAI-GS and GEN-GS remained at 12-h

232

culture, and the contents of these two compounds increased slightly with incubation

233

(Fig. 4c and 5c). Park et al. indicated that isoflavone glucoside was the only substrate

234

for the formation of succinyl derivatives by B. subtilis natto 25. However, we found

235

that B. subtilis BCRC 80517 could generate 6’’-O-succinyl conjugations from

236

corresponding aglycones, and we did not observe any peaks representing DAI-G and

237

GEN-G in the culture broth during the biotransformation process.

238 239

Biotransformation of individual DAI-G and GEN-G by B. subtilis BCRC 80517

240

We examined the bioconversion of individual DAI-G and GEN-G by B. subtilis

241

BCRC 80517 by sampling the culture broth at different incubation times to detect

242

changes in isoflavone content (Fig. 6 and 7, respectively). The contents of both

243

DAI-G and GEN-G remained at their original levels during the first 12 h, then

244

decreased rapidly in the exponential phase of growth, with predominantly increasing

245

contents of DAI-7P and GEN-7P after 12-h incubation. A small amount of DAI-GS

246

and GEN-GS was also detected in the 6-h culture broth and increased only slightly in 11



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247

the culture broth. These phenomena indicated that enzymatic interconversion of

248

isoflavone glucosides and the corresponding 6’’-O-succinylated derivatives occurred

249

in culture broth during the biotransformation process. At the end of 48-h incubation,

250

the content of GEN-7P reached 5.0 mM, for about 90% conversion rate (Fig. 7a-b).

251

However, only 3.8 mM of DAI-7P remained at 48 h, for a 68% bioconversion rate

252

(Fig. 6a-b). Both DAI-G and GEN-G seemed not to influence the growth of bacteria,

253

because the cell growth curves were almost identical to those of the control (Fig. 6b

254

and 7b). The phosphate conjugates formation was positively related to cell growth, so

255

the biosynthesis of phosphorylated isoflavones could be attributed to cell growth. In

256

addition, DAI-4’P and GEN-4’P were detected and the levels of these two new

257

compounds showed increasing trends during biotransformation (Fig. 6c and 7c). Kuo

258

et al. reported glucoside conjugates could be effectively hydrolyzed into their

259

aglycones from soybean fermented with B. subtilis natto 26. However, in the current

260

study, DAI and GEN contents were observed at 6-h incubation, then were maintained

261

at trace levels to the end of the biotransformation by B. subtilis BCRC 80517.

262 263

Proposed metabolic route of isoflavones by B. subtilis BCRC 80517

264

According to the changes in contents of isoflavones and derivatives in culture broth at

265

different incubation times, we propose a potent metabolic route for isoflavone

266

biotransformation by B. subtilis BCRC 80517 in Fig. 8. During the bioconversion,

267

aglycones are predominantly phosphorylated at the 7-O position, and a small amount

268

of 4’-O-phosphate conjugates is generated. In addition, aglycones are glycosylated

269

into glycosides and further converted into a small amount of 6’’-O-succinyl

270

glucosides. Glucosides are mainly deglycosylated into aglycones, followed by rapid

271

7-O-phosphorylation, along with a small amount of isoflavone 4’-O-phosphates 12


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272

produced. Moreover, glucosides are converted into a small amount of 6’’-O-succinyl

273

glucosides. Furthermore, 6’’-O succinyl glucosides may be de-succinylated by

274

esterase and then de-glycosylated by β-glucosidase to form aglycones, which can be

275

further converted into isoflavone phosphate conjugates. During the biotransformation,

276

various isoflavone derivatives in the metabolites pool could be converted mutually by

277

particular enzymes produced from B. subtilis BCRC 80517. Further intensive study is

278

needed to characterize specific enzymes, especially isoflavone phosphorylation

279

enzymes, to explore the detailed molecular mechanism of the biotransformation of

280

isoflavone by B. subtilis BCRC 80517.

281 282

Effect of isoflavones and their 7-O-phosphate derivatives on growth of B. subtilis

283

BCRC 80517

284

We found that GEN had higher phosphorylation efficiency for B. subtilis BCRC

285

80517 as a biotransformation substrate than did DAI. Nevertheless, CFUs were

286

severely inhibited with GEN in the culture broth, but DAI did not affect the growth of

287

bacteria. According to these production rates and growth patterns with GEN, B.

288

subtilis BCRC 80517 may be equipped with an isoflavone phosphorylation system for

289

xenobiotic metabolism, to avoid harmful effects. We performed initial

290

growth-inhibition experiments with DAI and GEN at 100 µM. The concentration was

291

chosen based on the solubility of aglycones in NB medium containing 1% DMSO for

292

measuring bacterial growth by an optical density method (Fig. 9). GEN maximally

293

prohibited growth of B. subtilis BCRC 80517 with incubation time 12 h. After 16 h of

294

incubation, the effect of GEN on cell viability declined because of the

295

phosphorylation of GEN in the culture medium. We investigated whether the

296

phosphorylated metabolites had less growth inhibition than the aglycones by applying 13



297

them at equimolar inhibitory concentrations as their corresponding compounds.

298

Indeed, as expected, GEN suppressed growth at the concentration used, with only a

299

minor effect of 100 and 500 µM GEN-7P on bacterial growth. By contrast, the cell

300

growth was not affected by the addition of 100 µM DAI or 100 or 500 µM DAI-7P.

Page 14 of 40

301 302 303

Discussion In this study, we propose a metabolism road map in biotransformation of

304

isoflavone by B. subtilis BCRC 80517 and identify two novel isoflavone derivatives,

305

DAI-4’P and GEN-4’P, from culture broth. The beneficial effect of isoflavones on

306

human health has received growing attention over the past few decades. However,

307

since both DAI and GEN have poor solubility and low permeability, they are

308

categorized into BCS class IV, representing the characteristic of low oral

309

bioavailability 13. To enhance their practical applications, many researchers have

310

concentrated on increasing the solubility of isoflavones by means of microbial

311

biotransformation, which offers an alternative method for modifying the chemical

312

structure, with the advantages of ease of cultivation and rapid growth, mild conditions,

313

stereo-selectivity and high production with few byproducts as compared with organic

314

synthesis 27.

315

To date, hydroxylated and glycosylated isoflavones have been the major

316

water-soluble derivatives produced by microbial biotransformation. Isoflavones are

317

commonly hydroxylated at the C3′ position of the B ring by microorganisms.

318

Streptomyces avermitilis MA-4680 hydroxylated DAI at the C3’ and C6 or C8

319

position via cytochrome P450 (CYP) CYP105D7 and CYP105D6 enzymes,

320

respectively 17-18. CYP107H1, known as fatty acid hydroxylase from B. subtilis 168,

321

exhibited ortho-specific hydroxylation activity toward DAI 16. Moreover, Esaki et al. 14


Page 15 of 40


322

revealed the formation mechanism of hydroxylated isoflavones during soybean

323

fermentation: isoflavone glycosides were gradually hydrolyzed into the corresponding

324

aglycones by β-glucosidase and further hydroxylated to form dihydro-isoflavone by

325

CYP monooxygenases from Aspergillus saitoi 28. Regarding glycosylation, Li et al.

326

used maltosyltransferase from Thermotoga maritima to obtain DAI conjugation with

327

multiglucoside 19 and used cyclodextrin glucanotransferase from Bacillus sp. I-5 to

328

create a cycloamylose GEN complex 20, all compounds showing about 104 times

329

higher solubility than their algycones.

330

Succinylated isoflavones were first isolated and identified in 1999. In the present

331

study, we found a small amount of DAI-GS and GEN-GS in culture broth during the

332

bioconversion of isoflavones. Toda et al. found DAI-GS and GEN-GS in soybean

333

foods (natto and cheonggukjang) fermented with B. subtilis 24. These two compounds

334

showed biological activities for preventing bone loss and bone-strength reduction

335

without uterine atrophy or bodyweight gain in an ovarian hormone-deficient rat model.

336

Very recently, a high yield of 6’’-O-succinyl isoflavones (> 90%) from aglycones was

337

achieved by using the resting cells of a solvent-tolerant strain, Bacillus licheniformis

338

ZSP0, in aqueous miscible organic medium (10% (v/v) DMSO) 29; the conventional

339

aqueous-phase biocatalysis by B. subtilis was incompetent for the accumulation of

340

succinyl-glucosides because of low substrate solubility in the aqueous phase.

341

Although broad biological methods have been used to discover the formation of

342

various water-soluble isoflavone derivatives, the microbial ability to form phosphate

343

conjugates is limited to a few bacterial strains. Several fungal strains, such as

344

Circinella muscae, Absidia cylindrospora and A. glauca were found to phosphorylate

345

compactin (ML-236B) and monacolin K at the C5’ hydroxyl group; these

346

water-soluble derivatives also showed high hypocholesterolemic activity in animal 15



Page 16 of 40

347

experiments 30. Lack et al. elucidated that formation of phenylphosphate is the first

348

step of phenol degradation under anaerobic conditions in denitrifying the bacteria

349

Pseudomonas sp. and Thauera aromatica 31. The incubation of puerarin, the

350

8-C-glycosdie of DAI, with Bacillus cereus NT02, generated 6’’-O-phosphate

351

conjugate, but the phosphate moiety was attached via a hydroxyl group in the 6’’-O

352

position on the glucose moiety and not directly to a phenolic hydroxyl group 32.

353

Recently, Zühlke et al. isolated Bacillus amyloliquefaciens from sewage sludge and

354

found that it could convert bisphenols to their phosphate conjugates; these derivatives

355

were less toxic and less estrogenically active than their parent bisphenols 33.

356

In this work, we purified and characterized DAI-4’P and GEN-4’P, two novel

357

metabolites of isoflavones, in aglycones-containing broth cultivated with B. subtilis

358

BCRC 80517. Moreover, we examined the effect of each isoflavone on growth of B.

359

subtilis BCRC 80517 to determine the relationship between cell growth and

360

bioconversion rate of isoflavone 7-O-phosphate. Although the chemical structure of

361

DAI is similar to that of GEN and only lacks the hydroxyl group at the 5-O position,

362

GEN had an inhibitory effect on B. subtilis BCRC 80517 growth relative to DAI. In

363

contrast, DAI-G and GEN-G, with similar chemical structures, had the same minor

364

effect on cell growth. Ulanowska et al. indicated that GEN inhibited the growth of B.

365

subtilis type culture strain 168 at concentrations in the culture broth up to 100 µM as

366

compared with other flavonoids 34. GEN negatively affected global synthesis of DNA,

367

RNA and protein by hampering the function of DNA topoisomerases type I and type

368

II, so it might influence DNA topology significantly and have strong negative effects

369

on DNA replication 35. Contrary to GEN, DAI has inhibitory activity against

370

topoisomerase I but inhibits topoisomerase II only weakly, thus having only weak

371

antibacterial activity against B. subtilis 168 34. Furthermore, we found that GEN was 16


Page 17 of 40


372

phosphorylated more efficiently by less cell number during biotransformation as

373

compared with DAI. GEN-7P applied at equimolar concentrations as GEN did not

374

inhibit bacterial growth. The main biological function of phosphorylation of

375

isoflavones by B. subtilis BCRC 80517 may be a mechanism to reduce the growth

376

inhibition of xenobiotic molecules by the bacteria strain. This viewpoint is similar to

377

antibiotic resistance via phosphorylation in global microbiota 36. Antibiotic kinases,

378

which include aminoglycoside and macrolide O-phosphotransferase, are widely

379

distributed among bacterial pathogens and pose a serious threat to currently used

380

antimicrobial therapies 37-38. Also, phosphorylation at hydroxyl groups in positions 21

381

and 23 of the ansa chain of rifampin has been reported in pathogenic bacteria of the

382

genera Nocardia and Rhodococcus; these phosphorylated products did not show

383

antimicrobial activity 39. Additionally, S. avermitilis MA4680 expresses

384

chloramphenicol 3’-O-phosphotransferase to inactivate corresponding antibiotic

385

molecules 40, and B. pumilus can form a phosphate ester with the 8’-O position of

386

amicoumacin 41. These microbial agents gain self-resistance against antibiotics by

387

phosphorylation.

388

Xenobiotic metabolism plays an essential role in the bacterial defense against

389

xenobiotic molecules, which might have harmful effects on biological systems 42. In

390

the current study, we demonstrated that phosphorylation modifications change the

391

physicochemical properties of GEN, which alters its biological and physiological

392

effects. However, the enzymes that specific catalyze the phosphorylation of

393

isoflavones have not been revealed. Further research is under way to reveal the

394

mysteries of the isoflavone-phosphorylation enzyme and catalysis mechanism.

395

17



Page 18 of 40

396

ASSOCIATED CONTENT

397

Supporting Information

398

The ESI-MS/MS spectra of DAI-GS and GEN-GS. The NMR spectra of DAI-4’P and

399

GEN-4’P.

400 401

ACKNOWLEDGEMENT

402

The MS spectra were obtained at the Joint Center for Instruments and Research,

403

College of Bioresources and Agriculture, National Taiwan University. The NMR

404

spectra were obtained at the Core Facility for Protein Structural Analysis supported by

405

the National Core Facility Program for Biotechnology at Academia Sinica, Taiwan.

406

We thank Laura Smales (BioMedEditing, Toronto, Canada) for English language

407

editing.

408 409

Author Information

410

Corresponding Author

411

*Phone: +886-2-33664819. Fax: +886-2-23632714. E-mail: snw@ ntu.edu.tw.

412

ORCID

413

Chen Hsu: 0000-0003-2620-8726

414

Nan-Wei Su: 0000-0002-1402-7349

415

Funding

18


Page 19 of 40


416

This work was a part of the research project, which was supported by the Ministry of

417

Science and Technology, Executive Yuan, Taiwan [Grant Number MOST

418

104-2320-B-002-027-MY3].

419

Notes

420

The authors declare no competing financial interest.

421

Abbreviations Used

422

DAI, daidzein; GEN, genistein; DAI-G, daidzin; GEN-G, genistin; DAI-GS,

423

6’’-O-succinyl daidzin; GEN-GS, 6’’-O-succinyl genistin; DAI-7P, daidzein

424

7-O-phosphate; GEN-7P, genistein 7-O-phosphate; DAI-4’P, daidzein 4’-O-phosphate;

425

GEN-4’P, genistein 4’-O-phosphate; I.S., internal standard; DMSO, dimethyl

426

sulfoxide; HPLC, high performance liquid chromatography; LC-MS, liquid

427

chromatography-mass spectrometry; SD, standard deviation; UV, ultraviolet.

428

429

References

430

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431

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432

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Biotransformed product, genistein 7-O-phosphate, enhances the oral bioavailability of

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genistein. J. Funct. Foods 2015, 13, 323-335.

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24. Toda, T.; Uesugi, T.; Hirai, K.; Nukaya, H.; Tsuji, K.; Ishida, H., New 6-O-acyl

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isoflavone glycosides from soybeans fermented with Bacillus subtilis (natto). I.

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6-O-succinylated isoflavone glycosides and their preventive effects on bone loss in

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ovariectomized rats fed a calcium-deficient diet. Biol. Pharm. Bull. 1999, 22 (11),

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Bacillus species. J. Food Sci. 2010, 75 (1), C128-C133.

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26. Ko, J. H.; Kim, B. G.; Joong-Hoon, A., Glycosylation of flavonoids with a 23



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bioactive flavonoids. Biotechnol. Adv. 2015, 33 (1), 214-223.

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mechanism for potent antioxidative o-dihydroxyisoflavones in soybeans fermented

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with Aspergillus saitoi. Biosci Biotechnol Biochem. 1999, 63 (5), 851-8.

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miscible organic medium. Biotechnol. Appl. Biochem. 2015, 62 (2), 255-259.

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38 (3), 328-332.

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the first step in anaerobic phenol metabolism in a denitrifying Pseudomonas sp.

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33. Zühlke, M.-K.; Schlüter, R.; Henning, A.-K.; Lipka, M.; Mikolasch, A.;

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Schumann, P.; Giersberg, M.; Kunze, G.; Schauer, F., A novel mechanism of

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conjugate formation of bisphenol A and its analogues by Bacillus amyloliquefaciens:

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Detoxification and reduction of estrogenicity of bisphenols. Int. Biodeterior.

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Biodegradation. 2016, 109, 165-173.

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34. Ulanowska, K.; Tkaczyk, A.; Konopa, G.; Węgrzyn, G., Differential antibacterial

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activity of genistein arising from global inhibition of DNA, RNA and protein

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synthesis in some bacterial strains. Arch. Microbiol. 2006, 184 (5), 271-278.

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35. Constantinou, A.; Mehta, R.; Runyan, C.; Rao, K.; Vaughan, A.; Moon, R.,

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Flavonoids as DNA topoisomerase antagonists and poisons: structure-activity

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relationships. J. Nat. Prod. 1995, 58 (2), 217-225.

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36. Wright, G. D., The antibiotic resistome: the nexus of chemical and genetic

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diversity. Nat. Rev. Microbiol. 2007, 5 (3), 175-186.

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37. Cundliffe, E., How Antibiotic-producing organisms avoid suicide. Annu. Rev.

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Microbiol. 1989, 43 (1), 207-233.

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38. Wright, G. D., Aminoglycoside phosphotransferases: proteins, structure, and

546

mechanism. Front Biosci 1999, 4 (1), 9-12.

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39. Yazawa, K.; Mikami, Y.; Maeda, A.; Morisaki, N.; Iwasaki, S., Phosphorylative

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1994, 33 (6), 1127-1135.

550

40. Rajesh, T.; Sung, C.; Kim, H.; Song, E.; Park, H.-Y.; Jeon, J.-M.; Yoo, D.; Kim,

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H. J.; Kim, Y. H.; Choi, K.-Y., Phosphorylation of chloramphenicol by a recombinant

552

protein Yhr2 from Streptomyces avermitilis MA4680. Bioorg. Med. Chem. Lett. 2013,

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23 (12), 3614-3619.

554

41. Hashimoto, M.; Taguchi, T.; Nishida, S.; Ueno, K.; Koizumi, K.; Aburada, M.;

555

Ichinose, K., Isolation of 8'-phosphate ester derivatives of amicoumacins:

556

structure-activity relationship of hydroxy amino acid moiety. J. Antibiot. 2007, 60

557

(12), 752.

558

42. Spanogiannopoulos, P.; Bess, E. N.; Carmody, R. N.; Turnbaugh, P. J., The

559

microbial pharmacists within us: a metagenomic view of xenobiotic metabolism. Nat.

560

Rev. Microbiol. 2016, 14 (5), 273.

561

26


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562

TABLE CAPTION

563

Table 1. Chemical Characteristics of Isoflavone Derivatives Generated from the

564

Biotransformation Process of Cultivating B. subtilis BCRC 80517 with DAI and

565

GEN.

566

FIGURE LEGENDS

567

Figure 1. High-performance liquid chromatograms of culture broth of the

568

biotransformation of GEN and DAI by B. subtilis BCRC 80517 at (a) 0 h and (b) 48 h

569

after incubation. The culture broth was incubated at 37 oC, 150 rpm. Peak: 1, DAI-7P;

570

2, DAI-4’P; 3, GEN-7P; 4, GEN-4’P; 5, DAI-GS; 6, GEN-GS; 7, DAI; 8, GEN.

571

Benzoic acid was used as I.S.

572

Figure 2. Mass and NMR spectra for DAI-4’P. (a) ESI-MS of protonated parent ion

573

(m/z 335) and (b-c) ESI-MS/MS of protonated fragment ions (m/z 255, 137, 119), (d)

574

2D HMQC 1H/31P (TPP in acetone-d6, 202 MHz for 31P NMR), (e) chemical structure

575

of DAI-4’P.

576

Figure 3. Mass and NMR spectra for GEN-4’P. (a) ESI-MS of protonated parent ion

577

(m/z 351) and (b-c) ESI-MS/MS of protonated fragment ions (m/z 271,153), (d) 2D

578

HMQC 1H/31P (TPP in acetone-d6, 202 MHz for 31P NMR), (e) chemical structure of

579

GEN-4’P.

580

Figure 4. Time course analysis of (a) biotransformation, (b) determination of cell 27



Page 28 of 40

581

growth and bioconversion rate during incubation with individual DAI by B. subtilis

582

BCRC 80517. (c) HPLC elution profiles of the biotransformation of DAI by B.

583

subtilis BCRC 80517 at (c1) 0 h, (c2) 24 h and (c3) 48 h after incubation. The culture

584

broth was incubated at 37 oC, 150 rpm. Control experiments involved NB medium

585

culture without DAI. Data are mean ± SD from 3 experiments. Benzoic acid was used

586

as I.S.

587

Figure 5. Time course analysis of (a) biotransformation, (b) determination of cell

588

growth and bioconversion rate during incubation with individual GEN by B. subtilis

589

BCRC 80517. (c) HPLC elution profiles of the biotransformation of GEN by B.

590


591


592

culture without GEN. Data are mean ± SD from 3 experiments. Benzoic acid was

593

used as I.S.

594


595

growth and bioconversion rate during incubation with individual DAI-G by B. subtilis

596

BCRC 80517. (c) HPLC elution profiles of the biotransformation of DAI-G by B.

597


598


599

culture without DAI-G. Data are mean ± SD from 3 experiments. Benzoic acid was 28


Page 29 of 40


600

used as I.S.

601


602

growth and bioconversion rate during incubation with individual GEN-G by B.

603

subtilis BCRC 80517. (c) HPLC elution profiles of the biotransformation of GEN-G

604

by B. subtilis BCRC 80517 at (c1) 0 h, (c2) 24 h and (c3) 48 h after incubation. The

605

culture broth was incubated at 37 oC, 150 rpm. Control experiments involved NB

606

medium without GEN-G. Data are mean ± SD from 3 experiments. Benzoic acid was

607

used as I.S.

608

Figure 8. A proposed roadmap of soy isoflavone biotransformation by B. subtilis

609

BCRC 80517.

610

Figure 9. Effect of individual isoflavone and their derivatives on growth of B. subtilis

611

BCRC 80517. Effect of (a) GEN (100 µM), GEN-7P (100 and 500 µM) and (b) DAI

612

(100 µM), DAI-7P (100 and 500 µM). Growth was monitored by measuring the OD600

613

values, and 1% DMSO as control was added to the culture broth for incubation at 37

614

o

C, 150 rpm for 24 h. Data are mean ± SD from 3 experiments.

29



Page 30 of 40

Table 1. Chemical Characteristics of Isoflavone Derivatives Generated from the Biotransformation Process of Cultivating B. subtilis BCRC 80517 with DAI and GEN.

Abbrev.

Formula

Rt (min)a

λmax (nm)

[M+H]+ (m/z)

MS2 Fragment ions (m/z)

Daidzein 7-O-phosphate

DAI-7P

C15H11O8P

8.8

251

335.2

317.2, 255.1

2

Daidzein 4’-O-phosphate

DAI-4’P

C15H11O8P

11.9

251

335.2

317.0, 255.2

3

Genistein 7-O-phosphate

GEN-7P

C15H11O8P

14.2

259

351.2

333.1, 271.1

4

Genistein 4’-O-phosphate

GEN-4’P

C15H11O8P

19.2

259

351.2

333.1, 271.1

5

6''-O-succinyl daidzin

DAI-GS

C25H24O12

27.3

258

516.4

499.0, 255.2

6

6''-O-succinyl genistin

GEN-GS

C25H24O13

37.4

260

532.1

515.0, 271.2

7

Daidzein

DAI

C15H10O4

39.8

249

255.0

227.2, 199.2, 137.1

8

Genistein

GEN

C15H10O5

51.2

262

271.0

243.2, 215.2, 153.0

Peak No.

Compound

1

name

a

HPLC condition: Column, YMC ODS-AM (250×4.6 mm, 5 µm); Mobile phase, Solvent A: 0.1% acetic acid in H2O, Solvent B: 0.1% acetic acid in acetonitrile; Gradient, 15%-20% in 20 min (B), 20%-24% in 10 min (B), 24%-24% in 6 min (B), 24%-35% in 8 min (B), 35%-35% in 6 min (B); Flow rate, 1.0 mL/min; Temperature, ambient. More details are described in Materials and methods.

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Figure 1.

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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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Figure 6.

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Figure 7.

37



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Figure 8.

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Figure 9.

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TABLE OF CONTENTS GRAPHICS

40


Phosphorylation of Isoflavones by Bacillus subtilis BCRC 80517 May

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