Use of a Promiscuous Glycosyltransferase from Bacillus subtilis 168 for the Enzymatic Synthesis of Novel Protopanaxatriol-Type Ginsenosides

Article abstract of DOI:10.1021/acs.jafc.7b03907

Ginsenosides are the principal bioactive ingredients of Panax ginseng and possess diverse notable pharmacological activities. UDP-glycosyltransferase (UGT)-mediated glycosylation of the C6-OH and C20-OH of protopanaxatriol (PPT) is the prominent biological modification that contributes to the immense structural and functional diversity of PPT-type ginsenosides. In this study, the glycosylation of PPT and PPT-type ginsenosides was achieved using a promiscuous glycosyltransferase (Bs-YjiC) from Bacillus subtilis 168. PPT was selected as the probe for the in vitro glycodiversification of PPT-type ginsenosides using diverse UDP-sugars as sugar donors. Structural analysis of the newly biosynthesized products demonstrated that Bs-YjiC can transfer a glucosyl moiety to the free C3-OH, C6-OH, and C12-OH of PPT. Five PPT-type ginsenosides were biosynthesized, including ginsenoside Rh1 and four unnatural ginsenosides. The present study suggests flexible microbial UGTs play an important role in the enzymatic synthesis of novel ginsenosides.

Full text of DOI:10.1021/acs.jafc.7b03907

Subscriber access provided by READING UNIV
Article
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
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 30
Journal of Agricultural and Food Chemistry
1
2
Promiscuous glycosyltransferase from Bacillus subtilis 168 for the enzymatic
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
Cai,a Caixia Dong,c Zhubo Dai,a Xueli Zhanga,* and Yuanxia Suna,*
5
6
a
7
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of
8
Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China
b University of Chinese Academy of Sciences, Beijing, China
9
c
10
11
12
13
14
15
16
17
18
19
Tianjin Key Laboratory on Technologies Enabling Development of Clinical
Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin
300070, China
* Correspondence author. Mailing address:
Xueli Zhang, Eꢀmail: zhang_xl@tib.cas.cn, Tel: +862284861983
Yuanxia Sun, Eꢀmail: sun_yx@tib.cas.cn, Tel: +862284861960
32 Xiqi Road, Tianjin Airport Economic Area, Tianjin, 300308, Peoples Republic of
China
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 30
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
ABSTRACT
Ginsenosides are the principally bioactive ingredients of Panax ginseng and
possess diverse notable pharmacological activities. UDPꢀglycosyltransferase
(UGT)ꢀmediated glycosylation of C6ꢀOH and/or C20ꢀOH of protopanaxatriol (PPT)
is the prominent biological modification that contributes to the immense structural
and functional diversity of PPTꢀtype ginsenosides. In this study, glycosylation of PPT
and PPTꢀtype ginsenosides was achieved using a promiscuous glycosyltransferase
(BsꢀYjiC) from Bacillus subtilis 168. PPT was selected as a probe for the in vitro
glycodiversification of PPTꢀtype ginsenosides using diverse UDPꢀsugars as sugar
donors. Structural analysis of the newly biosynthesized products demonstrated that
BsꢀYjiC can transfer a glucosyl moiety to the free C3ꢀOH, C6ꢀOH, and C12ꢀOH of
PPT. Five PPTꢀtype ginsenosides were biosynthesized, including ginsenoside Rh1 and
four unnatural ginsenosides. The present study suggests an important role of flexible
microbial UGTs for enzymatic synthesis of novel ginsenosides.
KEYWORDS: Protopanaxatriol, protopanaxatriolꢀtype ginsenosides, Bacillus
UDPꢀglycosyltransferase, glycosylation, chemical diversification
2
ACS Paragon Plus Environment

Page 3 of 30
Journal of Agricultural and Food Chemistry
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
INTRODUCTION
Ginseng (Panax ginseng C. A Meyer), a herbaceous perennial herb of the
Araliaceae family, is one of the most wellꢀknown and bestꢀselling oriental
medicines.1,2 This herb is frequently used in traditional medicine to strengthen
immunity, reduce fatigue, and provide nutrition for more than 2000 years in Eastern
Asia.3 The major pharmacologically active components of ginseng are glycosylated
dammaraneꢀtype tetracyclic triterpene saponins known as ginsenosides (ca. 2%–4% in
the dried roots and rhizomes of ginseng), which exhibit diverse and notable
pharmacological characteristics, such as antitumor, anticancer, antidiabetic,
antiꢀinflammatory, antiꢀaging, antifatigue, antihypertensive, and immune
systemꢀenhancing activities.4ꢀ7
More than 140 naturally occurring ginsenosides were isolated from different
ginseng species.8 Most of these natural products can be classified into
protopanaxadiol (PPD) and protopanaxatriol (PPT) groups according to the structure
of the triterpene aglycon.9 Currently, most of the key genes involved in PPDꢀ and
PPTꢀtype ginsenosides biosynthetic pathway have been functionally characterized.
The initial biosynthetic step is the cyclization of 2,3ꢀoxidosqualene to form
dammarenediolꢀII (DM), which is catalyzed by DM synthase (PgDDS).4,10 DM is
hydroxylated by PPD synthase (PgPPDS, CYP716A47) at its C12 position to form
PPD,11 and PPD is converted further to PPT after hydroxylation at its C6 position by
PPT
synthase
(PgPPTS,
CYP716A53v2).12
Additionally,
several
UDPꢀglycosyltransferases (UGTs) responsible for the glycosylation of C3ꢀOH and/or
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 30
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
C20ꢀOH of PPD, and C6ꢀOH and/or C20ꢀOH of PPT have been isolated from P.
ginseng in the past 3 years, and a number of PPDꢀtype ginsenosides (Rh2, CK, F2,
Rg3, and Rd) and PPTꢀtype ginsenosides (F1, Rh1, and Rg1) were biosynthesized by
introducing an engineered PPDꢀ or PPTꢀproducing pathway and ginseng UGT genes
into yeast cell factories.13ꢀ18
UGTꢀcatalyzed glycosylation acts as the final biosynthetic step for ginsenosides,
which present immense structural and functional diversity after the process. Some
UGTs, especially those isolated from microbes, show high substrate flexibility and
poor regiospecificity and thus can be exploited as effective biocatalysts for
glycorandomization of natural products with diverse structures.19 For example, UGT
OleD from Streptomyces antibioticus,20,21 MhGT1 from Mucor hiemalis,22 UGT58A1
from Absidia coerulea, UGT59A1 from Rhizopus japonicas,23 BlꢀYjiC from Bacillus
licheniformis,24,25 and BcGT1 from B. cereus26,27 are frequently applied to
glycodiversify a considerable number of important botanical natural products.
Recently, UGT51, which exhibits broad acceptor tolerance, was isolated from
Saccharomyces cerevisiae and demonstrated to be able to regiospecifically transfer a
glucosyl moiety to the free C3ꢀOH of PPD for the synthesis of ginsenoside Rh2.28
Additionally, several UGTs responsible for the biosynthesis of natural and unnatural
ginsenosides were isolated from several Bacillus strains, including UGT109A1
involved in biosynthesis of ginsenosides, such as
(3ꢀOꢀβꢀ ꢀglucopyranosylꢀ20(S)ꢀprotopanaxadiol),
12ꢀOꢀβꢀ
ginsenoside Rh2
D
D
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxadiol,
4
ACS Paragon Plus Environment

Page 5 of 30
Journal of Agricultural and Food Chemistry
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
3ꢀOꢀβꢀ
3ꢀOꢀβꢀ
D
ꢀglucopyranosylꢀ12ꢀOꢀβꢀ
D
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxadiol,
Dꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol,
12ꢀOꢀβꢀ
3ꢀOꢀβꢀ
involved
3ꢀOꢀβꢀ ꢀglucopyranosylꢀ6ꢀOꢀβꢀ
involved in
ꢀglucopyranosylꢀ20ꢀOꢀβꢀ
D
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol,
and
Dꢀglucopyranosylꢀ12ꢀOꢀβꢀ
D
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol;29 YjiC1
in
the
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol;30
biosynthesis of ginsenoside
biosynthesis
of
and
Ia
D
D
BSGT1
(3ꢀOꢀβꢀ
D
D
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol).31 Based
on these studies, we explored substrateꢀflexible UGTs from microbes for the
biosynthesis of novel ginsenosides for drug discovery.
BsꢀYjiC from Bacillus subtilis 168 is a promiscuous and robust UGT toward a
considerable number of structurally diverse types of natural and unnatural products,
including several structurally diverse types of triterpenes.32 Furthermore, BsꢀYiC can
glycosylate both the free C3ꢀOH and C20ꢀOH of PPD and PPDꢀtype ginsenosides to
synthesize a series of natural and unnatural ginsenosides (unpublished data). In this
study, the potential of BsꢀYjiC as a biocatalyst for glycosylation of PPT and PPTꢀtype
ginsenosides was explored further. PPT was selected as a probe for in vitro
glycodiversification of PPTꢀtype ginsenosides using diverse UDPꢀsugars as sugar
donors. Furthermore, regiospecificity, stereospecificity, and glycosylation patterns of
BsꢀYjiC toward PPT were elucidated by analysis of the structures of the glycosylated
products and the glycosylation process.
MATERIALS AND METHODS
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 30
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
Chemicals and reagents. PPT and PPTꢀtype ginsenosides (Rh1, F1, and Rg1)
were obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan,
China).
UDPꢀglucose
(UDPG),
UDPꢀgalactose
(UDPꢀGal),
UDPꢀNꢀacetylglucosamine (UDPꢀGlcNAc), and UDPꢀglucuronic acid (UDPꢀGlcA)
were purchased from Sigma–Aldrich (St. Louis, MO, USA).
Glycosylation of PPT and PPT-type ginsenosides. Gene BsꢀYjiC (NP_389104)
was inserted into the BamHI and SalI restriction sites of pET28a expression vector to
generate an Nꢀterminal His6ꢀtagged gene. Subsequent expression and purification of
BsꢀYjiC was carried out as described previously.32 Briefly, Escherichia coli BL21
(DE3) cells harboring recombinant pET28aꢀBsꢀYjiC were precultured at 37 °C on LB
1
medium containing 50 µg mL− of kanamycin. After the OD600 reached 0.6–0.8,
recombinant BsꢀYjiC was induced with 0.2 mM isopropylꢀβꢀD ꢀthiogalactopyranoside
(IPTG) and incubated further at 37 °C and 200 rpm for 6–8 h. The recombinant E. coli
cells containing recombinant BsꢀYjiC were collected by centrifugation, resuspended
in lysis buffer (50 mM TrisꢀHCl, pH 8.0, 150 mM NaCl, and 25 mM imidazole), and
then disrupted with a French Press. Cell debris was removed by centrifugation at
17,000×g for 30 min. The supernatant containing recombinant BsꢀYjiC was purified
with an AKTA Purifier system (GE Healthcare, Piscataway, NJ, USA) coupled with a
NiꢀNTA agarose affinity column.
Enzymatic assays (0.3 mL) containing 10 mM diverse UDPꢀsugars (UDPG,
UDPꢀGlcA, UDPꢀGal, or UDPꢀGlcNAc), 50 mM TrisꢀHCl (pH 8.0), 10 mM MgCl2, 1
µg purified BsꢀYjiC, and 2 mM PPT or PPTꢀtype ginsenosides were carried out at 35
6
ACS Paragon Plus Environment

Page 7 of 30
Journal of Agricultural and Food Chemistry
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
°C for 0.5 h. To determine the effect of UDPG concentrations (2, 4, 8, or 16 mM) on
the glycosylation patterns of BsꢀYjiC toward PPT, duplicate reactions were performed
in the presence of 2 mM PPT. To analyze the glycosylated products using products 1–
5 as aglycons, duplicate reactions were performed in the presence of 4 mM UDPG.
The reactions were terminated by adding an equal volume of methanol and then
analyzed by highꢀperformance liquid chromatography (HPLC) and HPLC coupled
with quantitative time of flightꢀhighꢀresolution electrospray ionizationꢀmass
spectrometry (HPLCꢀQꢀTOF/ESIꢀMS).
HPLC and HPLC-Q-TOF/ESI-MS analysis of the glycosylated products. A
total of 20 µL of reactants were examined by HPLC and HPLCꢀQꢀTOF/ESIꢀMS as
described in our previous study.33 The XBꢀC18 reverseꢀphase column (4.6 × 250 mm,
5 µm particle, Welch, Shanghai, China) connected to an Agilent 1260 HPLC system
was eluted with solvent A (water plus 0.1% formic acid) and solvent B (acetonitrile
plus 0.1% formic acid) by using a gradient program of 25%–85% B in 0–25 min. The
ESI probe was operated in positive ion mode.
Structural analysis of the glycosylated products of PPT. For the structural
analysis of products 1–5, a scaleꢀup reaction (200 mL) was prepared as described
above. The enzymatic reactions were terminated by adding an equal volume of
methanol. Subsequently, the reactants were condensed under reduced pressure
distillation, and the residues were resuspended in methanol (10 mL). The glycosylated
products were purified with an Agilent 1200 preparative HPLC system coupled with a
reverseꢀphase preparative C18 column (21.2 × 250 mm, 5 µm particle, Welch,
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 30
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
Shanghai, China). The preparative column was eluted with solvent A (water) and
solvent B (methanol) using a gradient program of 50%–85% B in 0–60 min. The flow
rate was 10 mL/min, and other HPLC conditions were described above. After vacuum
freeze drying, the purified products were dissolved in methanolꢀd4. 1D NMR (1H
NMR and 13C NMR) and 2D NMR spectra (heteronuclear multipleꢀbond correlation
spectroscopy [HMBC], heteronuclear singular quantum correlation [HSQC], and
homonuclear correlation spectroscopy [COSY]) were obtained using a Bruker
DMXꢀ600 NMR spectrometer.
Kinetic analysis of Bs-YjiC. For kinetic analysis of BsꢀYjiC toward PPT, Rh1,
F1, and Rg1, the reaction mixtures (0.3 mL) containing 50 mM TrisꢀHCl (pH 8.0), 10
mM MgCl2, 10 mM UDPG, 0.1 ꢁg of purified BsꢀYjiC, and varying concentrations of
PPT (50ꢀ600 ꢁM), Rh1 (50ꢀ600 ꢁM), F1(50ꢀ800 ꢁM), and Rg1 (50ꢀ600 ꢁM) were
incubated at 35 °C for 20 min. All the subsequent steps were performed as described
above. The kinetic parameters were calculated by nonlinear regression analysis using
GraphPad Prism 5.0 software. The kcat values were calculated using the predicted
molecular mass of 4.5 ×104 g mol−1 for BsꢀYjiC.
RESULTS AND DISCUSSION
Glycodiversification of PPT with Bs-YjiC. Nꢀterminal His6ꢀtagged BsꢀYjiC
was expressed in E. coli BL21 (DE3) and was easily purified to homogeneity
by oneꢀstep affinity chromatography on nickelꢀnitrilotriacetic acid (NiꢀNTA)ꢀagarose
(Figure S1). PPT is the common triterpene aglycon of PPTꢀtype ginsenosides.15
Therefore, PPT was selected as a probe for the in vitro glycodiversification of
8
ACS Paragon Plus Environment

Page 9 of 30
Journal of Agricultural and Food Chemistry
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
PPTꢀtype ginsenosides with UDPG as sugar donor (Figure 1). Five new products (1ꢀ5)
were identified from the BsꢀYjiCꢀcatalyzed reactant through HPLC analysis, whereas
no new product was obtained in the control reaction mixtures catalyzed by total lysate
from E. coli BL21 (DE3) expressing pET28a (Figure 2A). Further HPLCꢀQꢀTOF/
ESIꢀMS analysis confirmed that products 1 ([M+H]+ m/z+ ~963.5477), 2 ([M+H]+
m/z+ ~801.4994), 3 ([M+H]+ m/z+ ~801.4974), 4 ([M+H]+ m/z+ ~639.4437), and 5
([M+H]+ m/z+ ~639.4438) were the glycosylated derivatives of PPT (C30H52O4,
calculated molecular weight, [M+H]+ m/z+ ~ 477.3938) with 1–3 glucosyl moieties
attached to the PPT skeleton (Figure 2B).
For the elucidation of the regioꢀ and stereospecificity of BsꢀYjiC toward PPT,
products 1–5 were purified by preparative HPLC, and their structures were elucidated
on the base of 1D NMR (1H NMR and 13C NMR) and 2D NMR spectra (HMBC,
HSQC, and COSY) (Figures S2–S26). For product 5, the observation of significant
downfield shift (~11 ppm) of C3 suggested that a glucosyl moiety was attached to the
C3ꢀOH of PPT (Table S1).21,33 Furthermore, the HMBC correlations of the sugar
anomeric signal Hꢀ1ʹ (δH 4.34, d, J=7.80 Hz) with C3 (δC 90.6) confirmed that product
1
13
5 was 3ꢀOꢀβꢀ
Dꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol. The H and C NMR spectra
of product 4 were consistent with those of authentic ginsenoside Rh1 (Table S2).15
A
notably significant downfield 13Cꢀshift (~12 ppm, “glycosylation shift”) at δ 80.9 (C6)
indicated that a glucosyl moiety was attached to the C6ꢀOH of PPT. In the HMBC
data, longꢀrange correlations between the sugar anomeric signal Hꢀ1ʹ (δH 4.35, d,
J=7.80 Hz) with C6 (δC 80.9) suggested that product 4 was 6ꢀOꢀβꢀ
D
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 30
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol (ginsenoside Rh1). Product 3 exhibited
spectroscopic data similar to those of products 4 and 5 (Table S3). The observation of
significant downfield shift of C3 (~11 ppm) and C12 (~7 ppm) suggested that a
glucosyl moiety was attached to the C3ꢀOH and C12ꢀOH of PPT, respectively. The
HMBC correlations of sugar anomeric signal Hꢀ1ʹ (δH 4.35, d, J=7.80 Hz) with C3 (δC
90.5) and sugar anomeric signal Hꢀ1ʹʹ (δH 4.54, d, J=7.80 Hz) with C12 (δC 79.3)
suggested
that
product
3
was
For
3ꢀOꢀβꢀ ꢀglucopyranosylꢀ12ꢀOꢀβꢀ
D
Dꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol.
product 2, the 13CꢀNMR “glycosylation shift” (~11 ppm) of C3 (δC 91.0) and C6 (δC
80.8) indicated that a glucosyl moiety was attached to the C3ꢀOH and C6ꢀOH of PPT,
respectively (Table S4). In the HMBC data, the HMBC correlations of sugar anomeric
signals Hꢀ1ʹ (δH 4.37, d, J=7.80 Hz) with C3 (δC 91.0) and Hꢀ2ʹʹ (δH 4.35, d, J=7.80
Hz) with C6 (δC 80.8) suggested a βꢀglucosyl moiety attached to the C3ꢀOH and
C6ꢀOH of PPT, respectively. Thus, product
3ꢀOꢀβꢀ ꢀglucopyranosylꢀ6ꢀOꢀβꢀ
2
was determined to be
1
D
D
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol. The H and
13C NMR spectra of product 1 were highly similar to those of products 2, 3, 4, and 5
(Table S5). The observation of significant downfield 13C shift of the C3 (~12 ppm),
C6 (~12 ppm), and C12 (~7 ppm) of PPT skeleton indicated that a glucosyl moiety
was attached to the C3ꢀOH, C6ꢀOH and C12ꢀOH of PPT, respectively. Furthermore,
the HMBC correlations of the sugar anomeric signals Hꢀ1ʹ (δH 4.36, d, J=7.80 Hz)
with C3 (δC 90.9), Hꢀ1ʹʹ (δH 4.38, d, J=7.80 Hz) with C6 (δC 80.7), and Hꢀ1ʹʹʹ (δH 4.54,
d, J=7.80 Hz) with C12 (δC 79.3) demonstrated that product
1 was
10
ACS Paragon Plus Environment

Page 11 of 30
Journal of Agricultural and Food Chemistry
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
3ꢀOꢀβꢀDꢀglucopyranosylꢀ6ꢀOꢀβꢀDꢀglucopyranosylꢀ12ꢀOꢀβꢀDꢀglucopyranosylꢀ20(S)ꢀpr
otopanaxatriol. The observation of large anomeric protonꢀcoupling constants (J=7.80)
indicated all the sugar moieties attached to PPT skeleton by βꢀglycosidic bond and an
inverting mechanism of BsꢀYjiC. Thus, BsꢀYjiC was the first reported UGT that can
regiospecifically and stereospecifically glycosylate the free C3ꢀOH, C6ꢀOH, and
C12ꢀOH of PPT (Figure 1). Of these five glycosylated derivatives of PPT, products 1,
2, 3, and 5 were unnatural PPTꢀtype ginsenosides and products 1 and 3 were first
synthesized in this study. Similar to other natural PPTꢀtype ginsenoides, the newly
biosynthesized ginsenosides in this study should possess novel biological and
pharmacological activities.3 Notably, the deduced amino acid sequences of BsꢀYjiC
exhibited 94.39% identity with those of UGT109A1 from B. subtilis CTCC 63501.
However, UGT109A1 can only catalyze a continuous twoꢀstep glycosylation of the
free
C3ꢀOH
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol,
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol
ꢀglucopyranosylꢀ12ꢀOꢀβꢀ
ꢀglucopyranosylꢀ20(S)ꢀprotopanaxatriol.29
and
C12ꢀOH
of
PPT
to
produce
3ꢀOꢀβꢀ
D
12ꢀOꢀβꢀ
3ꢀOꢀβꢀ
D
and
D
D
Thus,
further study based on homologous modeling and siteꢀdirected mutagenesis should be
carried out to determine the key amino acids of BsꢀYjiC involved in the regiospecific
glycosylation of PPT.
Compared with UGTs isolated from plants, some microbial UGTs are more
flexible toward both the sugar donors and aglycon acceptors.34ꢀ37 Thus, the reactions
of PPT with UDPꢀGlcA, UDPꢀGal, and UDPꢀGlcNAc as sugar donors were
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 30
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
performed under identical conditions with that of PPT and UDPG (Figure 3). HPLC
analysis of the reactants confirmed that BsꢀYjiC could glycosylate PPT with UDPꢀGal
and UDPꢀGlcNAc as sugar donors, whereas no new product was observed in the
reactant incubating PPT with UDPꢀGlcA or control reactant without sugar donors.
When UDPꢀGal was used as sugar donor, products a ([M+H]+ m/z+ ~639.4434), b
([M+H]+ m/z+ ~639.4443), and c ([M+H]+ m/z+ ~639.4447) were identified as
monoꢀgalactosides of PPT (Figure S27), as indicated by the HPLCꢀQꢀTOF/ESIꢀMS
analysis. Conversely, when UDPꢀGlcNAc was used as sugar donor, products d
([M+H]+ m/z+ ~680.4697) and e ([M+H]+ m/z+ ~680.4709) were identified as
monoꢀNꢀacetylglucosaminide of PPT (Figure S28). Furthermore, when UDPꢀGal and
UDPꢀGlcNAc were used as sugar donors, the number of newly formed products,
number of attached sugar moieties, and conversion rates of PPT were considerably
lower than those obtained when UDPG was used (Figures 2 and 3). These results
suggested that the sugar moiety of UDPꢀsugars played an important role on the
glycosylation patterns and catalytic efficiencies of BsꢀYjiC.
Glycosylation patterns of Bs-YjiC toward PPT. The concentration of UDPG
plays an important role on the number and concentration ratio of UTGꢀcatalyzed
products.25 To determine the glycosylation patterns of BsꢀYjiC toward PPT when
UDPG is used as a glucosyl donor, duplicate reactions were carried out with various
UDPG concentrations (2, 4, 8, and 16 mM) in the presence of 2 mM PPT (Figure 4A).
At a low UDPG concentration (2 mM), products 3 (diglucoside) and 5
(monoglucoside) were the major products, indicating that BsꢀYjiC favorably
12
ACS Paragon Plus Environment

Page 13 of 30
Journal of Agricultural and Food Chemistry
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
glycosylated the C3ꢀOH and C12ꢀOH of PPT to form products 5 and 3. When UDPG
had a concentration twice as high of that of PPT, the concentration of product 5
decreased and only a trace amount of product 1 was detected, whereas the
concentrations of products 2 and 3 increased. When the concentration ratios of
UDPG/PPT increased to 4 or 8, the concentrations of products 1, 2, and 3 increased
and product 4 was not detected.
To confirm the glycosylation patterns of BsꢀYjiC, we performed duplicate
reactions using products 1, 2, 3, 4, and 5 as substrates (2 mM) in the presence of 4
mM UDPG (Figure 4B). When product 5 was used as substrate, product 3 was the
major product and only trace amounts of products 1 and 2 were detected, indicating
that BsꢀYjiC favorably glycosylated the C12ꢀOH of product 5 to form product 3
(Figure 1). Furthermore, the detection of products 1 and 2 suggested that BsꢀYjiC can
catalyze a continuous twoꢀstep glycosylation of the C6ꢀOH and C12ꢀOH of product 5
(Figure 4B). With products 2 or 4 as substrate, the identification of product 1
reconfirmed that it could be formed via a continuous twoꢀstep glycosylation reaction
of product 4. Moreover, analysis of the glycosylated products using products 2, 4 or 5
as substrates suggested that product 1 was mainly formed via a continuous twoꢀstep
glycosylation of the C3ꢀOH and C12ꢀOH of product 4 (Figure 1). Meanwhile, no new
product was observed using products 1 or 3 as substrates. This result was consistent
with the glycosylation patterns of BsꢀYjiC, as shown in Figure 1.
Glycosylation of ginsenosides Rh1, F1 and Rg1 with Bs-YjiC. We were
interested in glycosylation of other PPTꢀtype ginsenosides using BsꢀYjiC as a
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 30
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
biocatalyst. Thus, reactions of ginsenosides Rh1, F1, and Rg1 with UDPG as glucosyl
donor were performed under conditions identical to that of PPT (Figure 5). With
ginsenoside Rh1 (calculated molecular weight, [M+H]+ m/z+ ~639.4471), one
diglucoside (product R-1, retention time (RT) =10.3 min, [M+H]+ m/z+ ~801.4982)
and one triglucoside (product R-2, RT=6.5 min, [M+H]+ m/z+ ~963.5377) were
confirmed by HPLCꢀQꢀTOF/ESIꢀMS (Figure S29). In the case of ginsenoside F1
(calculated molecular weight, [M+H]+ m/z+ ~639.4471), one diglucoside (product
F-1, RT=10.6 min, [M+H]+ m/z+ ~801.4998), three triglucosides (product F-2,
RT=10.2 min, [M+H]+ m/z+ ~963.5526; product F-3, RT=9.0 min, [M+H]+ m/z+ ~
963.5520; product F-4, RT=7.0 min, [M+H]+ m/z+ ~963.5522), and two
tetraglucosides (product F-5, RT=6.4 min, [M+H]+ m/z+ ~1125.6059; product F-6,
RT=5.5 min, [M+H]+ m/z+ ~1125.6045) were detected (Figure S30). With Rg1, three
triglucosides (product R1, RT=7.0 min, [M+H]+ m/z+ ~963.5491; product R2, RT=6.8
min, [M+H]+ m/z+ ~963.5504; product R3, RT=6.4 min, [M+H]+ m/z+ ~963.5497),
two tetraglucosides (product R4, RT=5.9 min, [M+H]+ m/z+ ~1125.6016; product R5,
RT=5.6 min, [M+H]+ m/z+ ~1125.6053), and one pentaglucoside (product R6, RT=5.4
min, [M+H]+ m/z+ ~1287.6802) were produced (Figure S31). Given the regioꢀ and
stereospecificity of BsꢀYjiC toward PPT, most of the newly biosynthesized products
were novel PPTꢀtype ginsenosides.
Kinetic parameters of Bs-YjiC. The kinetic parameters of purified BsꢀYjiC for
PPT, ginsenosides Rh1, F1, and Rg1, were determined (Table 1) (Figure S32). The Km
values of BsꢀYjiC for PPT (103.60 ꢁM), Rh1 (50.34 ꢁM), F1 (211.80 ꢁM), and Rg1
14
ACS Paragon Plus Environment

Page 15 of 30
Journal of Agricultural and Food Chemistry
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
(107.40 ꢁM) were comparable with previously reported ginseng UGTs and microbial
UGTs involved in the biosynthesis of PPTꢀtype ginsenosides.15,29,31 The turnover
numbers (kcat) of BsꢀYjiC for PPT, Rh1, F1, and Rg1 were 23.16 s−1, 14.95 s−1, 22.45
s−1, and 23.09 s−1, respectively. The kcat values of BsꢀYjiC were much higher than
those of previously reported ginseng UGTs and microbial UGTs15,29,31,37 and thus the
catalytic efficiencies (kcat/Km) of BsꢀYjiC toward PPT (0.22 ꢁM−1 s−1), Rh1 (0.30
ꢁM−1 s−1), F1 (0.11 ꢁM−1 s−1), and Rg1 (0.21 ꢁM−1 s−1) were considerably high. The
high catalytic efficiencies of BsꢀYjiC toward PPT and PPTꢀtype ginsenosides and its
broad acceptors tolerance toward a considerable number of structurally diverse types
of natural and unnatural products were consistent with the previous notion that
naturally occurring UGTs with high catalytic proficiencies are generally more flexible
toward aglycons.20,32,37
In summary, BsꢀYjiC from B. subtilis 168 was the first reported UGT that can
transfer a glucosyl moiety to the free C3ꢀOH, C6ꢀOH, and C12ꢀOH of PPT. Our
findings provided a significant insight into the important roles of microbial UGTs for
the enzymatic glycodiversification of ginsenosides. Further pharmacological
properties of these newly biosynthesized unnatural PPTꢀtype ginsenosides should be
studied. Future structural study of BsꢀYjiC should be carried out to elucidate the
structure–function relationship. Furthermoere, it will be of particular interest to
introduce BsꢀYjiC or an engineered BsꢀYjiC into PPTꢀproducing chassis cells to
synthesize these natural and unnatural ginsenosides or a specific ginsenoside via
metabolic engineering.
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 30
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
Supporting Information
The 1Hꢀ and 13CꢀNMR spectral data for products 1ꢀ5 (Tables S1ꢀS5), some
experimental results (Figures S1 and S32), and HPLCꢀQꢀTOF/ESIꢀMS and NMR
analysis (Figures S2ꢀS31).
AUTHOR INFORMATION
Corresponding Authors
Tel.: +862284861983. Eꢀmail: zhang_xl@tib.cas.cn
Tel.: +862284861960. Eꢀmail: sun_yx@tib.cas.cn
Notes
The authors declare no competing financial interest.
Funding
This work was supported by National Natural Science Foundation of China (No.
21702226) and the Science and Technology Planning Project of Tianjin (No.
11ZCZDSY08900).
REFERENCES
(1) Choi, J.; Choi, T.; Lee, M.; Kim, T. Ginseng for Health Care: A Systematic
Review of Randomized Controlled Trials in Korean Literature. Plos One. 2013, 8,
697ꢀ702.
(2) Zhao C.; Gao X.; Liu X.; Wang Y.; Yang S.; Wang F.; Ren Y. Enhancing
Biosynthesis of a Ginsenoside Precursor by SelfꢀAssembly of Two Key Enzymes in
Pichia pastoris. J. Agric. Food Chem. 2016, 64, 3380ꢀ3385.
(3) Park, C.ꢀS.; Yoo, M.ꢀH.; Noh, K.ꢀH.; Oh, D.ꢀK. Biotransformation of
16
ACS Paragon Plus Environment

Page 17 of 30
Journal of Agricultural and Food Chemistry
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial
glycosidases. Appl. Microbiol. Biotechnol. 2010, 87, 9ꢀ19.
(4) Lee, M. H.; Han, J. Y.; Kim, H. J.; Kim, Y. S.; Huh, G. H.; Choi, Y. E.
DammarenediolꢀII production confers TMV tolerance in transgenic tobacco
expressing Panax ginseng dammarenediolꢀII synthase. Plant Cell physiol. 2012, 53,
173ꢀ82.
(5) Kang A.; Xie T.; Zhu D.; Shan J.; Di L.; Zheng X. Suppressive Effect of
Ginsenoside Rg3 against LipopolysaccharideꢀInduced DepressionꢀLike Behavior and
Neuroinflammation in Mice. J. Agric. Food Chem. 2017.
(6) Seo, J. Y.; Ju, S. H.; Oh, J.; Lee, S. K.; Kim, J. S. Neuroprotective and Cognition
Enhancing Effects of Compound K Isolated from Red Ginseng. J. Agric. Food Chem.
2016, 64, 2855.
(7) Li, L.; Shin, S. Y.; Lee, S. J.; Moon, J. S.; Im, W. T.; Han, N. S. Production of
ginsenoside F2 by using Lactococcus lactis with enhanced expression of
βꢀglucosidase gene from Paenibacillus mucilaginosus. J. Agric. Food Chem. 2016,
64, 2506.
(8) Pei, J.; Xie, J.; Yin, R.; Zhao, L.; Ding, G.; Wang, Z.; Xiao, W. Enzymatic
transformation of ginsenoside Rb1 to ginsenoside 20 (S)ꢀRg3 by GH3 βꢀglucosidase
from Thermotoga thermarum DSM 5069 T. J. Mol. Catal. B: Enzym. 2015, 113,
104ꢀ109.
(9) Ossoukhova, A.; Owen, L.; Savage, K.; Meyer, M.; Ibarra, A.; Roller, M.;
Pipingas, A.; Wesnes, K.; Scholey, A. Improved working memory performance
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 30
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
following administration of a single dose of American ginseng (Panax quinquefolius
L.) to healthy middleꢀage adults. Hum. Psychopharmacol. 2015, 30, 108ꢀ22.
(10)Han, J. Y.; Kwon, Y. S.; Yang, D. C.; Jung, Y. R.; Choi, Y. E. Expression and
RNA interferenceꢀinduced silencing of the dammarenediol synthase gene in Panax
ginseng. Plant Cell physiol. 2006, 47, 1653ꢀ62.
(11)Han, J. Y.; Kim, H. J.; Kwon, Y. S.; Choi, Y. E. The Cyt P450 enzyme
CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediolꢀII
during ginsenoside biosynthesis in Panax ginseng. Plant Cell physiol. 2011, 52,
2062ꢀ73.
(12)Han, J. Y.; Hwang, H. S.; Choi, S. W.; Kim, H. J.; Choi, Y. E. Cytochrome P450
CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol
during ginsenoside biosynthesis in Panax ginseng. Plant Cell physiol. 2012, 53,
1535ꢀ1545.
(13)Jung, S. C.; Kim, W.; Park, S. C.; Jeong, J.; Park, M. K.; Lim, S.; Lee, Y.; Im, W.
T.; Lee, J. H.; Choi, G. Two ginseng UDPꢀglycosyltransferases synthesize ginsenoside
Rg3 and Rd. Plant Cell physiol. 2014, 55, 2177ꢀ88.
(14)Wang, P.; Wei, Y.; Yun, F.; Liu, Q.; Wei, W.; Yang, C.; Lei, Z.; Zhao, G.; Yue, J.;
Xing, Y. Production of bioactive ginsenosides Rh2 and Rg3 by metabolically
engineered yeasts. Metab. Eng. 2015, 29, 97ꢀ105.
(15)Wei, W.; Wang, P.; Wei, Y.; Liu, Q.; Yang, C.; Zhao, G.; Yue, J. Characterization
of Panax ginseng UDPꢀGlycosyltransferases Catalyzing Protopanaxatriol and
Biosyntheses of Bioactive Ginsenosides F1 and Rh1 in Metabolically Engineered
18
ACS Paragon Plus Environment

Page 19 of 30
Journal of Agricultural and Food Chemistry
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
Yeasts. Mol. Plant. 2015, 8, 1412ꢀ1424.
(16)Yan, X.; Fan, Y.; Wei, W.; Wang, P.; Liu, Q.; Wei, Y.; Zhang, L.; Zhao, G.; Yue, J.;
Zhou, Z. Production of bioactive ginsenoside compound K in metabolically
engineered yeast. Cell Res. 2014, 24, 770ꢀ3.
(17)Dai, Z.; Liu, Y.; Zhang, X.; Shi, M.; Wang, B.; Wang, D.; Huang, L.; Zhang, X.
Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides.
Metab. Eng. 2013, 20, 146ꢀ156.
(18)Dai, Z.; Wang, B.; Liu, Y.; Shi, M.; Wang, D.; Zhang, X.; Liu, T.; Huang, L.;
Zhang, X. Producing aglycons of ginsenosides in bakers' yeast. Sci. Rep. 2014, 4,
3698.
(19)Thibodeaux, C. J.; Melan; ccedil; on, C. E.; Liu, H. W. Natural‐Product Sugar
Biosynthesis and Enzymatic Glycodiversification. Angew. Chem., Int. Ed. 2009, 47,
9814ꢀ59.
(20)Gantt, R. W.; Goff, R. D.; Williams, G. J.; Thorson, J. S. Probing the Aglycon
Promiscuity of an Engineered Glycosyltransferase. Angew. Chem., Int. Ed. 2008, 47,
8889ꢀ92.
(21)Zhou, M.; Hou, Y.; Hamza, A.; Pain, C.; Zhan, C. G.; Bugni, T. S.; Thorson, J. S.
Probing the regiospecificity of enzymeꢀcatalyzed steroid glycosylation. Org. Lett.
2012, 14, 5424ꢀ7.
(22)Feng, J.; Zhang, P.; Cui, Y.; Li, K.; Qiao, X.; Zhang, Y. T.; Li, S. M.; Cox, R. J.;
Wu, B.; Ye, M. Regio‐and Stereospecific O‐Glycosylation of Phenolic Compounds
Catalyzed by a Fungal Glycosyltransferase from Mucor hiemalis. Adv. Synth. Catal.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 30
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
2017, 359, 995ꢀ1006.
(23)Xie, K.; Dou, X.; Chen, R.; Chen, D.; Fang, C.; Xiao, Z.; Dai, J. Two Novel
Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus
japonicus. Appl. Environ. Microbiol. 2017, 83, e03103ꢀ16.
(24)Pandey, R. P.; Gurung, R. B.; Parajuli, P.; Koirala, N.; Le, T. T.; Sohng, J. K.
Assessing acceptor substrate promiscuity of YjiCꢀmediated glycosylation toward
flavonoids. Carbohydr. Res. 2014, 393, 26ꢀ31.
(25)Pandey, R. P.; Tai, F. L.; Kim, E. H.; Yamaguchi, T.; Yong, I. P.; Kim, J. S.;
Sohng, J. K. Enzymatic Synthesis of Novel Phloretin Glucosides. Appl. Environ.
Microbiol. 2013, 79, 3516ꢀ21.
(26)Chiu, H. H.; Hsieh, Y. C.; Chen, Y. H.; Wang, H. Y.; Lu, C. Y.; Chen, C. J.; Li, Y.
K., Three important amino acids control the regioselectivity of flavonoid
glucosidation in glycosyltransferaseꢀ1 from Bacillus cereus. Appl. Microbiol.
Biotechnol. 2016, 1ꢀ14.
(27)Chiu, H. H.; Shen, M. Y.; Liu, Y. T.; Fu, Y. L.; Chiu, Y. A.; Chen, Y. H.; Huang, C.
P.; Li, Y. K. Diversity of sugar acceptor of glycosyltransferase 1 from Bacillus cereus
and its application for glucoside synthesis. Appl. Microbiol. Biotechnol. 2016, 100,
1ꢀ13.
(28)Zhuang, Y.; Yang, G.ꢀY.; Chen, X.; Liu, Q.; Zhang, X.; Deng, Z.; Feng, Y.
Biosynthesis of plantꢀderived ginsenoside Rh2 in yeast via repurposing a key
promiscuous microbial enzyme. Metab. Eng. 2017, 42, 25ꢀ32.
(29)Liang, H.; Hu, Z.; Zhang, T.; Gong, T.; Chen, J.; Zhu, P.; Li, Y.; Yang, J.
20
ACS Paragon Plus Environment

Page 21 of 30
Journal of Agricultural and Food Chemistry
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
Production of a bioactive unnatural ginsenoside by metabolically engineered yeasts
based on a new UDPꢀglycosyltransferase from Bacillus subtilis. Metab. Eng. 2017.
(30)Luo, S.; Dang, L.; Zhang, K.; Liang, L.; Li, G. Cloning and heterologous
expression of UDP ‐ glycosyltransferase genes from Bacillus subtilis and its
application in the glycosylation of ginsenoside Rh1. Lett. Appl. Microbiol. 2015, 60,
72ꢀ78.
(31)Wang, D.ꢀD.; Jin, Y.; Wang, C.; Kim, Y.ꢀJ.; Perez, Z. E. J.; Baek, N. I.;
Mathiyalagan, R.; Markus, J.; Yang, D.ꢀC. Rare ginsenoside Ia synthesized from F1
by cloning and overexpression of the UDPꢀglycosyltransferase gene from Bacillus
subtilis: synthesis, characterization, and in vitro melanogenesis inhibition activity in
BL6B16 cells. J Ginseng Res. 2016.
(32)Dai, L.; Li, J.; Yao, P.; Zhu, Y.; Men, Y.; Zeng, Y.; Yang, J.; Sun, Y. Exploiting the
aglycon promiscuity of glycosyltransferase BsꢀYjiC from Bacillus subtilis and its
application in synthesis of glycosides. J. Biotechnol. 2017, 248, 69ꢀ76.
(33)Dai, L.; Liu, C.; Zhu, Y.; Zhang, J.; Men, Y.; Zeng, Y.; Sun, Y. Functional
Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase
Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii. Plant Cell physiol.
2015, 56, 1172.
(34)Gantt, R. W.; Peltierpain, P.; Singh, S.; Zhou, M.; Thorson, J. S. Broadening the
scope of glycosyltransferaseꢀcatalyzed sugar nucleotide synthesis. Proc. Natl. Acad.
Sci. USA. 2013, 110, 7648ꢀ53.
(35)Pandey, R. P.; Parajuli, P.; Koirala, N.; Park, J. W.; Sohng, J. K. Probing
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 30
454
455
456
457
458
459
460
3ꢀhydroxyflavone for in vitro glycorandomization of flavonols by YjiC. Appl.
Environ. Microbiol. 2013, 79, 6833ꢀ8.
(36)Qin, W.; Liu, Y.; Ren, P.; Zhang, J.; Li, H.; Tian, L.; Li, W. Uncovering a
Glycosyltransferase Provides Insights into the Glycosylation Step during Macrolactin
and Bacillaene Biosynthesis. Chembiochem 2014, 15, 2747–2753.
(37)Oberthür, M.; Leimkuhler, C.; Kruger, R. G.; Lu, W.; Walsh, C. T.; Kahne, D. A
systematic investigation of the synthetic utility of glycopeptide glycosyltransferases.
J. Am. Chem. Soc. 2005, 127, 10747ꢀ10752.
461
462
22
ACS Paragon Plus Environment

Page 23 of 30
Journal of Agricultural and Food Chemistry
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
Figure 1. Glycosylation patterns of BsꢀYjiC 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.
Figure 2. HPLCꢀQꢀTOF/ESIꢀMS analysis of the glycosylated products of PPT
catalyzed by BsꢀYjiC. (A) HPLC chromatograms of PPT and PPTꢀtype ginsenoside
standards, control reactant mixtures, and BsꢀYjiCꢀcatalyzed reactants. (B) MS spectra
for products 1 (a), 2 (b), and 3 (c), 4 (d), and 5 (e).
Figure 3. HPLC analysis of the glycosylated products of PPT with diverse
UDPꢀsugars as sugar donors.
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.
Figure 5. HPLC analysis of the glycosylated products of Rh1, F1, and Rg1 catalyzed
by BsꢀYjiC.
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 30
484
Table 1 Kinetic parameters of BsꢀYjiC towards PPT, ginsenosides Rh1, F1, and Rg1
Substrate
PPT
Km (ꢁM)
kcat (sꢀ1)
kcat/Km (sꢀ1 ꢁMꢀ1)
103.60±18.28
50.34±11.25
211.80±23.81
107.40±11.48
23.16±1.22
14.95±0.76
22.45±0.97
23.09±0.74
0.22
0.30
0.11
0.21
Rh1
F1
Rg1
485
24
ACS Paragon Plus Environment

Page 25 of 30
Journal of Agricultural and Food Chemistry
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)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 30
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-YjiC-
catalyzed reactants. (B) MS spectra for products 1 (a), 2 (b), and 3 (c), 4 (d), and 5 (e).
116x88mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 27 of 30
Journal of Agricultural and Food Chemistry
Figure 3. HPLC analysis of the glycosylated products of PPT with diverse UDP-sugars as sugar donors.
60x44mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 30
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)
ACS Paragon Plus Environment

Page 29 of 30
Journal of Agricultural and Food Chemistry
Figure 5. HPLC analysis of the glycosylated products of Rh1, F1 and Rg1 catalyzed by Bs-YjiC.
78x74mm (300 x 300 DPI)
ACS Paragon Plus Environment