Expanded acceptor substrates flexibility study of flavonol 7-O-rhamnosyltransferase, AtUGT89C1 from Arabidopsis thaliana

Article abstract of DOI:10.1016/j.carres.2015.09.010

Acceptor substrates flexibility of previously characterized flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from Arabidopsis thaliana was explored with an endogenous nucleotide diphosphate sugar and five different classes of flavonoids (flavonols, flavones,

Full text of DOI:10.1016/j.carres.2015.09.010

Carbohydrate Research 418 (2015) 13–19
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Expanded acceptor substrates flexibility study of flavonol 7-O-
rhamnosyltransferase, AtUGT89C1 from Arabidopsis thaliana
Prakash Parajuli †, Ramesh Prasad Pandey †, Nguyen Thi Huyen Trang, Tae Jin Oh,
Jae Kyung Sohng *
Institute of Biomolecule Reconstruction, Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-
myeon, Asan-si, Chungnam 336-708, Republic of Korea
A R T I C L E
I N F O
A B S T R A C T
Article history:
Received 22 July 2015
Received in revised form 17 September
2015
Accepted 19 September 2015
Available online 9 October 2015
Acceptor substrates flexibility of previously characterized flavonol 7-O-rhamnosyltransferase (AtUGT89C1)
from Arabidopsis thaliana was explored with an endogenous nucleotide diphosphate sugar and five dif-
ferent classes of flavonoids (flavonols, flavones, flavanones, chalcone and stilbenes) through a
biotransformation approach. In contrast to the previous reports, this study highlights the expanded ac-
ceptor substrate promiscuity of AtUGT89C1 for the regiospecific glycosylation of diverse class of flavonoids
at 7-hydroxyl position using microbial thymidine diphosphate (TDP)-l-rhamnose as sugar donor instead
of uridine diphosphate-l-rhamnose. We examine the biocatalytic potential of AtUGT89C1 using endog-
enous sugar (TDP-l-rhamnose) from E. coli to generate a library of flavonoid 7-O-rhamnosides.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Flavonoid 7-O-rhamnosides
AtUGT89C1
NDP-sugar
7-O-Rhamnosyltransferase
Substrate flexibility
Flavonoids include a large group of plant secondary metabolites.
They exhibit profound human health promoting activities (antioxi-
dant, anti-inflammatory, anti-angiogenic, anti-proliferative, antifungal,
antibacterial), and protective effects against neurodegenerative dis-
eases like Alzheimer’s and Parkinson’s diseases.1–4 Although more than
9000 known derivatives of flavonoids have been reported till 2005,5
and the number of new flavonoid derivatives including diverse sugar
(UDP-glucose, UDP-glucuronic acid, TDP-rhamnose, UDP-xylose, etc.)
conjugated products should have increased significantly by now. In
plants, they are present in various forms (aglycon, glycosides, acylated,
methoxylated, hydroxylated, prenylated, malonylated, sulfated, etc.).
Flavonoid glycosides are found significantly higher than other scaf-
folds and have gained considerable attention due to their promising
health beneficial activities.6 Nevertheless, other flavonoids were also
found to exhibit various activities. Glycosylation, the attachment of
bulky hydrophilic sugar moiety, is a key mechanism in determining
complexity and remarkable chemical diversity of flavonoids in plants,
where regioselective glycosyltransferases offer an important means
to overcome limitations in terms of structural diversity and their phar-
macological potency.7,8
A large number of uridine diphosphate (UDP)-glycosyltransferases
(UGTs) from different sources (plants, animals, and microbial) have
been reported to modify plant secondary metabolites like flavo-
noids, terpenoids, steroids, xanthonoids through glycosylation.9
Arabidopsis thaliana is one of the model plants, which includes fla-
vonoids as major secondary metabolites, henceforth flavonoid
scaffold biosynthesis has been extensively studied in this plant.10
Offering diversity of flavonoid scaffolds, A. thaliana contains 120
glycosyltransferases belonging to the GT1 family proteins among
which many of them are functionally characterized.10–12 Unlike mi-
crobial GTs (YjiC from Bacillus licheniformis DSM-13,13 OleD14 and
its variants from Streptomyces), only a few of the UGTs from
A. thaliana and other plants have been reported to exhibit flexibil-
ity in catalyzing glycosylation reactions and in recognizing different
classes of flavonoid as acceptor substrates and different NDP-
sugars as sugar donors (Table 1). In fact, most of the plant UGTs are
found to be highly regiospecific to donor as well as acceptor sub-
strates. Currently, application of these flexible GTs in microbial
biotransformation have attracted considerable interest in synthe-
sizing and modifying the existing flavonoids and yielding novel
flavonoid glycosides.6 But, the existence of limited studies on sub-
strate flexibility of these plant UGTs has restricted the biosynthesis
of novel flavonoid glycosides. Therefore, recently domain swap-
ping and mutagenesis studies have been carried out to expand the
substrate promiscuity of many UGTs.20
*
Corresponding author. Institute of Biomolecule Reconstruction, Department of
BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70 Sunmoon-ro
221, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea. Tel.:
+82(41)530 2246; fax: +82(41)544 2919.
In this report, we studied the substrate flexibility of AtUGT89C1
with respect to Escherichia coli endogenous sugar (TDP-l-rhamnose)
with various flavonoids. AtUGT89C1 has been previously
E-mail address: sohng@sunmoon.ac.kr (J.K. Sohng).
†These authors equally contributed.
http://dx.doi.org/10.1016/j.carres.2015.09.010
0008-6215/© 2015 Elsevier Ltd. All rights reserved.

14
P. Parajuli et al./Carbohydrate Research 418 (2015) 13–19
Table 1
Functionally characterized glycosyltransferases from A. thaliana showing flexibility in terms of polyphenols as acceptor substrate and their sugar donors for O-glycosylation
A. thaliana GTs
Substrates
Features of glycosylation and sugar donor
References
11, 15, 16
UGT78D1
Flavonols (quercetin, kaempferol)
3-O-Rhamnosylation/glucosylation
UDP/TDP-rhamnose/glucose
as sugar donor
ArGt-3
Flavonols (3-hydroxyflavone, kaempferol, fisetin,
quercetin, myricetin and morin)
3-O-Rhamnosylation
3-O-Xylosylation,
3-O-Allosylation,
3-O-Amino sugars
13, 17–19, 34
11
UGT73C6
Flavonol 3-O-glucoside
7-O-Glucosylation
Flavonol 3-O-rhamnoside
Flavonols, Anthocyanidins
Flavonoids (flavonols, flavanones, flavonols, flavones),
flavonol 3-O-glucosides
UDP-Glucose as sugar donor
UDP-Glucose/N-acetylglucosamine
Regiospecific O-rhamnosylation but flexible with diverse
flavonoids as substrates and TDP/UDP-rhamnose as sugar
donor.
UGT78D2
3, 7, 15
AtUGT89C1
This study, 7, 15, 19
characterized as flavonol 7-O-rhamnosyltransferase that only accepts
flavonol 3-O-glycosides as substrate to conjugate rhamnose moiety
at 7-hydroxyl position.7,19 Previous studies have also pointed out that
it accepts only UDP-l-rhamnose as the sugar donor to catalyze
glycosylation reaction.15,19 Together with UDP-l-rhamnose syn-
thase (AtRHM1) from A. thaliana, AtUGT89C1 was found to synthesize
rare flavonol bisrhamnoside.15
Bacteria are rich in nucleotide sugars and they contain UDP-d-
glucose, a precursor nucleotide sugar for its modified derivatives
(UDP-d-galactose, UDP-d-xylose, UDP-d-glucuronic acid); TDP-l-
rhamnose, and modified deoxy-sugars like TDP-d-allose, TDP-d-
talose, etc. Nevertheless, UDP-l-rhamnose pathway is absent in
bacteria.21 On the contrary, UDP-l-rhamnose is a ubiquitous sugar
present in plants, as many diverse polysaccharides and polyphe-
nols contain a rhamnose moiety in their structure.16,21 A. thaliana
plant contains AtRHM1, AtRHM2 and AtRHM3 (UDP-l-rhamnose syn-
thase) are bi-functional enzymes that catalyze the synthesis of UDP-
l-rhamnose from UDP-d-glucose.16 In contrast, TDP-l-rhamnose is
present and is biosynthesized only in bacteria from TDP-4-keto-6-
deoxy-d-glucose after successive actions of two enzymes, epimerase
and ketoreductase.21 Thus, to facilitate the biotransformation of fla-
vonoids in E. coli AtUGT89C1 was co-expressed with AtRHM1 to
produce UDP-l-rhamnose in the cytosol.15,19
catalyzed the biotransformation reaction to conjugate rhamnose
moiety in quercetin. However, in strain C1, though no (U/T)DP-l-
rhamnose biosynthesis pathway was overexpressed, UGT89C1
accepted endogenous TDP-l-rhamnose as sugar donor for the
glycosylation reaction. Although E. coli BL21 (DE3) cannot synthe-
size UDP-l-rhamnose, every Gram negative bacterial cell surface
polysaccharide contains rhamnose moiety and are derived from
TDP-L-rhamnose.16,22 The conversion of quercetin to quercetin rham-
noside was 22 and 24% in strain C1 and C1M1, respectively. Control
experiments were performed at the same time preparing reaction
samples by feeding quercetin in recombinant strain harboring only
pET41b vector without gene along with pET32b-AtRHM1, pET41b
and pET32b empty plasmids and pET28-ArGt-3 (pET28 vector
harboring a regiospecific flavonol-3-O-rhamnosyltransferase
from A. thaliana which has proven function to synthesize querce-
tin 3-O-α-l-rhamnoside).17 The distinct difference in retention time
on HPLC-PDA chromatogram of reaction samples catalyzed by ArGt-3
and AtUGT89C1 directs the conjugation of rhamnose moiety to
7-hydroxyl position of quercetin. The difference in UV maxima
of quercetin 3-O-rhamnoside (344.7347 nm) where ~350 nm
was reported previously23 and quercetin 7-O-rhamnoside
(371.7347 nm)24 further supports the sugar attachment at 7-OH po-
sition (Fig. 1c and d).
In this experiment, we studied the donor (TDP-l-rhamnose and
UDP-l-rhamnose) and acceptor (various classes of flavonoids) sub-
strate flexibilities of AtUGT89C1. To measure the capacity of
AtUGT89C1, different flavonoid aglycones were used as acceptor sub-
strate. At first, we assayed the acceptor substrate flexibility of
AtUGT89C1 by simple whole-cell biotransformation process. The
biocatalysis reaction sample was prepared using two recombinant
strains spent culture medium from the E. coli BL21 (DE3) harbor-
ing pET41b-AtUGT89C1 with pET32b-AtRHM1 (strain C1M1) and
without pET32b-AtRHM1 (strain C1) fed with quercetin (6) as an
acceptor substrate explained in experimental procedure. Both the
biotransformation samples were analyzed by high performance liquid
chromatography-photo diode array (HPLC-PDA). The analyses re-
vealed the generation of a new peak (retention time (tR: 17.1 min))
in both reactions, that was well resolved from the substrate peak
(tR: 19.2 min) (Fig. 1A). This peak was further analyzed by high res-
olution quadruple time-of-flight electrospray ionization mass
spectrometry (HR-QTOF-ESI/MS). The molecular mass of the product
matched with that of quercetin rhamnoside (calculated mass for
the molecular formula C21H21O11 [M + H]+ m/z+ 449.1084 and found
mass [M + H]+ m/z+ 449.1092) (Fig. 1B). In fact, this result revealed
that AtUGT89C1 can accept not only flavonol 3-O-glycosides but also
flavonol aglycon as acceptor substrates. Moreover, it was also evident
that GT is flexible enough to accept TDP-l-rhamnose as sugar donor
because strain C1 does not contain AtRHM1. The strain harboring
pET32b-AtRHM1 possibly synthesized UDP-l-rhamnose in E. coli and
We further studied flexibility of GT in terms of acceptor sub-
strates without using AtRHM1 through biotransformation under
identical condition. Seed cultures were prepared and transformed
into the flask containing 100 mL LB-medium. Biotransformation re-
action samples were prepared according to the methodology
explained in experimental section. Twelve different classes of fla-
vonoids – flavones: apigenin (1), chrysin (2), luteolin (3), 7,8-
dihydroxyflavone (4); flavonols: quercetin (5), myricetin (6), morin
(7), 3-hydroxyflavone (8); flavanones: hesperetin (9), naringenin (10);
Stilbene: resveratrol (11); Chalcone: phloretin (12) (Fig. 2) were fed
exogenously. Samples were prepared from in vivo reaction mix-
tures taking 500 μL culture broth, centrifuged to separate cell palette
from culture medium. Double volume of ethyl acetate was added
in culture medium for extraction of compound followed by drying
and dissolved in 100 μL methanol for analytical procedure. From each
reaction mixtures, 20 μL was injected into HPLC-PDA as described
in experimental section. The HPLC-PDA chromatogram generated
a single product peak from all the flavonoid reaction mixtures except
8, 11 and 12 (Fig. S1a and b). These reaction mixtures were then
subjected to high-resolution mass analyses using HR-QTOF-ESI/
MS in positive ion mode. In all the reaction mixtures of different
classes of flavonoids having 7-hydroxyl position flavonols (except
8), flavones and flavanones HPLC-PDA showing novel peak, we found
the exact mass spectrum matching with the calculated mass of each
flavonoid rhamnoside along with their daughter fragment of aglycons
(Fig. S2). Comparison of these mass spectra with standard

Fig. 1. A. HPLC-PDA chromatogram of biocatalysis reaction mixture of quercetin with AtRHM1 and without AtRHM1 catalyzed by AtUGT89C1 compared with standard quercetin including two controls (E. coli harboring only
pET41b without gene + pET32b-AtRHM1 and E. coli with two empty plasmids pET41b and pET32b) at the UV absorbance of 320 nm. However, product retention time of quercetin 3-O-rhamnoside catalyzed by ArGt-3 reported
previously17 was compared with quercetin 7-O-rhamnoside product. B. The product was further confirmed by high resolution mass analyses (upper spectrum). C. UV maxima of quercetin-7-O-rhamnoside. D. UV maxima of
quercetin 3-O-rhamnoside.

16
P. Parajuli et al./Carbohydrate Research 418 (2015) 13–19
Fig. 2. Structures of different classes of falvonoids used in this study. Flavones: apigenin (1), chrysin (2), luteolin (3) and 7, 8-dihydroxyflavone (4); flavonols: quercetin (5),
myricetin (6), morin (7) and 3-hydroxyflavone (8); flavanones: hesperetin (9) and naringenin (10); stilbene: resveratrol (11); chalcone: phloretin (12).
calculated mass reflected successful generation of different classes
of flavonoid 7-O-rhamnosides through whole-cell biotransforma-
tion using AtUGT89C1 glycosyltransferase. The HPLC-PDA data were
further analyzed by integrating each peak and quantified the amount
of product formed in culture medium based on the regression equa-
tion derived from the standard curve of each aglycon as apigenin
(~25%, ~20 mg/L), chrysin (~16%, ~12 mg/L), luteolin (~17%, ~13 mg/
L), 7,8-dihyroxyflavone (~14%, ~11 mg/L), quercetin (~24%, ~22.5 mg/
L), myricetin (~15%, ~13.7 mg/L), morin (~14%, ~12 mg/L), hesperitin
(~13%, ~12.3 mg/L) and naringenin (~14%, ~11.4 mg/L) (Fig. 3). The
above mentioned data show conversion percentage in culture
medium only. We further accessed the product conversion in cell
lysate (cells) considering each flavonoid from different classes as rep-
resentative. Since we are accessing the substrate promiscuity of
AtUGT89C1, 100 mL culture of strain C1 was made and flavonoids;
3, 5 and 10 from flavone, flavonol and flavanone classes respective-
ly were biotransformed in identical condition. Samples were prepared
from cell lysate and culture medium separately. Ethyl acetate ex-
traction was done for extraction of compound from culture medium
as explained above while cell lysate was extracted by methanol
(10 mL) as explained in previous paper.19 The HPLC-PDA data were
analyzed to quantify the product formation as explained above. Pro-
duction of each flavonoid rhamnoside in culture medium and cells
were not significantly different. Conversion of 3 that represents fla-
vones was ~19.8% (~13.5 mg/L) in cells and ~21% (~14.2 mg/L) culture
medium, 5 that represents flavonols was ~19.5% (~21 mg/L) in cells
but ~21% (~21.4 mg/L) in culture medium, whereas 9 that repre-
sents flavanones was ~13% (~12.3 mg/L) in cells and ~18.5%
(~16.4 mg/L) in culture medium (Fig. S3). This result predicts the
similar production profile of other flavonoids in cell lysate through
biotransformation reaction.
Microbial NDP-sugars are utilized as sugar donors by heterolo-
gously overexpressed dedicated GTs and exogenously supplemented
aglycons (flavonoids) are used as acceptor substrates in glycosylation
reactions during microbial biotransformations. During the process,
specific products of interest including novel flavonoid glycoside de-
rivatives could be generated. The AtUGT89C1 utilized the microbial
NDP-sugar (TDP-l-rhamnose) through the approach of biotrans-
formation, and different flavonoid rhamnosides were generated. Since
wild type E. coli BL21 (DE3) was used for the construction of re-
combinant strains and in biotransformation reaction, the production
of each flavonoid rhamnoside was not significant. In contrast, the
production of flavonoid rhamnoside can be enhanced by engineer-
ing the host strain through the approach of metabolic engineering,
synthetic biology, and overexpression of NDP-sugar biosynthetic
pathway genes and supplementation of external carbon source.18
But, this study was focused on substrate donor/acceptor specifici-
ties of AtUGT89C1 enzyme. Although, AtUGT89C1 can accept both
(T/U)DP-l-rhamnose as sugar donor, the strain C1M1 having
AtUGT89C1 and AtRHM1 should exhibit higher production com-
pared to the strain C1, but the difference in production of each
rhamnosides was relatively smaller (Fig. 3). The possible reasons
for having low conversion in glycoside synthesis could be the lower
availability of (T/U)DP-l-rhamnose in E. coli BL21 (DE3) cytosol or
catalytic efficacy of glycosyltransferase toward flavonoid aglycon as
substrate. Since the production of TDP-rhamnose or UDP-rhamnose
in E. coli was not monitored, the exact reason for lower glycoside
production is still unknown even though AtUGT89C1 accepts both
U/TDP-rhamnose as sugar donors. Although AtUGT89C1 expres-
sion was good in soluble form while performing the SDS–PAGE
(sodium dodecyl sulfate–polyacrylamide gel electrophoresis) anal-
yses (Fig. S4). However, previous reports have shown significant

P. Parajuli et al./Carbohydrate Research 418 (2015) 13–19
17
Fig. 3. Bioconversion of each class of flavonoids catalyzed by AtUGT89C1 in recombinant strain: C1-E. coli BL21 (DE3)-pET41b-AtUGT89C1 and C1M1-E. coli BL21 (DE3)-
pET41b-AtUGT89C1 along with pET32b-AtRHM1. Error bars represents the standard deviations obtained from three individual production data obtained under identical
reaction condition as described in experimental procedures.
production of bis-glycosides (67.4 mg/L of quercetin 3, 7-O-
bisrhamnoside and 67 mg/L of quercetin 3-O-glucoside-7-
rhamnoside) using AtUGT89C1 in association with AtRHM1/
AtRHM2 and other 3-O-glycosyltransferases (AtUGT78D1,
AtUGT78D2, AtUGT78D3).15,16 These were engineered E. coli strains
especially designed for better productivity of glycoside synthesis.
Since, this GT was previously characterized as 7-hydroxyl posi-
tion specific using various flavonol glycosides as substrates, all the
generated flavonoid rhamnosides catalyzed by AtUGT89C1 were pre-
sumed to be 7-O-rhamnosides. To investigate this, we carried out
biotransformation of different classes of flavonoid i.e., flavone (1)
which has OH group at 7th position using strain C1 and purified
the glycoside as described in experimental procedures using
preparative-HPLC. The product was analyzed by 1H and 13C nuclear
magnetic resonance (1D-NMR) followed by HMBC (heteronuclear
multiple bond correlation) (2D-NMR). While comparing the 1H NMR
of standard apigenin and reaction product, although the parent com-
pound has 3-hydroxyl groups, signals at δ 12.95 (1H, s, 5-OH) and
δ 10.46 (1H, s, 4′-OH) were detected but 7-OH was missing in re-
action product (Fig. S4C). The sugar moiety could have replaced
proton of this hydroxyl group. The anomeric proton (1′′-H) was con-
sistent with δ 5.55 (d, J = 1.8 Hz, 1H), however anomeric proton-
coupling constant (J = 1.8 Hz) confirms the conjugation of rhamnose
moiety to be in α-configuration (Table 2). To confirm the position
of sugar conjugation, we further analyzed HMBC (Fig. 4). Correla-
tion of anomeric proton (H-1′′) with C-7 in HMBC confirmed the
attachment of rhamnose moiety at 7th position of apigenin (Fig. 4).
Therefore, the final product was determined to be apigenin 7-O-
α-l-rhamnoside. However, while feeding 8 in identical conditions,
we were unable to detect product peak in HPLC-PDA. This analy-
sis supports the conjugation of rhamnose moiety at 7-hydroxyl
position. Therefore, we can conclude the notion that AtUGT89C1 not
only accepts the flavonol 3-O-glycosides as substrate but is also
capable of accepting diverse classes of flavonoid aglycones having
7th hydroxyl group as acceptor substrates. Furthermore, it has been
reported that very few bacteria possess UDP-l-rhamnose biosyn-
thesis pathway; however, E. coli lacks this pathway.21 To synthesize
UDP-l-rhamnose in E. coli and generate flavonol bis-glycosides, re-
searchers have used (UDP-l-rhamnose synthase) AtRHM1/
AtRHM215,16 from A. thaliana. Nevertheless, none of the reports have
mentioned about AtUGT89C1 accepting various classes of flavo-
noid aglycone as acceptor substrates and TDP-l-rhamnose as sugar
donor.
Glycosylation is typically a terminal step in flavonoid biosyn-
thesis in plants which significantly improves the biological activities,
solubility and stability of aglycones.25 GTs play pivotal role in the
synthesis of pharmacologically active plant natural product
Table 2
Comparison of 1H and 13C NMR spectra of apigenin and apigenin 7-O-α-L-rhamnoside
measured in DMSO (d6)
Positions
(H and C)
Apigenin (1)
Apigenin 7-O-α-l-rhamnoside
1H
13C
1H
13C
5-OH
4′-OH
7-OH
2
3
4
6
8
9
10
1′
2′
3′
5′
12.96
10.58
10.58
–
6.77
–
6.18
6.47
–
161.83
161.54
164.51
164.12
103.22
182.18
99.53
94.36
157.69
104.09
121.56
128.85
116.35
116.35
128.85
–
12.95
10.44
Not detected
–
6.86
–
6.44
6.86
–
–
–
7.96
6.93
6.93
7.96
161.8
161.6
162.1
164.7
103.5
182.4
98.8
95.2
157.4
105.5
121.20
128.7
116.5
116.5
128.7
100
70.7
72.1
70.3
70.6
–
–
7.91
6.92
6.92
7.91
–
6′
1″
2″
3″
4″
5″
6″-CH3
5.55 (d, J = 1.8 Hz, 1H)
(2′′-OH: 5.20), (2′′-H: 3.56)
(3′′-OH: 4.96), (3′′-H: 3.22)
(4′′-OH: 4.85), (4′′-H: 3.64)
3.85
–
–
–
–
–
–
–
–
–
1.12
18.4

18
P. Parajuli et al./Carbohydrate Research 418 (2015) 13–19
Fig. 4. HMBC of Apigenin 7-O-rhamnoside showing correlation of anomeric proton and C-7 of apigenin.
scaffolds by decorating their sugar units25,26 These enzymes
are involved in synthesizing oligosaccharides, polysaccharides
and glycoconjugates of pharmacologically important compounds.
However, among 97 GT families identified so far (http://
www.cazy.org/GT1.html), only few of the GT1 family proteins are
flexible in terms of accepting diverse donor and acceptor sub-
strates, which limits the biosynthesis of various sugars conjugated
flavonoids.26 This study expanded the possibility of synthesizing
regiospecific flavonoid 7-O-rhamnosides by simple biocatalysis re-
action using bacterial endogenous TDP-l-rhamnose as sugar donor.
Further strain engineering and overexpression of sugar pathway spe-
cific genes could be an alternative approach to enhance the glycoside
yield.
exploited as an active compound for antimalarial therapeutics.30
Another compound, kaempferitrin (kaempferol 3, 7-O-rhmanoside),
activates the insulin signaling pathway and also stimulates secre-
tion of adiponectin in 3T3-L1 adipocytes while tested in mice.
Activation of glucose uptake activities and translocation studies of
this compound could serve as food supplement for diabetic pa-
tients in the future.29 Most of the naturally occurring sugars are in
D-form but rhamnose occurs in L-form. Glycoconjugates deferring
in stereochemistry of sugar (D/L-configuration and α/β-configuration)
have great importance with respect to their biological activities.
Talosin A (genistein 7-O-α-l-taloside) and Talosin B (genistein 4′-
7-O-α-l-taloside) are glycosides of isoflavonoid containing
l-talopyranoside in α-configuration and considered to be strong an-
tifungal agents with low toxicity because of sugar appendage and
their stereochemistry.30,31 Most of the bioactive natural products con-
taining deoxy sugars are also in α-L-form.31 Thus, AtUGT89C1
producing l-rhamnose sugar conjugated flavonoids at 7-OH posi-
tion in α-configuration by using endogenous TDP-l-rhamnose from
E. coli BL21 (DE3) would certainly be of interest to many scientists
around the globe.
Unlike AtUGT89C1, other GTs (UGT78D3, ArGt-317 and UGT78D1)
from A. thaliana accept flavonols as acceptor substrates and trans-
fer rhamnose moiety from TDP-l-rhamnose to 3-hydroxyl position
of flavonols. Moreover, UGT78D3 is capable of transferring rham-
nose moiety at 7-OH position of flavonol 3-O-glycosides (Table 1).
However, accepting different classes of flavonoid aglycones as ac-
ceptor substrates to synthesize 7-O-rhamnosides utilizing bacterial
TDP-l-rhamnose as sugar donor has not been reported yet. This cata-
lytic flexibility of AtUGT89C1 would certainly benefit in developing
diverse novel compounds like flavonoids 7-O-rhamnoside and 3,7-
bisrhamnoside, which could exhibit novel biological potential.
Rhamnose is a naturally occurring deoxy-sugar found widely in
bacteria and plants.25 Conjugation of rhamnose moiety into dietary
flavonoids has attracted considerable attention because of its bio-
logical activities. Quercetin 7-O-rhamnoside has shown significant
activity (IC50 of 0.014 μg/mL) against porcine epidemic diarrhea virus,
which is a predominant cause of severe entero-pathogenic
diarrhea.27,28 Moreover, in the case of kaempferol 7-O-α-l-
rhamnoside, enhanced anti-inflammatory activity was seen as
compared with kaempferol aglycone29 and recently, it has also been
1. Experimental procedures
1.1. General
E. coli BL21 (DE3) (Stratagene, La Jolla, CA, USA) was used for the
biotransformation. For the selection and maintenance of plasmid,
E. coli strains were grown at 37 °C in Luria–Bertani (LB) broth or on
agar plates supplemented with the appropriate amount of antibi-
otics (kanamycin 50 μg/mL and ampicillin 100 μg/mL). The pET41b-
AtUGT89C1 and pET32b-AtRHM1 were obtained from Dr. Gale G.
Bozzo, University of Guelph, Canada.19 All the chemicals (metha-
nol, water, ethyl acetate, etc.) used in this study including flavonoids

P. Parajuli et al./Carbohydrate Research 418 (2015) 13–19
19
were purchased from either Sigma-Aldrich or another high grade
commercial source. Flavonoids used in this study are available in
our laboratory and are of more than 98% purity. All restriction
enzymes used for digestion confirmation were obtained from Takara
(Shiga, Japan). Recombinant strains were constructed as E. coli BL21
(DE3)-pET41b-AtUGT89C1 and E. coli BL21 (DE3)-pET41b-AtUGT89C1
and pET32b-AtRHM1 using heat shock plasmid transformation tech-
nique for whole-cell biotransformation.
(15–25 min), 50% (25–30 min), and 20% (20–35 min). NMR analy-
ses were performed on Varian Unity INOVA 700 MHz spectrometer
(Varian, USA).33
Acknowledgments
This work was supported by a grant from the Next-Generation
BioGreen 21 Program (SSAC, grant#: PJ011119012015), Rural De-
velopment Administration, Republic of Korea and by the “Leaders
in Industry-university Cooperation” Project, supported by the Min-
istry of Education (MOE).
1.2. Whole-cell biotransformation of flavonoids
Cultures were made to a volume of 200 mL in LB medium supple-
mented with respective antibiotics. In case of recombinant strain
harboring pET41b-AtUGT89C1 kanamycin 50 μg/mL was added in
culture broth while in recombinant strain harboring pET32b-
AtRHM1, ampicillin 100 μg/mL was added. After 3 hours, each culture
was aseptically transferred into 50 mL falcon tube and centri-
fuged for 10 min at 1000 RCF. Supernatant was discarded and cell
was re-suspended in autoclaved distilled water. Equal density (OD600
1.5) of cells was inoculated into 100 mL LB medium in shake flask
at 4 RCF and incubated at 37 °C until the final OD600 reached to 0.5–
0.7. The cultures were then induced by 0.1 mM isopropyl-1-thio-
β-D-galactopyranoside (IPTG) and incubated at 20 °C for 15 h to allow
protein expression. Flavonoids as substrates were added at a con-
centration of 0.2 mM by dissolving in dimethyl sulfoxide (DMSO)
in each flask for whole-cell biocatalysis reaction. The biotransfor-
mation reaction was carried out for 48 h at 20 °C.
Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.carres.2015.09.010.
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After 48 h incubation, 500 μL of aliquot culture from all flasks
was taken, centrifuged at 1000 RCF for separation of culture medium
and cell lysate. Double volume of ethyl acetate was added in culture
medium for compound extraction. The organic ethyl acetate frac-
tion was collected after centrifugation at 1000 RCF, evaporated and
the dried sample was reconstituted in 100 μL volume of metha-
nol. This sample was further analyzed by HPLC-PDA. The remaining
culture volumes were again mixed with double volume ethyl acetate;
the aqueous layer was fractionated, dried and dissolved in 1 mL
methanol for LC-QTOF-ESI/MS analyses.
The cultural extract dissolved in methanol was directly ana-
lyzed by reverse-phase HPLC-PDA connected with C18 column
(Mightysil RP-18 GP (4.6 × 250 mm, 5 μm)) in binary conditions of
H2O (0.05% trifluoroacetic acid buffer) and 100% acetonitrile (ACN)
at a flow rate of 1 mL/min for 30 minute. The ACN concentrations
were 10% (0–2 min), 70% (20–24 min), 100% (24–28 min), and 50%
(28–30). The exact mass of the compounds was analyzed using LC-
QTOF-ESI/MS [ACQUITY (UPLC, Waters, Mil-ford, MA)-SYNAPT G2-S
(Waters)] in the positive ion mode. The maximum UV absorbance
of each compound was used to monitor the chromatograms. Bio-
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prep-HPLC with a C18 column [YMC-Pack ODS-AQ (150 × 20 mm I.D.,
10 μm)] connected to a UV detector (308 nm) in binary condition
of H2O (0.05% trifluoroacetic acid buffer) and 100% acetonitrile (ACN)
at a flow rate of 10 mL/min for 35 minute. The ACN concentra-
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