Enzymatic Monoglucosylation of Rubusoside and the Structure-Sweetness/Taste Relationship of Monoglucosyl Derivatives

Article abstract of DOI:10.1021/acs.jafc.0c03236

Monoglucosylation of rubusoside not only could increase its structural diversity but may also improve its taste. To biosynthesize the monoglucosyl rubusosides, a series of glycosyltransferases and glycosynthases were screened to identify the enzymes capable of specifically glycosylating the hydroxyl groups of the 13-O-β-d-glucosyl and 19-COO-β-d-glucosyl moieties. After structural characterization, the effect of structure on sweetness and taste was established based on these rubusoside-derived analogues, including two first characterized compounds. β-Monoglucosylation of two 2-hydroxyl groups, as well as α-monoglucosylations of the 4- and 6-hydroxyl groups of the 13-glucosyl moiety, could significantly increase the relative sweetness of rubusoside to 140 while maintaining or improving the taste quality. In contrast, monoglucosylations of other hydroxyl groups in our study usually decreased the taste quality of the rubusoside. Additionally, the possibility of a negative influence of these monoglucosylated derivatives on the function of islets was preliminarily excluded, which should facilitate the development of rubusoside-derived sweeteners.

Full text of DOI:10.1021/acs.jafc.0c03236

Subscriber access provided by University of Birmingham
Functional Structure/Activity Relationships
Enzymatic Monoglucosylation of Rubusoside and the Structure/
sweetness-taste Relationship of Monoglucosyl Derivatives
Ling Zhao, Yao Wang, Zhenlin Li, Xiaonan Wang, Yijun Chen, and Xuri Wu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c03236 • Publication Date (Web): 20 Jul 2020
Downloaded from pubs.acs.org on July 23, 2020
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 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 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.
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 31
Journal of Agricultural and Food Chemistry
1
2
3
4
5
Enzymatic Monoglucosylation of Rubusoside and the
6
7
8
9
Structure/sweetness-taste Relationship of Monoglucosyl Derivatives
†
, #
†, #
‡
§
*,†
Ling Zhao, Yao Wang, Zhenlin Li, Xiaonan Wang, Yijun Chen, and
*
,†,§
Xuri Wu
†
1
1
0
1
Laboratory of Chemical Biology, College of Life Sciences and Technology, China
Pharmaceutical University, 639 Longmian Road, Nanjing, Jiangsu Province 211198,
1
1
2
3
PR China
‡
Department of Pharmaceutical Analysis and Metabolomics, Jiangsu Province
1
1
4
5
Academy of Traditional Chinese Medicine, 100 Shizi St. Hongshan Rd. Nanjing,
Jiangsu Province 210028, PR China.
§
1
1
6
7
Department of Biochemistry, College of Life Sciences and Technology, China
Pharmaceutical University, 639 Longmian Road, Nanjing, Jiangsu Province 211198,
PR China.
1
1
8
9
2
0
2
2
1
2
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 31
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
ABSTRACT
Monoglucosylation of rubusoside not only could increase its structural diversity,
but may also improve its taste. To biosynthesize the monoglucosyl rubusosides, a
series of glycosyltransferases and glycosynthases were screened to identify the
enzymes capable of specifically glycosylating the hydroxyl groups of the 13-O-β- and
19-COO-β-D-glucosyl moieties. After structural characterization, the effect of
structure on sweetness and taste was established based on these rubusoside-derived
analogues, including two first characterized compounds. β-Monoglucosylation of two
2-hydroxyl groups, as well as α-monoglucosylations of the 4- and 6-hydroxyl groups
of the 13-glucosyl moiety, could significantly increase the relative sweetness of
rubusoside to 140, while maintaining or improving taste quality. In contrast,
monoglucosylations of other hydroxyl groups in our study usually decreased the taste
quality of the rubusoside. Additionally, the possibility of a negative influence of these
monoglucosylated derivatives on the function of islets was preliminarily excluded,
which should facilitate the development of rubusoside-derived sweeteners.
KEYWORDS: rubusoside, enzymatic monoglucosylation, monoglucosyl rubusoside,
Structure/sweetness-taste relationship, low-calorie sweetener
ACS Paragon Plus Environment

Page 3 of 31
Journal of Agricultural and Food Chemistry
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Rubusoside (13-O-β-glucosyl-19-O-β-glucosyl steviol) is a highly potent and
slightly bitter low-calorie sweetener found in the leaves of Rubus suavissimus S. Lee
1
(Rosaceae). Rubusoside is 114 times sweeter than 0.025% sucrose, has a slight
aftertaste, and can potentially be used as a natural sweetener like stevioside in foods,
2
beverages, and oral medicines or as an intermediate in the preparation of other sweet
3
additives. Additionally, rubusoside also shows a variety of pharmacological
functions,
including
antihyperglycemic,
antitumor,
anticariogenic
and
2
,4-6
anti-inflammatory effects.
Interestingly, rubusoside exhibits an advantageous
solubilizing property in that it can enhance the aqueous solubility of a water-insoluble
7
compound. Consequently, rubusoside has attracted extensive attention in recent
years.
Glycosylation is a valuable way to improve the physic-chemical properties of
8
-10
natural products, such as water solubility, physical stability, and taste quality.
In the
case of rubusoside, to eliminate the bitter taste and probe the structure–sweetness
relationship, the addition of a single sugar unit to a specific position has been
3
,11
suggested as a good solution, depending upon the position. Traditionally, chemical
methods have been used to attempt the monoglycosylation of rubusoside by
introducing glycosyl moieties at different hydroxyl groups of the 13-O-β- and
19-COO-β-glucosyl moieties. However, due to the similar reactivity of all the
different hydroxyl groups in rubusoside, chemical or even chemo-enzymatic
monoglycosylation of rubusoside has been unsuccessful and remains a great challenge.
In contrast, biocatalytic glycosylation has become a major player to selectively
prepare monoglycosyl rubusosides given the excellent regioselectivity and broad
substrate specificity of the enzymes.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 31
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Up to now, enzymatic monoglycosylation of rubusoside, including glucosylation,
1
2-14
15
16
fructosylation, and galactosylation, has been attempted occasionally to
improve rubusoside’s sweetness and taste quality. However, a systematic relationship
of structure with taste and sweetness of monoglycosylated rubusosides has yet to be
thoroughly established to guide the development of new rubusoside-derived
sweeteners. The lack of such a relationship is due in large part to the extreme
difficulty of selective monoglucosylation of rubusoside, which is caused by its large
structure and its 8 hydroxyl groups. As a result, to the best of our knowledge there
have been no reports on the generation of a full range of monoglucosylated analogues.
There have been some biocatalytic examples of preparing a limited number of
monoglucosyl rubusosides. For example, using cyclodextrin glucanotransferases
(CGTase) from Bacillus, two α-1,4-monoglucosyl analogues were biosynthesized by
respectively glucosylating two 4-hydroxyl groups of both glucosyl moieties in
rubusoside, but a series of unexpected byproducts, including di-, tri- and
1
2,13
tert-glucosylated derivates, were simultaneously generated.
In another example,
UDP-glucose dependent glycosyltransferase UGT91D2 was shown to indeterminately
add glucose to C13- or C19-position via the formation of a β-1,2-linkage, thus
3
producing a mixture of 1,2-stevioside and rebaudioside E. In addition to UGT and
CGTase, dextransucrase, with an α-D-glucosyl moiety of sucrose as donor, is another
type of alternative enzyme that can indeterminately carry out the
1
4
mono-transglucosylation of rubusoside.
Perhaps because of the difficulty in regioselectively conducting mono-
glucosylation, the influence of the hydroxyl groups in rubusoside on its taste and
sweetness intensity has not yet been precisely determined, thereby hindering the
development of rubusoside-derived low-calorie sweeteners. Because of the superior
ACS Paragon Plus Environment

Page 5 of 31
Journal of Agricultural and Food Chemistry
9
9
9
9
6
7
8
9
operability and feasibility over other glycosylation methods, regioselective enzymatic
glucosylation of hydroxyl groups in rubusoside could be expected to generate
structurally diverse monoglucosyl derivatives. In this study, a variety of
glycosyltransferases and glycosynthases were screened and used to conduct
mono-glucodiversification of rubusoside. With the production and the structural
characterization of the monoglucosyl rubusosides, followed by evaluation of their
sweetness and taste quality, for the first time the relationship of
structure-sweetness/taste was established preliminarily. Additional experiments
showed that the contingent risk of these derivatives on insulin secretion of islets was
precluded, thus increasing their possibility of becoming new, high-intensity
sweeteners.
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
MATERIALAND METHODS
Materials and Reagents. E. coli BL21 (DE3) and DH5α were purchased from
Vazyme Biotech Co., Ltd. (Nanjing, China). The genes of OleD, YjiC, UGT76G1,
Bgl3 E383A, Abg-2F6, GtfD, GtfR and Maltogenic amylase were chemically
synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (Suzhou, China). HPLC
grade trifluoroacetic acid (TFA) and formic acid were obtained from Aladdin
Bio-Chem Technology Co., Ltd. (Shanghai, China). Acetonitrile (HPLC grade) was
purchased from TEDIA (USA). UDP-glucose (>95%) was obtained from Suzhou
Ruixin Biotechnology Co., Ltd. (Suzhou, China). Rubusoside (>95%) was purchased
from Shanghai Tongtian Biotechnology Co., Ltd. (Shanghai, China). Monoglucosyl
rubusosides 5 and 6 were obtained from Chengdu DeSiTe Biological Technology Co.,
Ltd. (Chengdu, China). 1-Fluorinated glucose (F-1-glucose) was synthesized by the
Medical and Chemical Institute of China Pharmaceutical University (Nanjing, China)
1
7
according to the literature. All other chemicals with analytical purity were
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 31
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
commercially available, unless otherwise stated.
Soluble Expression of Enzymes in E. coli. The genes of OleD, YjiC, UGT76G1,
Bgl3 E383A, Abg-2F6, GtfD, GtfR and Maltogenic amylase were synthesized and
inserted into the corresponding pET vectors, including pET-22b (+), pET-33b (+) and
pET-302, with specific restriction endonucleases (Table S1, Supporting Information).
Then, the constructed plasmids were transformed into E. coli BL21 for expression. As
an example, for the expression of glycosyltransferase OleD, pET22b-oled containing
an OleD gene was transformed into E. coli BL21 (DE3) by the CaCl transformation
2
method. After screening and sequencing, the resulting E. coli cell harboring
pET22b-oled was inoculated in 50 mL LB medium supplemented with 100 μg/mL
ampicillin and grown overnight at 37 °C. Then, the seed cultures were inoculated into
200 mL fresh LB medium containing 100 μg/mL ampicillin with an OD600 of 0.30 and
cultivated at 37 °C to an OD600 of 0.9 ± 0.1. The optimal conditions for the expression
of OleD were identified in terms of the concentration of IPTG (0.1–1 mM),
temperature (15–30 °C), and induction time (0–18 h). For all groups, the soluble
expression of OleD was determined by SDS-PAGE analysis. With the same procedure
described above, the optimal expression conditions of other enzymes were obtained
and are listed in Table S1 (Supporting Information).
HPLC and LC/MS Analytical Methods. HPLC analyses of rubusoside and its
glucosylated derivatives were conducted on a YMC-Pack ODS column (5 µm, 250 ×
4.6 mm) with various gradients of mobile phase A (0.1% TFA in water) and mobile
phase B (0.1% TFA in acetonitrile) at 35 °C with UV detection at 210 nm. 10 μL of
sample solution was analyzed by HPLC with a flow rate of 0.8 mL/min and a mobile
phase mix of A and B as follows: 10%-95% B (0–30 min), 95% B (30–38 min).
For the LC/MS analysis, the mobile phase used for HPLC separation consisted of
ACS Paragon Plus Environment

Page 7 of 31
Journal of Agricultural and Food Chemistry
1
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
0.1% HCOOH in water (A) and 0.1% HCOOH in acetonitrile (B). The HPLC setup
was the same as that for HPLC analysis of glucosylated rubusosides, and the LC/MS
parameters were set as shown in Table S2 (Supporting Information).
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Screening of Enzymes for the Biosynthesis of Monoglucosyl Rubusosides.
Taking as an example the OleD-catalyzed glucosylation of rubusoside, 2 mL of the
reaction mixture consisted of 2 mg/mL of rubusoside (3.12 mM), 15.6 mM
UDP-glucose, 1 mL of crude OleD (5.2 mg/mL), and 50 mM Tris-HCl (pH 8.0),
1
8
following reports in the literature (Supporting Information, Table S3). For the
reaction without UDP-glucose, a sugar donor for OleD served as the control. All
reactions were initiated and conducted at 30 °C while stirring at 200 rpm for 12 h, and
then quenched by adding 2 mL of methanol. After vortexing and centrifugation, the
resulting supernatants were subjected to HPLC and LC/MS analysis. With the same
operational procedure described above, the glucosylation of rubusoside catalyzed by
3
,19-25
other enzymes was investigated using previously reported reaction conditions.
Optimization of Reaction Conditions for the Biosynthesis of Monoglucosyl
Rubusosides. With the production of monoglucosylated rubusoside verified, the
reaction conditions were examined to improve catalytic efficiency. 50 mM sodium
acetate buffer, 50 mM potassium phosphate buffer, 100 mM Tris-HCl buffer, 50 mM
glycine-NaOH buffer, and 50 mM KCl-NaOH buffer were employed to find the
optimal reaction pH values. Then, the molar ratio of rubusoside to sugar donor, in the
range of 1:1–1:10, was optimized. For the optimization of reaction time, all enzymatic
reactions were monitored over 24 h. Samples were taken at 2 h intervals and subjected
to HPLC analysis. The reaction temperature for each enzymatic system was set
according to processes described in the literature (Supporting Information, Table
1
8-26
S4).
All reactions were repeated three times.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 31
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
Preparation and Identification of Monoglucosyl Rubusosides. The scalable
preparation of each monoglucosylated rubusoside was performed at 0.5 L using the
optimized reaction conditions. After the reaction time, the reaction mixture was boiled
for 5 min and centrifuged at 12,000 g for 20 min. The resulting supernatant was
concentrated and subjected to semi-preparative HPLC for the purification of the
monoglucosyl derivative. The semi-preparative HPLC purification was performed on
a Waters preparative ODS column (5 µm, 250 × 30 mm) with an injection volume of
0.5 mL and a flow rate of 15 mL/min at room temperature. Acetonitrile containing 0.1%
TFA and water containing 0.1% TFA were respectively used as mobile phase A and B.
The chromatographic procedure was as follows: 20–33% B (0–5 min), 33–40% B (5–
25 min), 40–90% B (25–30 min). Fractions containing monoglucosyl product with a
purity greater than 95% were combined and a rotary vacuum evaporator was used to
remove the acetonitrile. Subsequently, the resulting aqueous phase was lyophilized to
give monoglucosyl rubusoside as a white powder. Lastly, the structural
characterizations of all the purified derivatives were performed on a Bruker Advance
AMX500 NMR spectrometer (Rheinstetten, Germany).
Sensory Evaluation of Monoglucosyl Rubusosides. Sensory evaluation of
highly purified monoglucosyl compounds (> 95%) was performed using sucrose as a
control along with rubusoside. Volunteers, 15 women and 20 men, evaluated both
taste quality and sweetness. Taste quality was evaluated with all compounds dissolved
in water in 0.5% solutions. The volunteers were randomly chosen to taste each
solution and record the taste quality as “sweet, slightly sweet, tasteless, slightly bitter,
or bitter”. To evaluate sweetness, rubusoside and its derivatives were each dissolved
in 0.1, 0.05, 0.025, 0.017, 0.0125, 0.01, and 0.0083% (w/v) aqueous solutions to
1
3
assess their sweetness relative to a 0.025% (w/v) sucrose solution.
ACS Paragon Plus Environment

Page 9 of 31
Journal of Agricultural and Food Chemistry
1
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
Effects of Monoglucosyl Rubusosides on Glucose-stimulated Insulin Secretion.
2
7
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
Following a reported procedure, the islets of Langerhans were isolated from male
BALB/c mice using collagenase P (Roche) digestion. Isolated islets were cultured in
RPMI 1640 medium supplemented with 20% FBS, 100 U/mL penicillin, and 100
μg/mL streptomycin overnight at 37 °C. Then, 10 islets per well were placed in
-6
24-well plates containing KRBH buffer. The islets were treated with 10 M of
monoglucosyl rubusosides for 12 h and transferred into KRBH buffer containing 0,
2.8, and 22.2 mM glucose, in turn for 1.5 h each. The insulin concentration in all
groups of supernatants was determined by the ELISA method (Millipore, Billerica,
USA). All experiments involving handling of animals were performed in accordance
with the guidelines and regulations set and approved by the Animal Care Committee
of China Pharmaceutical University.
Statistical Analysis. All experiments were performed with more than three
independent duplicates. Data was expressed as the mean ± standard deviation.
Statistical significance between two groups was determined using student t-test
(Tukey), and multiple comparisons between groups were performed by one-way
2
2
12
analysis of variance (ANOVA). Significance was set at p < 0.05.
13 RESULTS AND DISCUSSION
2
2
2
2
2
2
14
15
16
17
18
19
Biosynthesis of β-1,2-Monoglucosyl Rubusosides. Although numerous enzymes,
such as glycosyltransferases and glycosynthases, have been explored to glucosylate
8
,9
various natural products, such as flavonoids, terpenoids, and alkaloids, the vast
majority of 1,2-glucosidic linkages were formed with β-stereo-selectivity. On account
of this, we focused on the search for enzymes with broad substrate specificity and
excellent β-1,2-regioselectivty in glucosylation to prepare two β-1,2-monoglucosyl
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 31
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
rubusosides. Consequently, two well-studied microbial UGTs, OleD from
1
8
19
Streptomyces antibioticus and YjiC from Bacillus licheniformis , were selected as
candidates to form a β-1,2-monotransglucosylated rubusoside with UDP-glucose as
the sugar donor.
As shown in Table S1 (Supporting Information), the genes of YjiC and OleD were
inserted into plasmid pET302 and pET22b (+), respectively, for the soluble expression
in E. coli cells. The optimized expression conditions enabled both enzymes to be
expressed in soluble form (Figure S1, Supporting Information). Then, the cell extract
of E. coli cells expressing YjiC or OleD was utilized as the biocatalyst using UDPG
as the donor to glycosylate the 2-hydroxyl group of the 13-O-β- or
19-COO-β-glucosyl moiety in rubusoside. The formation of glucosylated rubusosides
was monitored and quickly verified by LC/MS. For OleD, a single product with a
-
better hydrophilicity than rubusoside was observed with a m/z = 803.3729 [M-H] ,
indicating that monoglucosylation of rubusoside occurred (Figure 1). After systematic
optimization, the yield of this monoglucosylated product reached 96.4% at 15 mg/mL
of rubusoside, without any undesired by-products at any of the tested pH values.
NMR analysis displayed that the monoglucosylated analogue produced by OleD was
identical to rebaudioside KA (monoglucosyl rubusoside 1), a trace ingredient from
2
8
Stevia rebaudiana, with a β-1,2-linkage at the 19-position of rubusoside (Figure 2
and Table S5, Supporting Information). On account of the difficulty in the scalable
preparation from Stevia rebaudiana, the sensory quality of rebaudioside KA had not
been previously evaluated. However, OleD-catalyzed monoglucosylation provided an
enzymatic approach to prepare the necessary amount rebaudioside KA, enabling the
assessment of sweetness in this study. Additionally, a feasible and economical cell
factory was constructed by redesigning the glucose metabolic flux in E. coli to
ACS Paragon Plus Environment

Page 11 of 31
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
45
produce rebaudioside KA at a preparative scale, without using exogenous
2
9
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
UDP-glucose as a sugar donor.
In contrast to OleD, for all of the investigated pH values there were two
monoglucosylated rubusosides with the same molecular weight produced in the
YjiC-catalyzed reaction (Figure 1). Both derivatives were purified to elucidate their
structures after optimizing the reaction conditions. As shown in Figure 1, 2 and Table
S6 (Supporting Information), the major monoglucosylated rubusoside at 13.2 min
+
with a m/z = 827.3691 [M+Na] was stevioside (monoglucosyl rubusoside 2), a
3
well-studied sweet compound from Stevia rebaudiana, with a glucose directly
bound to the 2-hydroxyl group of the 13-O-β-glucosyl moiety by a β-1,2-glucosidic
3
linkage. Surprisingly, apart from glycosyltransferase UGT91D2, YjiC-catalyzed
synthesis of stevioside was not previously reported, which would have provided an
environmental-friendly alternative to obtain this commercial sweetener.
Biosynthesis
of
β-1,3-Monoglucosyl
Rubusosides.
To
date,
1,3-monoglucosylated rubusosides have rarely been discovered and reported in the
literatures, regardless of α- or β-stereochemistry. Intriguingly, in addition to stevioside
+
(2), YjiC also provided compound 3 (m/z = 827.3689 [M+Na] ), which was
structurally identified as an uncharacterized monoglucosyl rubusoside with a
β-1,3-glucosidic linkage in the 19-COO-β-glucosyl moiety (Figure 2 and Table S7,
Supporting Information) at an optimized conversion of 12.8%. Previously, YjiC was
3
0
usually presumed to form mainly a β-1,2-glucosidic linkage. The biosynthesis of
new compound 3 not only provided an analogue to investigate the structure-taste
relationship of rubusoside, but also revealed the latent β-1,3-glucosylation function
and substrate specificity of YjiC in glycosylating natural products. A structural
comparison of monoglucosyl derivatives 2 and 3 suggested that rubusoside primarily
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 31
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
preferred to enter the active site pocket of YjiC with the 13-O-β-glucosyl moiety in
the front, which might result in more 2 than 3 in the reaction. However, the reason
why YjiC preferentially transfers a glucose to the 13- and 19-glucosyl moieties of
rubusoside requires further investigation in the future.
Regarding the 1,3-monoglycosylation of the 13-position in rubusoside,
2
0
UDPG-glycosyltransferase
UGT76G1
from
Stevia
rebaudiana
and
1-fluorinated-glucose (F-1-glucose) dependent glycosynthase Bgl3 E383A (a mutant
2
1
of β-glucosidase Streptomyces sp. ) were selected because of their β-1,
3-regioselective glucosylation activity and their broad substrate specificity. Unlike
with glycosyltransferase, the active sugar donor for Bgl3 E383A-catalyzed
1
7
glucosylation was F-1-glucose, which could be chemically obtained from glucose.
After soluble expressions in E. coli cells (Figure S1, Supporting Information),
UGT76G1 as well as Bgl3 E383A in cell extracts were each used to conduct the
glucosylation of rubusoside under optimized reaction conditions. As shown in the
LC/MS profile (Figure 1), both biocatalysts exclusively generated a single
monoglucosylated derivative with a yield > 92%, without any excessive glucosylation
at any of the tested reaction parameters. Subsequently, two monoglucosyl analogues
were purified and structurally determined to be the same, a known sweet compound,
rebaudioside G (monoglucosyl rubusoside 4), with a glucose unit bound to
13-O-β-glucosyl moiety by a β-1,3-linkage (Figure 2 and Table S8, Supporting
Information). Interestingly, enzymatic preparation of rebaudioside G from rubusoside
by UGT76G1 was described previously and showed the emergence of a
3
di-glycosylated derivative, whereas excessive glycosylation did not take place in this
study. Therefore, it is worthwhile to determine the possible factors for
UGT76G1-catalyzed glycosylation of natural products in the future. Additionally,
ACS Paragon Plus Environment

Page 13 of 31
Journal of Agricultural and Food Chemistry
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
95
Bgl3 E383A-catalyzed β-1,3-monoglucosylation of rubusoside with excellent yield
was disclosed for the first time, which not only provided a new and efficient approach
to synthesize rebaudioside G, but also showed that glycosynthase was able to carry
out selective glucosylation of tetracylic diterpenoids harboring a large backbone.
Biosynthesis of α-1,6-Monoglucosyl Rubusosides. Because there were only a
few examples of executing enzymatic β-1,4- and β-1,6-glucosylation of natural
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
3
1
terpenoids, we expected that -stereoselective biocatalysts, many of them
non-Leloir glycosyltransferases, would yield 1,4- and 1,6- mono-glycosyl rubusosides
in the work presented here. In order to site-specifically conduct monoglucosylation of
both 4-hydroxyl groups in rubusoside, our focus was placed on screening reported
CGTases that possessed obvious α-1,4-transglucosylation activity over a broad
substrate spectrum. We found two reported α-1,4-monoglucosylated derivatives (5
1
3
and 6) that were regioselectively prepared by CGTase from Bacillus circulans, with
the association of structure and sensory quality illustrated in detail, which were
included in our construction of the structure–sweetness relationship of rubusoside
derivatives.
1,6-Monoglucosylation of natural tetracyclic diterpenoids by chemical or
3
1
enzymatic means was previously described only minimally, which made the
bio-preparation of α-1,6-monoglycosylated rubusosides more complicated. Taking
regioselectivity as well as substrate specificity into account, maltogenic amylase from
25
Bacillus stearothermophilus, with the capability of forming a 1,6-glucosidic linkage,
was selected to attempt α-1,6-monoglucosylation of rubusoside. However, based on
the HPLC analysis of the reaction mixtures, maltogenic amylase failed to give any
rubusoside-derived products for any of the investigated reaction conditions.
Alternatively, GtfR from Streptococcus oralis,
a
versatile non-Leloir
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 31
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
glycosyltransferase with sucrose as a glucose source, exhibited the possibility to
glucosylate 6-hydroxyl groups of rubusoside on account of its broad α-1,2-, α-1,3-
2
6
and α-1,4-transglucosylation activities towards several flavonoids. As shown in
Figure S1 (Supporting Information), an obviously soluble expression of recombinant
GtfR in E. coli cell could provide sufficient enzyme proteins to cause the
glucosylation of rubusoside. Surprisingly, the combination of LC/MS and NMR
analyses showed that GtfR (Figures 1 and 3), with a slight regio-preference for the
19-β-glucosyl moiety, simultaneously biosynthesized two α-1,6-monoglucosyl
derivatives (7 and 8) without the appearance of other multi- glycosylated analogues
(Table S9 and S10, Supporting Information) at any of the investigated parameters.
With the optimized reaction parameters, the yields of 7 and 8 reached 14.0 and 23.0%,
respectively, at 35 °C and pH 6.0 for 6 h.
Compound 7, with an α-1,6-glucosidic bond at the 13-β-glucosyl moiety of
rubusoside, was biosynthesized using dextransucrase from Leuconostoc citreum in
1
4
2016, whereas α-1,6-monoglucosylation of the 19-COO-β-glucosyl moiety was first
characterized with the creation of compound 8 in this work. The production of 7 and 8
not only increased the structural diversity of the monoglucosylated rubusosides for the
illustration of the structure–sweetness relationship, but also provided the first
demonstration of the utility of GtfR for the biosynthesis of the α-1,6-glucosidic bond.
The α-1,6-transglucosylation function of GtfR reveals it to be a versatile biocatalyst in
synthesizing all types of α-glucosidic linkages, which should contribute to the
glycodiversification of natural products.
Relationship of Structure with Sweetness and Taste of Monoglucosyl
Rubusosides. Sensory evaluation of biosynthesized monoglucosyl rubusosides was
1
3,14
performed according to previous methods,
including the mouth-feel properties of
ACS Paragon Plus Environment

Page 15 of 31
Journal of Agricultural and Food Chemistry
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
45
both α-1,4- monoglucosylated analogues (5 and 6). As shown in Table 1, no
significant difference in taste profile was observed for each derivative between
selected volunteers. However, monoglucosidic modification of all free hydroxyl
groups in both glucosyl residues of rubusoside, irrespective of their stereochemistry
and sites, had a significant influence on sweetness and taste quality. For example, two
β-1,2-monoglucosidic derivatives, rebaudioside KA (1) and stevioside (2), exhibited
at least 1.2 higher sweetness intensity than rubusoside, while only
β-1,2-mono-transglucosylation at the 19-COO-glucosyl moiety could improve the
taste quality of rubusoside. However, previous work has shown that transferring an
additional glucose to the 2-hydroxyl group of the 13-glucosyl moiety in rebaudioside
KA (1) by forming a β-1,2-glucosidic bond perceptibly degraded its taste quality but
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
2
8
increased its sweetness. In contrast, α-1,4- and α-1,6- addition of a glucosyl unit to
the 13-glucosyl moiety of rubusoside not only brought about a remarkable increase in
the sweetness intensity by more than 20%, but also upgraded the taste quality. The
resulting two monoglucosyl derivatives (compounds 5 and 7) were almost identical to
rebaudioside KA in sensory properties, as shown in Table 1. Therefore, in terms of
improved sweetness and taste, rebaudioside KA (1), 5, and 7, resembling siamenoside
3
2
I and mogroside V from Siraitia grosvenorii, could potentially be applied in the
replacement of sucrose and synthetic sweeteners, as they overcome some sensory
drawbacks of commercial natural sweeteners such as stevioside and rubusoside.
In another example, a significant decrease in the relative sweetness was observed
for compounds 3 and 4, accompanied with the appearance of bitterness (Table 1). It
appeared that β-selective monoglucosylation of both free 3-hydroxyl groups of 13-
and 19-glucosyl moieties of rubusoside might decrease the sweetness and taste quality,
whereas α-stereoselective addition of a glucosyl moiety to the 3-hydroxyl group of the
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 31
1
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
glucosyl moiety at the 13-position improved the sweetness and taste quality. Similar
to 3 and 4, compound 8, with α-1,6-monotransglucosylation at the 19-glucosyl moiety,
displayed a mild loss in sweetness and flavor (Table 1), indicating that a free
6-hydroxyl group of the 19-glucosyl moiety may be necessary for maintaining the
sweetness quality of rubusoside. From compound 6, it was also found that
α-1,4-monoglucosylation of the 19-glucosyl moiety made no difference in the
sweetness and taste quality of rubusoside, but β-selective addition of a glucose unit to
1
3
the 4-hydroxyl group at the 19-position evidently worsened its sensory quality.
In brief, β-monoglucosylation of the 2-hydroxyl group of the 19-glucosyl moiety,
as well as α-monoglucosylation of the 4- and 6-hydroxyl groups at the 13-position,
could efficiently improve the sweetness intensity and taste quality of rubusoside. In
contrast,
β-monoglucosylation
of
the
two 3-hydroxyl
groups and
α-monoglucosylation of the 6-hydroxyl group of the 19-glucosyl moiety could
thoroughly degrade the sweetness of rubusoside, resulting in bitterness. Without
regard to the stereochemistry, monoglucosylation of the other two hydroxyl groups (2
and 6) had no significant impact on the improvement of sensory quality. Thus, the
structure–sweetness relationship of the monoglucosyl derivatives suggest that
selectively glycosylated elongation of the 13- and 19-glucosyl moieties would
facilitate the development of rubusoside-derived low-calorie sweeteners with
improved taste profiles.
Influence of Monoglucosyl Rubusosides on Glucose-stimulated Insulin
Secretion of Islets. It is widely assumed that the possibility of influencing islet
function is a crucial safety index to determine if a sweet substance can be developed
3
3
as a low-calorie sweetener. Hence, it is important to check if pretreatment of
monoglucosyl rubusosides affects the glucose-stimulated insulin secretion of islets,
ACS Paragon Plus Environment

Page 17 of 31
Journal of Agricultural and Food Chemistry
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
95
especially for 1, 5, and 7, which have improved sweetness and taste compared to
rubusoside. As shown in Figure 4, no significant difference was found in the insulin
secretion between any of the monoglucosyl derivatives and either the control or
rubusoside (p > 0.05) in the 2.8 mM and 22.2 mM glucose groups. Therefore, the
treatments using compounds 1–8 had no obvious effect on the insulin secretion in
response to glucose stimulation, preliminarily indicating that monoglucosylation does
not change islet function nor decrease the safety of rubusoside to the islets. The lack
of an effect of the monoglucosyl rubusosides on insulin secretion should facilitate the
development of 1, 5, and 7 as natural sweeteners as alternatives to sugar in food and
beverages. Importantly, the biosynthetic approaches described in this study also
provide possible solutions to ensure sufficient supplies of monoglucosyl rubusosides,
overcoming the inferiority of chemical catalysts in the site-specific
mono-transglucosylation of molecules with similar active hydroxyl groups.
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
Therefore, in consideration of the elimination of the bitter taste of rubusoside,
site-specific monoglucosylation of both glucosyl moieties was explored with several
screened glycosyltransferases and glycosynthases to give a series of β-1,2-, β-1,3- and
α-1,6- monoglucosylated derivatives, including two compounds characterized for the
first time whereas chemical catalysts possessed insurmountable shortcomings in the
synthesis of these derivatives due to poor regioselectivity. After sensory evaluation,
the relationship of structure with sweetness and taste of these analogues was
established and combined with two reported α-1,4-monoglucosyl derivatives. The
combined data should be helpful for the creation of rubusoside-derived low-calorie
sweeteners with improved taste. Plus, three of these monoglucosyl rubusosides (1, 5,
and 7), because of the increased sweetness, improved taste quality, and safety with
regards to insulin secretion, are expected to become new sweeteners as substitutes for
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 31
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
sugar and artificial sweeteners. Moreover, further studies regarding monoglucosyl
rubusosides are currently being performed to determine the relationship between the
stereochemistry of monotransglucosylation and sweetness or taste.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI: xxxxxxx.
Soluble expression conditions of glycosyltransferases and glycosynthases; MS
parameters; Reaction conditions for the biosynthesis of monoglucosyl rubusosides;
SDS-PAGE analysis of the soluble expressions of enzymes in E. coli. NMR data of
monoglucosyl rubusosides1-4, 7 and 8; NMR spectra of compound 2-4, 7 and 8.
AUTHOR INFORMATION
Corresponding Author
*E-mail: xuriwu@cpu.edu.cn (X.W.); yjchen@cpu.edu.cn (Y.C.)
ORCID
Xuri Wu: 0000-0002-0933-5902
Yijun Chen: 0000-0002-4920-152X
Author Contributions
#
L.Z. and Y.W. contributed equally to this work.
Funding
This work was supported by the MOST (2018YFA0902000 and 2019ZX09721001),
the “111 Project” from the Ministry of Education of China and the State
Administration of Foreign Expert Affairs of China (111-2-07), Natural Science
Foundation of Jiangsu Province (BK20181334), and the PAPD of Jiangsu Province.
ACS Paragon Plus Environment

Page 19 of 31
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
45
Notes
46
47
48
49
50
51
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors are grateful to 35 volunteers for their help with sensory evaluation.
REFERENCES
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
(1) Tanaka, T.; Kohda, H.; Tanaka, O.; Chen, F. H.; Chou, W. H.; Leu, J. L.
Rubusoside (β-D-glucosyl ester of 13-O-β-D-glucosyl-steviol), a sweet principle of
Rubus chingii Hu (Rosaceae). Agric. Biol. Chem. 1981, 45, 2165-2166.
(2) Chu, J. P.; Zhang, T. T.; He, K. X. Cariogenicity features of Streptococcus
mutans in presence of rubusoside. BMC Oral Health 2016, 16, 54-59.
(3) Olsson, K.; Carlsen, S.; Semmler, A.; Simon, E.; Mikkelsen, M. D.; Moller, B. L.
Microbial production of next-generation stevia sweeteners. Microb. Cell Fact. 2016,
15. 207.
(4) Thompson, A. M. G.; Iancu, C. V.; Nguyen, T. T. H.; Kim, D.; Choe, J. Y.
Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights
into transport specificity. Sci. Rep. 2015, 5. 12804.
(5) Kim, J.; Nguyen, T. T. H.; Jin, J. H.; Septiana, I.; Son, G. M.; Lee, G. H.; Jung, Y.
J.; Qureshi, D.; Mok, I. K.; Pal, K.; Yang, S. Y.; Kim, S. B.; Kim, D. Anti-cariogenic
characteristics of rubusoside. Biotechnol. Bioproc. E. 2019, 24, 282-287.
(6) Liu, Z. J.; Schwimer, J.; Liu, D.; Lewis, J.; Greenway, F. L.; York, D. A.;
Woltering, E. A. Gallic acid is partially responsible for the antiangiogenic activities of
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 31
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
Rubus leaf extract. Phytother. Res. 2006, 20, 806-813.
(7) Zhang, J.; Chou, G. X.; Liu, Z. J.; Liu, M. Employing rubusoside to improve the
solubility and permeability of antitumor compound betulonic acid. Nanomedicine
2016, 11, 2829-2844.
(8) Huang, G. L.; Mei, X. Y. Synthetic glycosylated natural products have
satisfactory activities. Curr. Drug Targets. 2014, 15, 780-784.
(9) Huang, G. L.; Lv, M. J.; Hu, J. C.; Huang, K. L.; Xu, H. Glycosylation and
activities of natural products. Mini Rev. Med. Chem. 2016, 16, 1013-1016.
(10) Guo, Q. B.; Zhang, T. T.; Wang, N. F.; Xia, Y. M.; Zhou, Z. Y.; Wang, J. R.;
Mei, X. F. RQ3, A natural rebaudioside D isomer, was obtained from glucosylation of
rebaudioside A catalyzed by the CGTase toruzyme 3.0 L. J. Agric. Food Chem. 2019,
67, 8020-8028.
(11)Kochikyan, V. T.; Markosyan, A. A.; Abelyan, L. A.; Balayan, A. M.; Abelyan,
V. A. Combined enzymatic modification of stevioside and rebaudioside A. Appl.
Biochem. Micro+. 2006, 42, 31-37.
(12)Ohtani, K.; Aikawa, Y.; Ishikawa, H.; Kasai, R.; Kitahata, S.; Mizutani, K.; Doi,
S.; Nakaura, M.; Tanaka, O. Further study on the 1,4-alpha-transglucosylation of
rubusoside, a sweet steviol-bisglucoside from Rubus suavissimus. Agric. Biol. Chem.
1991, 55, 449-53.
(13)Darise, M.; Mizutani, K.; Kasai, R.; Tanaka, O.; Kitahata, S.; Okada, S.; Ogawa,
S.; Murakami, F.; Chen, F. H. Enzymic transglucosylation of rubusoside and the
structure-sweetness relationship of steviol-bisglycosides. Agric. Biol. Chem. 1984, 48,
ACS Paragon Plus Environment

Page 21 of 31
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
90
2483-2488.
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
(14)Ko, J. A.; Ryu, Y. B.; Park, J. Y.; Kim, C. Y.; Kim, J. S.; Nam, S. H.; Lee, W. S.;
Kim, Y. M. Glucosyl rubusosides by dextransucrases improve the quality of taste and
sweetness. J. Microbiol. Biotechnol. 2016, 26, 493-497.
(15)Ishikawa, H.; Kitahata, S.; Ohtani, K.; Ikuhara, C.; Tanaka, O. Production of
stevioside and rubusoside derivatives by transfructosylation of β-fructofuranosidase.
Agric. Bioi. Chem. 1990, 54, 3137-3143.
(16)Kitahata, S.; Ishikawa, H.; Miyata, T.; Tanaka, O. Production of rubusoside
derivatives by transgalactosylation of various α-galactosidases. Agric. Biol. Chem.
1989, 53, 2929-2934.
(17)Yokoyama, M. Methods of synthesis of glycosyl fluorides. Carbohydr. Res. 2000,
327, 5-14.
(18)Williams, G. J.; Zhang, C.; Thorson, J. S. Expanding the promiscuity of a
natural-product glycosyltransferase by directed evolution. Nat. Chem. Biol. 2007, 3,
657-662.
(19)Gurung, R. B.; Kim, E. H.; Oh, T. J.; Sohng, J. K. Enzymatic synthesis of
apigenin glucosides by glucosyltransferase (YjiC) from Bacillus licheniformis DSM
13. Mol. Cells 2013, 36, 355-361.
(20)Dewitte, G.; Walmagh, M.; Diricks, M.; Lepak, A.; Gutmann, A.; Nidetzky, B.;
Desmet, T. Screening of recombinant glycosyltransferases reveals the broad acceptor
specificity of stevia UGT-76G1. J. Biotechnol. 2016, 233, 49-55.
(21)Faijes, M.; Saura-Valls, M.; Pérez, X.; Conti, M.; Planas, A. Acceptor-dependent
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 31
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
regioselectivity of glycosynthase reactions by Streptomyces E383A β-glucosidase.
Carbohydr. Res. 2006, 341, 2055-2065.
(22)Kim, Y. W.; Lee, S. S.; Warren, R. A. J.; Withers, S. G. Directed evolution of a
glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and
expands its substrate repertoire. J. Biol. Chem. 2004, 279, 42787-42793.
(23)Liang, C. N.; Zhang, Y.; Jia, Y.; Wang, W. Z.; Li, Y. H.; Lu, S. K.; Jin, J. M.;
Tang, S. Y. Engineering a carbohydrate-processing transglycosidase into
glycosyltransferase for natural product glycodiversification. Sci. Rep. 2016, 6, 21051.
(24)Swistowska, A. M.; Wittrock, S.; Collisi, W.; Hofer, B. Heterologous
hyper-expression of a glucansucrase-type glycosyltransferase gene. Appl. Microbiol.
Biotechnol. 2008, 79, 255-261.
(25)Lee, S. J.; Kim, J. C.; Kim, M. J.; Kitaoka, M.; Park, C. S.; Lee, S. Y.; Ra, M. J.;
Moon, T. W.; Robyt, J. F.; Park, K. H. Transglycosylation of naringin by Bacillus
stearothermophilus maltogenic amylase to give glycosylated naringin. J. Agric. Food
Chem. 1999, 47, 3669-3674.
(26)Overwin, H.; Wray, V.; Seeger, M.; Sepúlveda-Boza, S.; Hofer, B. Flavanone and
isoflavone glucosylation by non-Leloir glycosyltransferases. J. Biotechnol. 2016, 233,
121-128.
(27)Yu, L. T.; Li, X.; Zhang, Z. Y.; Du, P.; Liu, J. L.; Li, Y. J.; Yin, T. Q.; Yu, W. H.;
Sun, H.; Wang, M.; Luo, C. Dimorphic autoantigenic and protective effects of Reg2
peptide in the treatment of diabetic beta-cell loss. Diabetes Obes. Metab. 2019, 21,
1209-1222.
ACS Paragon Plus Environment

Page 23 of 31
Journal of Agricultural and Food Chemistry
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
34
(28)Ibrahim, M. A.; Rodenburg, D. L.; Alves, K.; Fronczek, F. R.; Mcchesney, J. D.;
Wu, C. M.; Nettles, B. J.; Venkataraman, S. K.; Jaksch, F. Minor diterpene glycosides
from the leaves of Stevia rebaudiana. J. Nat. Prod. 2014, 77, 1231-1235.
(29)Zhang, Y. Y.; Xu, S. H.; Jin, Y.; Dai, Y.; Chen, Y. J.; Wu, X. R. Efficient
biocatalytic preparation of rebaudioside KA: Highly selective glycosylation coupled
with UDPG rgeneration. Sci. Rep. 2020, 10, 6230.
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
(30)Dai, L. H.; Li, J.; Yang, J. G.; Men, Y.; Zeng, Y.; Cai, Y.; Sun, Y. X. Enzymatic
synthesis of novel glycyrrhizic acid glucosides using a promiscuous Bacillus
glycosyltransferase. Catalysts 2018, 8, 615.
(31)Rivas, F.; Parra, A.; Martinez, A.; Garcia-Granados, A. Enzymatic glycosylation
of terpenoids. Phytochem. Rev. 2013, 12, 327-339.
(32) Chiu, C. H.; Wang, R. B.; Lee, C. C.; Lo, Y. C.; Lu, T. J. Biotransformation of
mogrosides from Siraitia Grosvenorii swingle by Saccharomyces cerevisiae. J. Agric.
Food Chem. 2013, 61, 7127-7134.
(33)Geeraert, B.; Crombe, F.; Hulsmans, M.; Benhabiles, N.; Geuns, J. M.; Holvoet,
P. Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant
defense in obese insulin-resistant mice. Int. J. Obesity 2010, 34, 569-577.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 31
Figure Captions
Figure 1. LC-MS profile of the monoglucosyl rubusosides.
A) LC-MS chromatograms (UV detector) of reaction mixtures containing
(
monoglucosyl rubusosides. Ru, rubusoside; compounds 1–4, 7 and 8, monoglucosyl
rubusosides. i) reaction mixture of rubusoside and UDP-glucose by OleD; ii) reaction
mixture of rubusoside and UDP-glucose by YjiC; iii) reaction mixture of rubusoside
and UDP-glucose by UGT76G1; iv) reaction mixture of rubusoside and F-1-glucose
by Bgl3 E383A; v) reaction mixture of rubusoside and sucrose by GtfR. (B) Mass
spectra of monoglucosyl rubusosides. a) Mass spectrum for compound 1 produced by
OleD; b) and c) Mass spectra for compounds 2 and 3, respectively, produced by YjiC;
d) Mass spectrum for compound 4 produced by UGT76G1; e) Mass spectrum for
compound 4 produced by Bgl3 E383A; f) and g) Mass spectra for compounds 7 and 8,
respectively, produced by GtfR. Asterisks represent the molecular ion peaks of
compounds 1–4, 7 and 8.
Figure 2. Enzymatic synthesis of monoglucosyl rubusosides 1–4.
OleD from Streptomyces antibioticus, YjiC from Bacillus licheniformis, and
UGT76G1 from Stevia rebaudiana are UDP-glucose dependent glycosyltransferases.
Bgl3 E383A is a F-1-glucose dependent engineered glycosynthase from glycosidase
Bgl3.
Figure 3. Enzymatic synthesis of monoglucosyl rubusosides 7 and 8 by GtfR.
GtfR from Streptococcus oralis is a non-Leloir glycosyltransferase with sucrose as a
sugar donor.
Figure 4. Effects of monoglucosyl rubusosides on glucose-stimulated insulin
secretion in the islets.
(A) Insulin secretion in the isolated islets incubated with compounds 1–8 at 2.8 mM
ACS Paragon Plus Environment

Page 25 of 31
Journal of Agricultural and Food Chemistry
glucose. (B) Insulin secretion in the isolated islets incubated with compounds 1–8 at
2
2.2 mM glucose. Islets were isolated from male 12-week-old BALB/c mice. Ten
-6
islets in each group were each treated with 10 M of rubusoside or its monoglucosyl
derivatives, followed by an insulin determination. Compounds 1–8: the eight types of
monoglucosyl rubusosides in Table 1; Ru: rubusoside; C: control, an untreated group.
No significant difference was found between treated groups and controls (p > 0.05).
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 31
Table
Table 1. Relative sweetness and quality of taste of rubusoside and monoglucosyl
derivatives.
a
RS
Quality of
taste
Compound
R1
β-glc
R2
β-glc (2→1) β-glc
β-glc
b
Reference
This study
This study
This study
This study
13
1
2
3
4
140
143
-50
-73
142
113
140
89
+3
0
β-glc (2→1) β-glc
β-glc
β-glc (3→1) β-glc
β-glc
-1
-1
+3
0
β-glc (3→1) β-glc
β-glc (4→1) α-glc
β-glc
c
5
β-glc
c
6
β-glc (4→1) α-glc
β-glc
13
7
8
β-glc (6→1) α-glc
β-glc
+2
-1
0
This study
This study
13
β-glc (6→1) α-glc
β-glc
Rubusoside
β-glc
114
a
RS: relative sweetness to sucrose (on a weight basis)
b
Quality of taste relative to 3: +3, fairly better; +2, better; +1, slightly better; 0,
almost the same, -1, worse.
c
13
Data of compounds 5, 6 and rubusoside are from the literature.
ACS Paragon Plus Environment

Page 27 of 31
Journal of Agricultural and Food Chemistry
Figure 1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 31
Figure 2
2
8
ACS Paragon Plus Environment

Page 29 of 31
Journal of Agricultural and Food Chemistry
Figure 3
2
9
ACS Paragon Plus Environment