Promiscuous glycosyltransferase from Bacillus subtilis 168 for the

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Promiscuous glycosyltransferase from Bacillus subtilis 168 for the enzymatic synthesis of novel protopanaxatriol-type ginsenosides Longhai Dai, Jiao Li, Jiangang Yang, Yueming Zhu, Yan Men, Yan Zeng, Yi Cai, Caixia Dong, Zhubo Dai, Xueli Zhang, and Yuanxia Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03907 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018

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Journal of Agricultural and Food Chemistry

1

Promiscuous glycosyltransferase from Bacillus subtilis 168 for the enzymatic

2

synthesis of novel protopanaxatriol-type ginsenosides

3 4

Longhai Dai,a Jiao Li,a,b Jiangang Yang,a Yueming Zhu,a Yan Men,a Yan Zeng,a Yi

5

Cai,a Caixia Dong,c Zhubo Dai,a Xueli Zhanga,* and Yuanxia Suna,*

6 7

a

8

Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

9

b

10

c

11

Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin

12

300070, China

National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of

University of Chinese Academy of Sciences, Beijing, China Tianjin Key Laboratory on Technologies Enabling Development of Clinical

13 14

* Correspondence author. Mailing address:

15

Xueli Zhang, E-mail: [email protected], Tel: +862284861983

16

Yuanxia Sun, E-mail: [email protected], Tel: +862284861960

17

32 Xiqi Road, Tianjin Airport Economic Area, Tianjin, 300308, Peoples Republic of

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China

19

1

ACS Paragon Plus Environment


20 21

ABSTRACT Ginsenosides are the principally bioactive ingredients of Panax ginseng and

22

possess

diverse

notable

pharmacological

activities.

23

(UGT)-mediated glycosylation of C6-OH and/or C20-OH of protopanaxatriol (PPT)

24

is the prominent biological modification that contributes to the immense structural

25

and functional diversity of PPT-type ginsenosides. In this study, glycosylation of PPT

26

and PPT-type ginsenosides was achieved using a promiscuous glycosyltransferase

27

(Bs-YjiC) from Bacillus subtilis 168. PPT was selected as a probe for the in vitro

28

glycodiversification of PPT-type ginsenosides using diverse UDP-sugars as sugar

29

donors. Structural analysis of the newly biosynthesized products demonstrated that

30

Bs-YjiC can transfer a glucosyl moiety to the free C3-OH, C6-OH, and C12-OH of

31

PPT. Five PPT-type ginsenosides were biosynthesized, including ginsenoside Rh1 and

32

four unnatural ginsenosides. The present study suggests an important role of flexible

33

microbial UGTs for enzymatic synthesis of novel ginsenosides.

34

KEYWORDS: Protopanaxatriol, protopanaxatriol-type ginsenosides, Bacillus

35

UDP-glycosyltransferase, glycosylation, chemical diversification

2


UDP-glycosyltransferase

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INTRODUCTION

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Ginseng (Panax ginseng C. A Meyer), a herbaceous perennial herb of the

38

Araliaceae family, is one of the most well-known and best-selling oriental

39

medicines.1,2 This herb is frequently used in traditional medicine to strengthen

40

immunity, reduce fatigue, and provide nutrition for more than 2000 years in Eastern

41

Asia.3 The major pharmacologically active components of ginseng are glycosylated

42

dammarane-type tetracyclic triterpene saponins known as ginsenosides (ca. 2%–4% in

43

the dried roots and rhizomes of ginseng), which exhibit diverse and notable

44

pharmacological characteristics,

such as antitumor,

45

anti-inflammatory,

antifatigue,

46

system-enhancing activities.4-7

anti-aging,

anticancer,

antihypertensive,

antidiabetic,

and

immune

47

More than 140 naturally occurring ginsenosides were isolated from different

48

ginseng species.8 Most of these natural products can be classified into

49

protopanaxadiol (PPD) and protopanaxatriol (PPT) groups according to the structure

50

of the triterpene aglycon.9 Currently, most of the key genes involved in PPD- and

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PPT-type ginsenosides biosynthetic pathway have been functionally characterized.

52

The initial biosynthetic step is the cyclization of 2,3-oxidosqualene to form

53

dammarenediol-II (DM), which is catalyzed by DM synthase (PgDDS).4,10 DM is

54

hydroxylated by PPD synthase (PgPPDS, CYP716A47) at its C12 position to form

55

PPD,11 and PPD is converted further to PPT after hydroxylation at its C6 position by

56

PPT

57

UDP-glycosyltransferases (UGTs) responsible for the glycosylation of C3-OH and/or

synthase

(PgPPTS,

CYP716A53v2).12

3


Additionally,

several


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C20-OH of PPD, and C6-OH and/or C20-OH of PPT have been isolated from P.

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ginseng in the past 3 years, and a number of PPD-type ginsenosides (Rh2, CK, F2,

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Rg3, and Rd) and PPT-type ginsenosides (F1, Rh1, and Rg1) were biosynthesized by

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introducing an engineered PPD- or PPT-producing pathway and ginseng UGT genes

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into yeast cell factories.13-18

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UGT-catalyzed glycosylation acts as the final biosynthetic step for ginsenosides,

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which present immense structural and functional diversity after the process. Some

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UGTs, especially those isolated from microbes, show high substrate flexibility and

66

poor regiospecificity and thus can be exploited as effective biocatalysts for

67

glycorandomization of natural products with diverse structures.19 For example, UGT

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OleD from Streptomyces antibioticus,20,21 MhGT1 from Mucor hiemalis,22 UGT58A1

69

from Absidia coerulea, UGT59A1 from Rhizopus japonicas,23 Bl-YjiC from Bacillus

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licheniformis,24,25 and BcGT1 from B. cereus26,27 are frequently applied to

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glycodiversify a considerable number of important botanical natural products.

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Recently, UGT51, which exhibits broad acceptor tolerance, was isolated from

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Saccharomyces cerevisiae and demonstrated to be able to regiospecifically transfer a

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glucosyl moiety to the free C3-OH of PPD for the synthesis of ginsenoside Rh2.28

75

Additionally, several UGTs responsible for the biosynthesis of natural and unnatural

76

ginsenosides were isolated from several Bacillus strains, including UGT109A1

77

involved

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(3-O-β-D-glucopyranosyl-20(S)-protopanaxadiol),

79

12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol,

in

biosynthesis

of

ginsenosides,

such

4


as

ginsenoside

Rh2

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3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol,

81

3-O-β-D-glucopyranosyl-20(S)-protopanaxatriol,

82


83

3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol;29

84

involved

85

3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol;30

86

BSGT1

87

(3-O-β-D-glucopyranosyl-20-O-β-D-glucopyranosyl-20(S)-protopanaxatriol).31 Based

88

on these studies, we explored substrate-flexible UGTs from microbes for the

89

biosynthesis of novel ginsenosides for drug discovery.

in

involved

the

in

biosynthesis

and

biosynthesis

of

ginsenoside

YjiC1 of and Ia

90

Bs-YjiC from Bacillus subtilis 168 is a promiscuous and robust UGT toward a

91

considerable number of structurally diverse types of natural and unnatural products,

92

including several structurally diverse types of triterpenes.32 Furthermore, Bs-YiC can

93

glycosylate both the free C3-OH and C20-OH of PPD and PPD-type ginsenosides to

94

synthesize a series of natural and unnatural ginsenosides (unpublished data). In this

95

study, the potential of Bs-YjiC as a biocatalyst for glycosylation of PPT and PPT-type

96

ginsenosides was explored further. PPT was selected as a probe for in vitro

97

glycodiversification of PPT-type ginsenosides using diverse UDP-sugars as sugar

98

donors. Furthermore, regiospecificity, stereospecificity, and glycosylation patterns of

99

Bs-YjiC toward PPT were elucidated by analysis of the structures of the glycosylated

100

products and the glycosylation process.

101

MATERIALS AND METHODS 5



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Chemicals and reagents. PPT and PPT-type ginsenosides (Rh1, F1, and Rg1)

103

were obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan,

104

China).

105

UDP-N-acetylglucosamine (UDP-GlcNAc), and UDP-glucuronic acid (UDP-GlcA)

106

were purchased from Sigma–Aldrich (St. Louis, MO, USA).

UDP-glucose

(UDPG),

UDP-galactose

(UDP-Gal),

107

Glycosylation of PPT and PPT-type ginsenosides. Gene Bs-YjiC (NP_389104)

108

was inserted into the BamHI and SalI restriction sites of pET28a expression vector to

109

generate an N-terminal His6-tagged gene. Subsequent expression and purification of

110

Bs-YjiC was carried out as described previously.32 Briefly, Escherichia coli BL21

111

(DE3) cells harboring recombinant pET28a-Bs-YjiC were precultured at 37 °C on LB

112

medium containing 50 µg mL−1 of kanamycin. After the OD600 reached 0.6–0.8,

113

recombinant Bs-YjiC was induced with 0.2 mM isopropyl-β-D -thiogalactopyranoside

114

(IPTG) and incubated further at 37 °C and 200 rpm for 6–8 h. The recombinant E. coli

115

cells containing recombinant Bs-YjiC were collected by centrifugation, resuspended

116

in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 25 mM imidazole), and

117

then disrupted with a French Press. Cell debris was removed by centrifugation at

118

17,000×g for 30 min. The supernatant containing recombinant Bs-YjiC was purified

119

with an AKTA Purifier system (GE Healthcare, Piscataway, NJ, USA) coupled with a

120

Ni-NTA agarose affinity column.

121

Enzymatic assays (0.3 mL) containing 10 mM diverse UDP-sugars (UDPG,

122

UDP-GlcA, UDP-Gal, or UDP-GlcNAc), 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1

123

µg purified Bs-YjiC, and 2 mM PPT or PPT-type ginsenosides were carried out at 35 6


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124

°C for 0.5 h. To determine the effect of UDPG concentrations (2, 4, 8, or 16 mM) on

125

the glycosylation patterns of Bs-YjiC toward PPT, duplicate reactions were performed

126

in the presence of 2 mM PPT. To analyze the glycosylated products using products 1–

127

5 as aglycons, duplicate reactions were performed in the presence of 4 mM UDPG.

128

The reactions were terminated by adding an equal volume of methanol and then

129

analyzed by high-performance liquid chromatography (HPLC) and HPLC coupled

130

with quantitative time of flight-high-resolution electrospray ionization-mass

131

spectrometry (HPLC-Q-TOF/ESI-MS).

132

HPLC and HPLC-Q-TOF/ESI-MS analysis of the glycosylated products. A

133

total of 20 µL of reactants were examined by HPLC and HPLC-Q-TOF/ESI-MS as

134

described in our previous study.33 The XB-C18 reverse-phase column (4.6 × 250 mm,

135

5 µm particle, Welch, Shanghai, China) connected to an Agilent 1260 HPLC system

136

was eluted with solvent A (water plus 0.1% formic acid) and solvent B (acetonitrile

137

plus 0.1% formic acid) by using a gradient program of 25%–85% B in 0–25 min. The

138

ESI probe was operated in positive ion mode.

139

Structural analysis of the glycosylated products of PPT. For the structural

140

analysis of products 1–5, a scale-up reaction (200 mL) was prepared as described

141

above. The enzymatic reactions were terminated by adding an equal volume of

142

methanol. Subsequently, the reactants were condensed under reduced pressure

143

distillation, and the residues were resuspended in methanol (10 mL). The glycosylated

144

products were purified with an Agilent 1200 preparative HPLC system coupled with a

145

reverse-phase preparative C18 column (21.2 × 250 mm, 5 µm particle, Welch, 7



146

Shanghai, China). The preparative column was eluted with solvent A (water) and

147

solvent B (methanol) using a gradient program of 50%–85% B in 0–60 min. The flow

148

rate was 10 mL/min, and other HPLC conditions were described above. After vacuum

149

freeze drying, the purified products were dissolved in methanol-d4. 1D NMR (1H

150

NMR and

151

spectroscopy [HMBC], heteronuclear singular quantum correlation [HSQC], and

152

homonuclear correlation spectroscopy [COSY]) were obtained using a Bruker

153

DMX-600 NMR spectrometer.

13

C NMR) and 2D NMR spectra (heteronuclear multiple-bond correlation

154

Kinetic analysis of Bs-YjiC. For kinetic analysis of Bs-YjiC toward PPT, Rh1,

155

F1, and Rg1, the reaction mixtures (0.3 mL) containing 50 mM Tris-HCl (pH 8.0), 10

156

mM MgCl2, 10 mM UDPG, 0.1 µg of purified Bs-YjiC, and varying concentrations of

157

PPT (50-600 µM), Rh1 (50-600 µM), F1(50-800 µM), and Rg1 (50-600 µM) were

158

incubated at 35 °C for 20 min. All the subsequent steps were performed as described

159

above. The kinetic parameters were calculated by nonlinear regression analysis using

160

GraphPad Prism 5.0 software. The kcat values were calculated using the predicted

161

molecular mass of 4.5 ×104 g mol−1 for Bs-YjiC.

162

RESULTS AND DISCUSSION

163

Glycodiversification of PPT with Bs-YjiC. N-terminal His6-tagged Bs-YjiC

164

was expressed in E. coli BL21 (DE3) and was easily purified to homogeneity

165

by one-step affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA)-agarose

166

(Figure S1). PPT is the common triterpene aglycon of PPT-type ginsenosides.15

167

Therefore, PPT was selected as a probe for the in vitro glycodiversification of 8


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PPT-type ginsenosides with UDPG as sugar donor (Figure 1). Five new products (1-5)

169

were identified from the Bs-YjiC-catalyzed reactant through HPLC analysis, whereas

170

no new product was obtained in the control reaction mixtures catalyzed by total lysate

171

from E. coli BL21 (DE3) expressing pET28a (Figure 2A). Further HPLC-Q-TOF/

172

ESI-MS analysis confirmed that products 1 ([M+H]+ m/z+ ~963.5477), 2 ([M+H]+

173

m/z+ ~801.4994), 3 ([M+H]+ m/z+ ~801.4974), 4 ([M+H]+ m/z+ ~639.4437), and 5

174

([M+H]+ m/z+ ~639.4438) were the glycosylated derivatives of PPT (C30H52O4,

175

calculated molecular weight, [M+H]+ m/z+ ~ 477.3938) with 1–3 glucosyl moieties

176

attached to the PPT skeleton (Figure 2B).

177

For the elucidation of the regio- and stereospecificity of Bs-YjiC toward PPT,

178

products 1–5 were purified by preparative HPLC, and their structures were elucidated

179

on the base of 1D NMR (1H NMR and

180

HSQC, and COSY) (Figures S2–S26). For product 5, the observation of significant

181

downfield shift (~11 ppm) of C3 suggested that a glucosyl moiety was attached to the

182

C3-OH of PPT (Table S1).21,33 Furthermore, the HMBC correlations of the sugar

183

anomeric signal H-1ʹ (δH 4.34, d, J=7.80 Hz) with C3 (δC 90.6) confirmed that product

184

5 was 3-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. The 1H and 13C NMR spectra

185

of product 4 were consistent with those of authentic ginsenoside Rh1 (Table S2).15 A

186

notably significant downfield 13C-shift (~12 ppm, “glycosylation shift”) at δ 80.9 (C6)

187

indicated that a glucosyl moiety was attached to the C6-OH of PPT. In the HMBC

188

data, long-range correlations between the sugar anomeric signal H-1ʹ (δH 4.35, d,

189

J=7.80 Hz) with C6 (δC 80.9) suggested that product 4 was 6-O-β-D

13

C NMR) and 2D NMR spectra (HMBC,

9



Page 10 of 30

190

-glucopyranosyl-20(S)-protopanaxatriol (ginsenoside Rh1). Product 3 exhibited

191

spectroscopic data similar to those of products 4 and 5 (Table S3). The observation of

192

significant downfield shift of C3 (~11 ppm) and C12 (~7 ppm) suggested that a

193

glucosyl moiety was attached to the C3-OH and C12-OH of PPT, respectively. The

194

HMBC correlations of sugar anomeric signal H-1ʹ (δH 4.35, d, J=7.80 Hz) with C3 (δC

195

90.5) and sugar anomeric signal H-1ʹʹ (δH 4.54, d, J=7.80 Hz) with C12 (δC 79.3)

196

suggested

197

3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol.

198

product 2, the 13C-NMR “glycosylation shift” (~11 ppm) of C3 (δC 91.0) and C6 (δC

199

80.8) indicated that a glucosyl moiety was attached to the C3-OH and C6-OH of PPT,

200

respectively (Table S4). In the HMBC data, the HMBC correlations of sugar anomeric

201

signals H-1ʹ (δH 4.37, d, J=7.80 Hz) with C3 (δC 91.0) and H-2ʹʹ (δH 4.35, d, J=7.80

202

Hz) with C6 (δC 80.8) suggested a β-glucosyl moiety attached to the C3-OH and

203

C6-OH

204

3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. The 1H and

205

13

206

(Table S5). The observation of significant downfield

207

C6 (~12 ppm), and C12 (~7 ppm) of PPT skeleton indicated that a glucosyl moiety

208

was attached to the C3-OH, C6-OH and C12-OH of PPT, respectively. Furthermore,

209

the HMBC correlations of the sugar anomeric signals H-1ʹ (δH 4.36, d, J=7.80 Hz)

210

with C3 (δC 90.9), H-1ʹʹ (δH 4.38, d, J=7.80 Hz) with C6 (δC 80.7), and H-1ʹʹʹ (δH 4.54,

211

d, J=7.80 Hz) with C12 (δC 79.3) demonstrated that product 1 was

that

of

PPT,

product

respectively.

Thus,

product

was

3

2

was

determined

For

to

be

C NMR spectra of product 1 were highly similar to those of products 2, 3, 4, and 5 13

C shift of the C3 (~12 ppm),

10


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212

3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-pr

213

otopanaxatriol. The observation of large anomeric proton-coupling constants (J=7.80)

214

indicated all the sugar moieties attached to PPT skeleton by β-glycosidic bond and an

215

inverting mechanism of Bs-YjiC. Thus, Bs-YjiC was the first reported UGT that can

216

regiospecifically and stereospecifically glycosylate the free C3-OH, C6-OH, and

217

C12-OH of PPT (Figure 1). Of these five glycosylated derivatives of PPT, products 1,

218

2, 3, and 5 were unnatural PPT-type ginsenosides and products 1 and 3 were first

219

synthesized in this study. Similar to other natural PPT-type ginsenoides, the newly

220

biosynthesized ginsenosides in this study should possess novel biological and

221

pharmacological activities.3 Notably, the deduced amino acid sequences of Bs-YjiC

222

exhibited 94.39% identity with those of UGT109A1 from B. subtilis CTCC 63501.

223

However, UGT109A1 can only catalyze a continuous two-step glycosylation of the

224

free

225


226

12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol

227

3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxatriol.29

228

further study based on homologous modeling and site-directed mutagenesis should be

229

carried out to determine the key amino acids of Bs-YjiC involved in the regiospecific

230

glycosylation of PPT.

C3-OH

and

C12-OH

of

PPT

to

produce

and Thus,

231

Compared with UGTs isolated from plants, some microbial UGTs are more

232

flexible toward both the sugar donors and aglycon acceptors.34-37 Thus, the reactions

233

of PPT with UDP-GlcA, UDP-Gal, and UDP-GlcNAc as sugar donors were 11



234

performed under identical conditions with that of PPT and UDPG (Figure 3). HPLC

235

analysis of the reactants confirmed that Bs-YjiC could glycosylate PPT with UDP-Gal

236

and UDP-GlcNAc as sugar donors, whereas no new product was observed in the

237

reactant incubating PPT with UDP-GlcA or control reactant without sugar donors.

238

When UDP-Gal was used as sugar donor, products a ([M+H]+ m/z+ ~639.4434), b

239

([M+H]+ m/z+ ~639.4443), and c ([M+H]+ m/z+ ~639.4447) were identified as

240

mono-galactosides of PPT (Figure S27), as indicated by the HPLC-Q-TOF/ESI-MS

241

analysis. Conversely, when UDP-GlcNAc was used as sugar donor, products d

242

([M+H]+ m/z+ ~680.4697) and e ([M+H]+ m/z+ ~680.4709) were identified as

243

mono-N-acetylglucosaminide of PPT (Figure S28). Furthermore, when UDP-Gal and

244

UDP-GlcNAc were used as sugar donors, the number of newly formed products,

245

number of attached sugar moieties, and conversion rates of PPT were considerably

246

lower than those obtained when UDPG was used (Figures 2 and 3). These results

247

suggested that the sugar moiety of UDP-sugars played an important role on the

248

glycosylation patterns and catalytic efficiencies of Bs-YjiC.

249

Glycosylation patterns of Bs-YjiC toward PPT. The concentration of UDPG

250

plays an important role on the number and concentration ratio of UTG-catalyzed

251

products.25 To determine the glycosylation patterns of Bs-YjiC toward PPT when

252

UDPG is used as a glucosyl donor, duplicate reactions were carried out with various

253

UDPG concentrations (2, 4, 8, and 16 mM) in the presence of 2 mM PPT (Figure 4A).

254

At a low UDPG concentration (2 mM), products 3 (diglucoside) and 5

255

(monoglucoside) were the major products, indicating that Bs-YjiC favorably 12


Page 12 of 30

Page 13 of 30


256

glycosylated the C3-OH and C12-OH of PPT to form products 5 and 3. When UDPG

257

had a concentration twice as high of that of PPT, the concentration of product 5

258

decreased and only a trace amount of product 1 was detected, whereas the

259

concentrations of products 2 and 3 increased. When the concentration ratios of

260

UDPG/PPT increased to 4 or 8, the concentrations of products 1, 2, and 3 increased

261

and product 4 was not detected.

262

To confirm the glycosylation patterns of Bs-YjiC, we performed duplicate

263

reactions using products 1, 2, 3, 4, and 5 as substrates (2 mM) in the presence of 4

264

mM UDPG (Figure 4B). When product 5 was used as substrate, product 3 was the

265

major product and only trace amounts of products 1 and 2 were detected, indicating

266

that Bs-YjiC favorably glycosylated the C12-OH of product 5 to form product 3

267

(Figure 1). Furthermore, the detection of products 1 and 2 suggested that Bs-YjiC can

268

catalyze a continuous two-step glycosylation of the C6-OH and C12-OH of product 5

269

(Figure 4B). With products 2 or 4 as substrate, the identification of product 1

270

reconfirmed that it could be formed via a continuous two-step glycosylation reaction

271

of product 4. Moreover, analysis of the glycosylated products using products 2, 4 or 5

272

as substrates suggested that product 1 was mainly formed via a continuous two-step

273

glycosylation of the C3-OH and C12-OH of product 4 (Figure 1). Meanwhile, no new

274

product was observed using products 1 or 3 as substrates. This result was consistent

275

with the glycosylation patterns of Bs-YjiC, as shown in Figure 1.

276

Glycosylation of ginsenosides Rh1, F1 and Rg1 with Bs-YjiC. We were

277

interested in glycosylation of other PPT-type ginsenosides using Bs-YjiC as a 13



278

biocatalyst. Thus, reactions of ginsenosides Rh1, F1, and Rg1 with UDPG as glucosyl

279

donor were performed under conditions identical to that of PPT (Figure 5). With

280

ginsenoside Rh1 (calculated molecular weight, [M+H]+ m/z+ ~639.4471), one

281

diglucoside (product R-1, retention time (RT) =10.3 min, [M+H]+ m/z+ ~801.4982)

282

and one triglucoside (product R-2, RT=6.5 min, [M+H]+ m/z+ ~963.5377) were

283

confirmed by HPLC-Q-TOF/ESI-MS (Figure S29). In the case of ginsenoside F1

284

(calculated molecular weight, [M+H]+ m/z+ ~639.4471), one diglucoside (product

285

F-1, RT=10.6 min, [M+H]+ m/z+ ~801.4998), three triglucosides (product F-2,

286

RT=10.2 min, [M+H]+ m/z+ ~963.5526; product F-3, RT=9.0 min, [M+H]+ m/z+ ~

287

963.5520; product F-4, RT=7.0 min, [M+H]+ m/z+ ~963.5522), and two

288

tetraglucosides (product F-5, RT=6.4 min, [M+H]+ m/z+ ~1125.6059; product F-6,

289

RT=5.5 min, [M+H]+ m/z+ ~1125.6045) were detected (Figure S30). With Rg1, three

290

triglucosides (product R1, RT=7.0 min, [M+H]+ m/z+ ~963.5491; product R2, RT=6.8

291

min, [M+H]+ m/z+ ~963.5504; product R3, RT=6.4 min, [M+H]+ m/z+ ~963.5497),

292

two tetraglucosides (product R4, RT=5.9 min, [M+H]+ m/z+ ~1125.6016; product R5,

293

RT=5.6 min, [M+H]+ m/z+ ~1125.6053), and one pentaglucoside (product R6, RT=5.4

294

min, [M+H]+ m/z+ ~1287.6802) were produced (Figure S31). Given the regio- and

295

stereospecificity of Bs-YjiC toward PPT, most of the newly biosynthesized products

296

were novel PPT-type ginsenosides.

297

Kinetic parameters of Bs-YjiC. The kinetic parameters of purified Bs-YjiC for

298

PPT, ginsenosides Rh1, F1, and Rg1, were determined (Table 1) (Figure S32). The Km

299

values of Bs-YjiC for PPT (103.60 µM), Rh1 (50.34 µM), F1 (211.80 µM), and Rg1 14


Page 14 of 30

Page 15 of 30


300

(107.40 µM) were comparable with previously reported ginseng UGTs and microbial

301

UGTs involved in the biosynthesis of PPT-type ginsenosides.15,29,31 The turnover

302

numbers (kcat) of Bs-YjiC for PPT, Rh1, F1, and Rg1 were 23.16 s−1, 14.95 s−1, 22.45

303

s−1, and 23.09 s−1, respectively. The kcat values of Bs-YjiC were much higher than

304

those of previously reported ginseng UGTs and microbial UGTs15,29,31,37 and thus the

305

catalytic efficiencies (kcat/Km) of Bs-YjiC toward PPT (0.22 µM−1 s−1), Rh1 (0.30

306

µM−1 s−1), F1 (0.11 µM−1 s−1), and Rg1 (0.21 µM−1 s−1) were considerably high. The

307

high catalytic efficiencies of Bs-YjiC toward PPT and PPT-type ginsenosides and its

308

broad acceptors tolerance toward a considerable number of structurally diverse types

309

of natural and unnatural products were consistent with the previous notion that

310

naturally occurring UGTs with high catalytic proficiencies are generally more flexible

311

toward aglycons.20,32,37

312

In summary, Bs-YjiC from B. subtilis 168 was the first reported UGT that can

313

transfer a glucosyl moiety to the free C3-OH, C6-OH, and C12-OH of PPT. Our

314

findings provided a significant insight into the important roles of microbial UGTs for

315

the enzymatic glycodiversification of ginsenosides. Further pharmacological

316

properties of these newly biosynthesized unnatural PPT-type ginsenosides should be

317

studied. Future structural study of Bs-YjiC should be carried out to elucidate the

318

structure–function relationship. Furthermoere, it will be of particular interest to

319

introduce Bs-YjiC or an engineered Bs-YjiC into PPT-producing chassis cells to

320

synthesize these natural and unnatural ginsenosides or a specific ginsenoside via

321

metabolic engineering. 15



322

Supporting Information

323

The

324

experimental results (Figures S1 and S32), and HPLC-Q-TOF/ESI-MS and NMR

325

analysis (Figures S2-S31).

326

AUTHOR INFORMATION

327

Corresponding Authors

328

Tel.: +862284861983. E-mail: [email protected]

329

Tel.: +862284861960. E-mail: [email protected]

330

Notes

331

The authors declare no competing ﬁnancial interest.

332

Funding

333

This work was supported by National Natural Science Foundation of China (No.

334

21702226) and the Science and Technology Planning Project of Tianjin (No.

335

11ZCZDSY08900).

336

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22


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463

Figure 1. Glycosylation patterns of Bs-YjiC toward PPT and intermediates. PgUGT1,

464

PgUGT100, and PgUGT101 involved in the biosynthesis of natural ginsenosides Rh1,

465

F1, and Rg1 were isolated from ginseng in previous studies. Bold black arrows

466

represent the major catalytic steps of the intermediates.

467 468

Figure 2. HPLC-Q-TOF/ESI-MS analysis of the glycosylated products of PPT

469

catalyzed by Bs-YjiC. (A) HPLC chromatograms of PPT and PPT-type ginsenoside

470

standards, control reactant mixtures, and Bs-YjiC-catalyzed reactants. (B) MS spectra

471

for products 1 (a), 2 (b), and 3 (c), 4 (d), and 5 (e).

472 473

Figure 3. HPLC analysis of the glycosylated products of PPT with diverse

474

UDP-sugars as sugar donors.

475 476

Figure 4. HPLC analysis of the glycosylated products using different concentrations

477

of UDPG and different intermediates. (A) HPLC analysis of the glycosylated products

478

using different concentrations of UDPG (2, 4, 8, and 16 mM) in the presence of 2 mM

479

PPT; (B) HPLC analysis of the glycosylated products using products 1–5 as

480

substrates.

481 482

Figure 5. HPLC analysis of the glycosylated products of Rh1, F1, and Rg1 catalyzed

483

by Bs-YjiC.

23



484

Page 24 of 30

Table 1 Kinetic parameters of Bs-YjiC towards PPT, ginsenosides Rh1, F1, and Rg1 Substrate

Km (µM)

kcat (s-1)

kcat/Km (s-1 µM-1)

PPT

103.60±18.28

23.16±1.22

0.22

Rh1

50.34±11.25

14.95±0.76

0.30

F1

211.80±23.81

22.45±0.97

0.11

Rg1

107.40±11.48

23.09±0.74

0.21

485

24


Page 25 of 30


Figure 1. Glycosylation patterns of Bs-YjiC toward PPT and intermediates. PgUGT1, PgUGT100, and PgUGT101 involved in the biosynthesis of natural ginsenosides Rh1, F1, and Rg1 were isolated from ginseng in previous studies. Bold black arrows represent the major catalytic steps of the intermediates. 168x70mm (300 x 300 DPI)



Figure 2. HPLC-Q-TOF/HR-ESI-MS analysis of the glycosylated products of PPT catalyzed by Bs-YjiC. (A) HPLC chromatograms of PPT and PPT-type ginsenoside standards, control reactant mixtures, and Bs-YjiCcatalyzed reactants. (B) MS spectra for products 1 (a), 2 (b), and 3 (c), 4 (d), and 5 (e). 116x88mm (300 x 300 DPI)


Page 26 of 30

Page 27 of 30


Figure 3. HPLC analysis of the glycosylated products of PPT with diverse UDP-sugars as sugar donors. 60x44mm (300 x 300 DPI)



Figure 4. HPLC analysis of the glycosylated products using different concentrations of UDPG and different intermediates. (A) HPLC analysis of the glycosylated products using different concentrations of UDPG (2, 4, 8, and 16 mM) in the presence of 2 mM PPT; (B) HPLC analysis of the glycosylated products using products 1–5 as substrates. 106x137mm (300 x 300 DPI)


Page 28 of 30

Page 29 of 30


Figure 5. HPLC analysis of the glycosylated products of Rh1, F1 and Rg1 catalyzed by Bs-YjiC. 78x74mm (300 x 300 DPI)



Table of Contents Graphic 83x36mm (300 x 300 DPI)


Page 30 of 30

Promiscuous glycosyltransferase from Bacillus subtilis 168 for the

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