Published Ahead of Print 6 April 2012.

10.1128/AEM.00275-12.
2012, 78(12):4256. DOI: Appl. Environ. Microbiol.
Youhoon Chong and Joong-Hoon Ahn
Jeong-A Yoon, Bong-Gyu Kim, Woo Ju Lee, Yoongho Lim,

Escherichia coli
through Metabolic Engineering of
Production of a Novel Quercetin Glycoside
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Production of a Novel Quercetin Glycoside through Metabolic
Engineering of Escherichia coli
Jeong-A Yoon, Bong-Gyu Kim, Woo Ju Lee, Yoongho Lim, Youhoon Chong, and Joong-Hoon Ahn
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, Republic of Korea
Most avonoids exist as sugar conjugates. Naturally occurring avonoid sugar conjugates include glucose, galactose, glucu-
ronide, rhamnose, xylose, and arabinose. These avonoid glycosides have diverse physiological activities, depending on the type
of sugar attached. To synthesize an unnatural avonoid glycoside, Actinobacillus actinomycetemcomitans gene tll (encoding
dTDP-6-deoxy-L-lyxo-4-hexulose reductase, which converts the endogenous nucleotide sugar dTDP-4-dehydro-6-deoxy-L-man-
nose to dTDP-6-deoxytalose) was introduced into Escherichia coli. In addition, nucleotide-sugar dependent glycosyltransferases
(UGTs) were screened to nd a UGT that could use dTDP-6-deoxytalose. Supplementation of this engineered strain of E. coli
with quercetin resulted in the production of quercetin-3-O-(6-deoxytalose). To increase the production of quercetin 3-O-(6-de-
oxytalose) by increasing the supplement of dTDP-6-deoxytalose in E. coli, we engineered nucleotide biosynthetic genes of E. coli,
such as galU (UTP-glucose 1-phosphate uridyltransferase), rffA (dTDP-4-oxo-6-deoxy-D-glucose transaminase), and/or rfbD
(dTDP-4-dehydrorahmnose reductase). The engineered E. coli strain produced approximately 98 mg of quercetin 3-O-(6-de-
oxytalose)/liter, which is 7-fold more than that produced by the wild-type strain, and the by-products, quercetin 3-O-glucose
and quercetin 3-O-rhamnose, were also signicantly reduced.
M
any secondary metabolites found in nature exist as glycones
(26). Subtle alteration of the sugars in natural products
could change their molecular and cellular specicity, leading to
changes in pharmacological properties (16). Glycones are biolog-
ically synthesized using nucleotide sugars as a sugar donor (25).
Because the number of commercially available nucleotide sugars
is limited, the creation of diverse glycosylated secondary metabo-
lites is challenging. However, the unusual nucleotide sugars found
in some bacteria could be a valuable resource for extending the
diversity of glycones.
The outer membrane of Gram-negative bacteria is composed
of lipopolysaccharide(s) (LPS). LPS acts as an endotoxin and trig-
gers strong immune responses in animals. Oantigen is a repetitive
glycan polymer that is part of LPS (19). The building blocks of O
antigen in Escherichia coli are UDP-D-galacto-1,4-furanose,
dTDP--L-rhamnose, and UDP--N-acetyl-D-glucosamine. The
genes and biosynthetic pathway of O-antigen are well dened and
widespread in Gram-negative bacteria. dTDP-sugars, derived
fromglucose 1-phosphate, are involved in this pathway. In E. coli,
biosynthesis of dTDP-L-rhamnose from glucose 1-phosphate in-
volves in four genes; rfbA (dTDP-glucose pyrophosphorylase),
rfbB (dTDP-4-glucose 4,6-dehydratase), rfbC (dTDP-4-dehy-
drorhamnose 3,5-epimerase), and rfbD (dTDP-4-dehydrorham-
nose reductase). However, in some bacteria, other sugars are
present. For example, in certain serotypes of Actinobacillus actino-
mycetemcomitans, O antigen contains D-fucose and 6-deoxytalose
(2, 23). In A. actinomycetemcomitans, tll encodes dTDP-6-deoxy-
L-lyxo-4-hexulose reductase for the biosynthesis of dTDP-6-de-
oxytalose and fcd encodes dTDP-6-deoxy-D-xylo-4-hexulose re-
ductase for the biosynthesis of dTDP-D-fucose (15). Neither gene
is found in E. coli. Although A. actinomycetemcomitans can syn-
thesize unique types of nucleotide sugars, common intermediates
are found in the biosynthesis of dTDP-6-deoxytalose and dTDP-
D-fucose (Fig. 1). Therefore, engineering this nucleotide biosyn-
thesis pathway in E. coli could lead to production of dTDP-6-
deoxytalose or dTDP-D-fucose, whichcouldserve as substrates for
the production of glycones. In particular, dTDP-6-deoxy-L-lyxo-
4-hexulose reductase encoded by tll uses dTDP-4-dehydro-6-de-
oxy-L-mannose, which is also a common substrate for dTDP-4-
dehydrorhamnose reductase (Fig. 1). Therefore, the expression of
tll into E. coli would produce dTDP-6-deoxytalose.
The class of secondary metabolites known as avonoids is syn-
thesized mainly in plants, and most avonoids exist as glycones.
More than 350 glycones of quercetin have been found in various
plants (4). However, arabinose, galactose, glucose, glucuronic
acid, rhamnose, and xylose are the major sugars that are attached
to avonoids, because nucleotide sugar conjugates of these sugars
are commonly found in plants (22). Attachment of sugars to a-
vonoids is catalyzed by UDP-dependent glycosyltransferase
(UGT) (3). UGTs have specicity for sugar donors (nucleotide
sugars) and for sugar acceptors. The PSPG(plant secondary prod-
uct GT) motif at the Cterminus of UGTis known to be critical for
the recognition of the sugar donor (18). However, the basis sugar-
donor selectivity is not yet known. Therefore, it is not easy to
rationally design the attachment of a novel sugar to a avonoid.
Glycosylationof avonoids using E. coli harboring UGTresults
in the formation of avonoid glucose because most UGTs have
specicity for dUDP-glucose, and E. coli has endogenous dUDP-
glucose (9, 13). However, a recent study by our group showed that
the nucleotide sugar specicity of UGT could be changed when
the preferred nucleotide sugar is not available (7). This suggests
that unnatural avonoid glycosides could be synthesized through
modication of the nucleotide sugar metabolic pathway in the
Received 30 January 2012 Accepted 26 March 2012
Published ahead of print 6 April 2012
Address correspondence to Joong-Hoon Ahn, jhahn@konkuk.ac.kr.
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.00275-12
4256 aem.asm.org Applied and Environmental Microbiology p. 42564262 June 2012 Volume 78 Number 12

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host and by rational selection of the appropriate UGT. Nucleotide
sugars serve as the sugar donor for UGT. The nucleotide sugar
biosynthesis pathway could be engineered to synthesize a nucleo-
tide that is not normally present inE. coli. We wantedto synthesize
dTDP-6-deoxy-L-talose. Most steps of dTDP-6-deoxy-L-talose
biosynthesis are present in the dTDP-L-rhamnose biosynthetic
pathway in E. coli. dTDP-4-dehydro-6-deoxy-L-mannose is con-
verted to dTDP-L-rhamnose by rfbD at the nal step of dTDP-L-
rhamnose biosynthesis; however, it is converted to dTDP-6-de-
oxytalose by tll (Fig. 1).
The biological activities of avonoid glycosides are more often
modulated by the sugar than by the avonoid (5, 24). Therefore,
we want to synthesize a novel avonoid glycoside, which might
have a novel biological activity. Here, we showthat by introducing
a new gene for the biosynthesis of a nucleotide sugar, and screen-
ing for a UGT that could utilize the novel nucleotide sugar, an
unnatural avonoid glycoside, quecetin 3-O-(6-deoxytalose) can
be synthesized. In addition, through engineering the nucleotide
biosynthetic pathway of E. coli, the productivity of an unnatural
avonoid glycoside can be increased.
MATERIALS AND METHODS
DNA manipulation. The tll gene was synthesized based on the sequence
published by Nakano et al. (15). An EcoRI site was added to the 5= end of
the gene, and a NotI site was added to the 3= end of the gene to facilitate
cloning. To optimize the expression of tll in E. coli, codon optimization
based on E. coli codon usage was performed prior to synthesis. The syn-
thesized tll gene was veried by DNA sequencing. To express tll in E. coli,
EcoRI/NotI-digested tll DNA was subcloned into the corresponding
EcoRI/NotI site of the E. coli expression vectors pACYCDuet, pCDFDuet,
and pETDuet. The resulting constructs were named pA-tll, pC-tll, and
pE-tll, respectively (Table 1).
UGT genes from various sources were cloned into pGEX vector as
described previously (810) and transformed into E. coli BL21(DE3),
which was already transformed with pA-tll. As control, each UGT gene
was transformed into E. coli BL21(DE3) containing empty pACYCDuet.
Overnight cultures of each transformant were inoculated in 2 ml of Luria-
Bertani (LB) medium containing 50 g of ampicillin and chlorampheni-
col/ml. Induction, biotransformation, and analysis of reaction product
were performed as described below.
AtUGT78D1 from Arabidopsis thaliana was cloned by reverse tran-
scription-PCR. Briey, total RNA was isolated from 2-week-old A. thali-
FIG 1 Schematic diagram of the nucleotide pathway and the production of
quercetin 3-O-(6-deoxytalose) in E. coli. galU, UTP-glucose 1-phosphate uri-
dyltransferase; rffA, dTDP-4-oxo-6-deoxy-D-glucose transaminase; rfbA,
dTDP-glucose pyrophosphorylase; rfbB, dTDP-glucose 4,6-dehydratase; rfbC,
(dTDP-4-dehydrorhamnose 3,5-epimerase; rfbD, dTDP-4-dehydrorahmnose
reductase; tll, dTDP-6-deoxy-L-lyxo-4-hexulose reductase; GT, glycosyltrans-
ferase.
TABLE 1 Plasmids and strains used in this study
Plasmid or strain Relevant properties or genetic marker
a
Source or reference
Plasmids
pACYCDuet P15A ori; Cm
r
Novagen
pCDFDuet CloDE13 ori; Spe
r
Novagen
pETDuet f1 ori; Amp
r
Novagen
pGEX 5X-1 pRR322 ori; Amp
r
GE Healthcare Life Science
pG-D1 pGEX 5X-2 AtUGT78D1 from A. thaliana This study
pA-D1 pACYCDuet AtUGT78D1 from A. thaliana
pA-tll pACYCDuet tll from A. actinomycetemcomitans This study
pC-tll pCDFDuet tll from A. actinomycetemcomitans This study
pE-tll pETDuet tll from A. actinomycetemcomitans This study
pA-tll-D1 pACYCDuet tll from A. actinomycetemcomitans AtUGT78D1 from A. thaliana This study
pC-tll-D1 pCDFDuet tll from A. actinomycetemcomitans AtUGT78D1 from A. thaliana This study
pE-tll-D1 pETDuet tll from A. actinomycetemcomitans AtUGT78D1 from A. thaliana This study
E. coli strains
BL21 F

ompT hsdS
B
(r
B

m
B

) gal dcm lon (DE3) Novagen

BrfbD BL21(DE3) rfbD-FRT-Kan
r
This study
BrffA BL21(DE3) rffA-FRT-Kan
r
This study
BgalU-rfbD BL21(DE3) galU::FRT rfbD-FRT-Kan
r
This study
BgalU-rfbD-rffA BL21(DE3) galU::FRT rfbD::FRT rffA-FRT-Kan
r
This study
B11 BL21(DE3) galU::FRT rfbD::FRT rffA-FRT-Kan
r
harboring pA-tll-D1 This study
B12 BL21(DE3) galU::FRT rfbD::FRT rffA-FRT-Kan
r
harboring pE-tll-D1 This study
B13 BL21(DE3) galU::FRT rfbD::FRT rffA-FRT-Kan
r
harboring pC-tll and pA-D1 This study
B14 BL21(DE3) galU::FRT rfbD::FRT rffA::FRT-Kan
r
-FRT harboring pE-tll and pA-D1 This study
a
Cm
r
, chloramphenicol resistance; Amp
r
, ampicillin resistance; Spe
r
, spectinomycin resistance; Kan
r
, kanamycin resistance.
Quercetin 3-O-(6-Deoxytalose) Production in E. coli
June 2012 Volume 78 Number 12 aem.asm.org 4257

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ana whole plants. cDNA was synthesized as described in Kim et al. (6).
PCR was carried out using Hotstart DNA polymerase (Qiagen, Valencia,
CA). Two primers5=-ATGAATTCATGACCAAATTCTCCGA-3= and
5=-CATGCGGCCGCTAAACTTTCACAATTTCGT-3=were designed
based on the published sequence (At1g30530). For the expression in E.
coli, AtUGT78D1 was subcloned into pGEX5X-1. The resulting construct
was named pG-D1. The AtUGT78D1 gene was subcloned into the second
cloning site (NdeI/EcoRV) of pA-tll, pC-tll, and pE-tll, and the resulting
constructs were named pA-tll-D1, pC-tll-D1, and pE-tll-D1, respectively
(Table 1).
Deletion of the rfbD (dTDP-4-dehydrorhamnose reductase), galU
(UTP-glucose 1-phosphate uridyltransferase), and rffA (dTDP-4-oxo-6-
deoxy-D-glucose transaminase) genes from E. coli BL21(DE3) was done
using the Quick & Easy conditional knockout kit (Gene Bridges, Heidel-
berg, Germany). The primers used were listed in Table 2. Electroporation
was performed with the Bio-Rad MicroPulser electroporation apparatus
(Bio-Rad, Hercules, CA). The galUrfbDdouble mutant (strain BgalU-
rfbD in Table 1) and the galU rfbD rffA triple mutant (strain BgalU-
rfbD-rffA in Table 1) were created by deleting the selection marker using
plasmid 780FLPe.
Molecular modeling of AtUGT78D1. Homology modeling software
PRIME incorporated into the Schrodinger modeling software suite was
used to generate a structure of the AtUGT78D1 that was homologous to
the crystallographic structure of the avonoid 3-O-glucosyltransferase
(PDB 2C1X) (17, 20). The optimal model was selected based on bond
angle stereochemistry using PROCHECK (11). UDP (uridine-5=-diphos-
phate)-glucose was taken fromthe template (2C1X) and modeled into the
corresponding binding site in the homology structure of AtUGT78D1,
and then the glucose moiety was modied to 6-deoxy-L-talose or L-rham-
nose, respectively. After renement of the loop structures, the model was
subjected to energy minimization and molecular-dynamics (MD) simu-
lations in order to obtain a stable, low-energy conformation. Energy min-
imization was performed using a conjugate gradient minimize (0.05 con-
vergence criteria), the OPLS-AA force eld, and GB/SA continuum water
model. MD simulations were performed by pre-equilibration for 100 ps
and simulation for 1 ns at 300K with a 1-fs time step and SHAKE applied
to all hydrogen bonds. With the optimized model structures, docking of
quercetin was carried out. We used the protein preparation utilities in
Maestro to assign the charge state of ionizable residues, add hydrogens,
and carry out energy minimization. The ligand quercetin was then docked
into the homology models using the GLIDE (Schrodinger) module incor-
porated in Maestro. The default setting of the extreme precision mode of
GLIDE was used for the docking, and up to 10 poses were saved for anal-
ysis.
Production of quercetin 3-O-(6-deoxytalose) in E. coli. In order to
compare the production of quercetin 3-O-(6-deoxytalose) in different E.
coli strains, both pA-tll and pG-D1 were transformed into strains BL21,
BrfbD, BrffA, BgalU-rfbD, and BgalU-rfbD-rffA (Table 1). An overnight
culture of each transformant was inoculated into LB medium containing
the appropriate antibiotics (50 g/ml). Cells were grown at 37Cuntil the
optical density at 600 nm (OD
600
) reached 0.8, and then 1 mM IPTG
(isopropyl--D-thiogalactopyranoside) was added to induce protein ex-
pression. Cells were then incubated at 18C for 18 h. The cells were har-
vested and resuspended in M9 containing 2%glucose. At this step, the cell
density was adjusted to OD
600
of 3. Quercetin (100 M) was added to the
reaction mixture, which was then incubated at 30C for 4 h. The reaction
product was extracted twice with ethyl acetate, and the organic layer was
dried by evaporation. Analysis of reaction product using high-perfor-
mance liquid chromatography (HPLC) was performed as described by
Kim et al. (7). The production of quercetin 3-O-(6-deoxytalose) was cal-
culated using quercetin 3-O-glucose as a standard molecule because quer-
cetin 3-O-(6-deoxytalose) is not commercially available.
To select the best vector combination for the production of quercetin
3-O-(6-deoxytalose), vector combinations were transformed into BgalU-
rffA-rfbD. The resulting E. coli strains (B11 to B14 [Table 1]) were in-
duced, and biotransformation was performed as described above. To de-
termine the production of quercetin 3-O-(6-deoxytalose) using E. coli
B12, the cells were induced as described above. The reaction volume was
10 ml. Initially, 100 M quercetin was added, and then 50 M was added
at 8, 12, and 36 h. Therefore, a total of 250 M(75.5 mg/liter) of quercetin
was added. The reaction product (300 l) was collected at 2, 4, 6, 8, 10, 12,
24, 36, 48, 72, and 96 h. Three independent experiments were performed.
Nuclear magnetic resonance (NMR) experiments were performed on
a Bruker Avance 400 (Karlsruhe, Germany). The fraction containing the
reaction product was collected from several HPLC runs. The collected
sample was dried by evaporation and was dissolved in 150 l of dimethyl
sulfoxide (DMSO)-d6 and transferred into 2.5-mm NMR tube. The
NMR data for quercetin-3-O-(6-deoxytalose) were as follows:
1
HNMR
(DMSO-d6, 400 MHz): 6.22 (d, J 1.9 Hz, H-6), 6.41 (d, J 1.9 Hz,
H-8), 7.31 (d, J 2.0 Hz, H-2=), 6.90 (d, J 8.3 Hz, H-5=), 7.24 (dd, J
8.3, J 2.0 Hz, H-6=), 5.39 (d, J 1.1 Hz, H-1), 3.90 (m, H-2), 3.66 (t, J
2.9 Hz, H-3), 3.32 (m, H-4), 3.45 (q, J 6.4 Hz, H-5), 0.79 (d, J 6.4
Hz, H-6), 12.62 (5-OH);
13
CNMR(DMSO-d6, 100 MHZ): 157.4 (C-2),
134.1 (C-3), 177.7 (C-4), 161.1 (C-5), 98.8 (C-6), 164.5 (C-7), 93.8 (C-8),
156.6 (C-9), 104.1 (C-10), 120.8 (C-1=), 115.9 (C-2=), 145.3 (C-3=), 148.6
(C-4=), 115.6 (C-5=), 121.0 (C-6=), 102.2 (C-1), 69.9 (C-2), 65.2 (C-3),
72.3 (C-4), 68.9 (C-5), and 16.5 (C-6). NMR data are provided in Fig.
S1 in the supplemental material.
RESULTS
Production of quercetin 3-O-(6-deoxytalose) in E. coli. To syn-
thesize a novel avonoid glycoside in E. coli, at least two things
need to be provided: a novel nucleotide sugar and UGT. To syn-
thesize a nucleotide sugar donor, dTDP-6-deoxytalose in E. coli,
the tll gene was overexpressed in the E. coli strain BL21(DE3). As a
control, empty pACYCDuet vector was also transformed. The
production of dTDP-6-deoxytalose in E. coli was examined, but
we did not detect the production of dTDP-6-deoxytalose. It seems
that the amount of dTDP-6-deoxytalose was tiny so that it could
not be detected. This has also been reported that for another en-
dogenous nucleotide sugar, dTDP-L-rhamnose, which is pro-
duced from the same substrate as dTDP-6-deoxytalose but was
TABLE 2 Primers used for gene deletions in this study
Gene Primer Sequence (5=3=)
rfbD rfbD-forward TGGCATCATGAGCGAGATGCAAAAATTTGTTAAATTGCCGTAGTCGTAAAAATTAACCCTCACTAAAGGGCG
rfbD-reverse GGTGCTTATCAGTCGTGGATTGAACAGAACTATGAGGGCCGCCAGTAATGTAATACGACTCACTATAGGGCTC
rffA rffA-forward TCCACAAGGACGCTTTTGCCAACGACATATCAGGAAAAGTAGTTCAACAATGGACAGCAAGCGAACCGGAATTGC
rffA-reverse TGGTGCGAATGTAGAAAGCACCGCGTACTGGTTATACAGGTGATCACATGTCAGAAGAACTCGTCAAGAAGGCG
glaU glaU-forward ATGGCTGCCATTAATACGAAAGTCAAAAAAGCCGTTATCCCCGTTGCGGGAATTAACCCTCACTAAAGGGCG
galU-reverse AGTCATGGCTCTTCCCTTTCATATGATAGGCTTCAACCGTTTCTTTTTCGTAATACGACTCACTATAGGGCTC
Yoon et al.
4258 aem.asm.org Applied and Environmental Microbiology

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also not detected because only a tiny amount of dTDP-L-rham-
nose is present in E. coli (14).
We also monitored production of a new avonol glycoside.
Because UGT has nucleotide sugar specicity, 50 UGTs from
Oryza sativa, Glycine max, Bacillus subtilis, and Arabidopsis thali-
ana were screened to nd a UGT that could use quercetin as a
sugar acceptor and dTDP-6-deoxytalose as a sugar donor. Each
UGT gene used had previously been subcloned into the E. coli
expression vector pGEX (810). Each UGT gene was transformed
into BL21(DE3) containing pA-tll or the pACYCDuet vector, and
the biotransformation of quercetin by each transformant was
evaluated. Among the 50 transformants harboring different
UGTs, one transformant expressing AtUGT78D1, yielded a new
peak that was not present in the transformant not expressing tll
(Fig. 2A and B). The molecular mass of the new peak was at 448.1
Da, indicating that a 164.1-Da sugar was attached to quercetin
(302 Da). Using NMR, the structure of this new peak was deter-
mined to be quercetin 3-O-(6-deoxytalose). The peak at 12.0 min
observed in Fig. 2A was quercetin 3-O-rhamnose, and the peak at
11.1 min was quercetin 3-O-glucose. That is, quercetin 3-O-
rhamnose and quercetin 3-O-glucose were produced also by co-
expression of AtUGT78D1. Ideally, the production of these two
compounds should be reduced. Therefore, we engineered the nu-
cleotide-sugar biosynthesis pathway of E. coli.
AtUGT78D1 was previously characterized as a avonol 3-O-
rhamnosyltransferase (4). Because dTDP-6-deoxytalose is not
commercially available, molecular docking of dTDP-rhamnose
or dTDP-6-deoxytalose toAtUGT78D1was performed. BothdTDP-
6-deoxytalose and quercetinwere docked to AtUGT78D1, and the
docking structure was compared to that of dTDP-L-rhamnose and
quercetin (Fig. 3). Quercetin forms a hydrogen bond with His150,
and the 3-hydroxy group of quercetin is located close to His24,
which serves as a base for deprotonation from the 3-hydroxy
group. dTDP-6-deoxytalose was tted into AtUGT78D1 by form-
ing hydrogen bonds with Asp374 and Asn375. Like dTDP-6-de-
oxytalose, dTDP-L-rhamnose formed hydrogen bonds between
the 3-hydroxy group of rhamnose and Asn354 of AtUGT78D1
and between the 4-hydroxy group of rhamnose and Asp374 of
AtUGT78D1. The key reason why AtUGT78D1 showed higher
afnity for dTDP-L-rhamnose than dTDP-6-deoxytalose is that
the methyl group ts well in the substrate-binding pocket of
AtUGTD1. A methyl group of 6-deoxytalose could be involved
in hydrophobic interactions with Ala23, Trp353, and Gly352;
however, these interactions were not effective because the dis-
tance between the methyl group and these amino acids were not
close enough. In contrast, the methyl group of rhamnose went
into the hydrophobic pocket formed by Met282, Ala373, and
Leu372. Therefore, although overall, dTDP-L-rhamnose bound to
AtUGT78D1 better than dTDP-6-deoxytalose, AtUGT78D1
could use dTDP-6-deoxytalose as a sugar donor.
Engineering E. coli to increase the production of quercetin
3-O-(6-deoxytalose). Glucose 1-phosphate is a central molecule
in the nucleotide sugar biosynthetic pathway of E. coli. Glucose
1-phosphate leads to the synthesis of dUDP-glucose and dTDP-
glucose. Eventually, dUDP-glucose is converted to dUDP-galactose
and to dUDP-L-4-deoxy-4-formamido-L-arabinose via dUDP-
glucuronic acid. In addition, dTDP-glucose is converted into
dTDP-fucosamine and dTDP-rhamnose (21).
To increase the production of quercetin 3-O-(6-deoxytalose)
through the increasing the level of substrate for the production of
dTDP-6-dexoytalose, it would be necessary to modulate the nu-
cleotide sugar biosynthesis pathway. Three genes were selected;
the rst was galU (UTP-glucose 1-phosphate uridyltransferase),
which is at the branch point between dUDP-glucose and dTDP-
FIG2 HPLCproling of reaction product fromdifferent E. coli strains. (A) BL21 harboring pG-D1; (B) BL21 harboring pG-D1 and pA-tll; (C) BrfbDharboring
pG-D1 and pA-tll; (D) BgalU-rfbD-rffA harboring pG-D1 and pA-tll. S, quercetin; P1, quercetin 3-O-glucose; P2, quercetin 3-O-rhamnose; P3, quercetin
3-O-(6-deoxytalose).
Quercetin 3-O-(6-Deoxytalose) Production in E. coli
June 2012 Volume 78 Number 12 aem.asm.org 4259

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glucose. The second was rffA (dTDP-4-oxo-6-deoxy-D-glucose
transaminase), which leads to the synthesis of dTDP-fucosamine
fromdTDP-4-dehydro-6-deoxy-D-glucose. Finally, the third gene
was rfbD (dTDP-4-dehydrorhamnose reductase), which com-
petes for dTDP-4-dehydro-6-deoxy-L-mannose with tll. Combi-
nations of these three genes were deleted (Fig. 3) and, as a result,
four mutants, BrfbD, BrffA, BgalU-rfbD, and BgalU-rfbD-rffA,
were made (Table 1). pG-D1 and pA-tll were transformed into
each mutant. The HPLC proles of the reaction products from
each transformant differed (Fig. 2). After a 4 h of incubation, BL21
produced 18 mg of quercetin 3-O-glucose, 14 mg of quercetin
3-O-rhamnose, and 3.5 mg of quercetin 3-O-(6-deoxytalose)/li-
ter. BrffA produced a higher concentration of quercetin 3-O-(6-
deoxytalose) (6.5 mg/liter) than did wild type but still produced
almost the same concentration of quercetin 3-O-glucose and
quercetin 3-O-rhamnose as BL21. In contrast, BrfbD produced a
higher concentration of quercetin 3-O-(6-deoxytalose) (12 mg/
liter). In this strain, production of quercetin 3-O-rhamnose was
signicantly reduced. However, a high amount of quercetin 3-O-
glucose was still produced. The BgalU-rfbD double mutant pro-
duced 5-fold higher levels of quercetin 3-O-(6-deoxytalose) (17
mg/liter) than the wild-type BL21. In addition, the production of
quercetin 3-O-glucose and quercetin 3-O-rhamnose was reduced
in this mutant. More specically, only a negligible amount of
quercetin 3-O-glucose was detected, which seemed to result from
deletion of galU. The triple mutant, BgalU-rfbD-rffA, produced
the highest amount of quercetin 3-O-(6-deoxytalose) (23 mg/li-
ter), and the smallest amount of by-products among the tested
strains. The production of quercetin 3-O-(6-deoxytalose) pro-
duction in this strain was 6.5-fold higher than that in wild-type
BL21. Taken together, BgalU-rffA-rfbDnot only produced higher
concentration of quercetin 3-O-(6-deoxytalose) but also pro-
duced fewer by-products such as quercetin 3-O-glucose and quer-
cetin 3-O-rhamnose. The relative amount of reaction products
produced by the different E. coli strains is summarized in Fig. 4.
Production of quercetin 3-O-(6-deoxytalose). The produc-
tion of quercetin 3-O-(6-deoxytalose) was optimized. Different
copies and combinations of E. coli cloning vectors were tested to
determine which plasmid was best for the production of quercetin
3-O-(6-deoxytalose). The tll and AtUGT78D1 genes were both
cloned into pACYCDuet, and pETDuet, which have different
copy numbers in E. coli. The resulting constructs, pA-tll-D1 and
pE-tll-D1, were transformed into BgalU-rfbD-rffA to make
strains B11 and B12, respectively. tll was subcloned into plasmids
having higher copy number in E. coli vectors pCDFDuet and
pETDuet than the plasmid expressing AtUGT78D1, which was
pACYCDuet. Both plasmids containing tll and AtUGT78D1 were
transformed into BgalU-rfbD-rffA to create strains B13 and B14.
Using these four strains, production of quercetin 3-O-(6-deoxy-
talose) was examined. B12 produced 70 mg of quercetin 3-O-
(6-deoxytalose)/liter, B11 produced 35 mg/liter, B13 produced 13
mg/liter, and B14 produced 7 mg/liter (Fig. 5). The copy number
of construct was not related with the productivity of quercetin
3-O-(6-deoxytalose). It seemed that the highest copy number
gave a higher metabolic load to E. coli. On the other hand, a lower
copy number of the construct did not produce enough proteins
to convert quercetin into quercetin 3-O-(6-deoxytalose).
Therefore, the best construct was selected to maximize the pro-
duction of quercetin 3-O-(6-deoxytalose). Using strain B12,
the production of quercetin 3-O-(6-deoxytalose) was moni-
tored. A nal concentration of 250 M quercetin (75.5 mg/
liter) was added. Conversion of quercetin into quercetin 3-O-
(6-deoxytalose) dramatically increased until 24 h. After 96 h,
98 mg of quercetin 3-O-(6-deoxytalose)/liter was produced
(Fig. 6).
FIG 3 Docking of AtUGT78D1 with dTDP-L-rhamnose (A) or dTDP-6-deoxytalose (B).
FIG 4 Relative production of three quercetin 3-O-glycoses in different mu-
tant strains harboring pG-D1 andpA-tll. Three independent experiments were
carried out. Error bars represent the standard deviation.
Yoon et al.
4260 aem.asm.org Applied and Environmental Microbiology

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DISCUSSION
By engineering the nucleotide biosynthetic pathway in E. coli, the
production of quercetin 3-O-(6-deoxytalose) in BgalU-rfbD-rffA
increased 7-fold compared to the wild-type strain. In addition,
the production of the by-products was also signicantly reduced.
This demonstrates that it is possible to increase the levels of a
particular reaction product by metabolic engineering of the nu-
cleotide sugar pathway. We also optimized the plasmid vectors to
maximize the production of quercetin 3-O-(6-deoxytalose). The
production of 3-O-(6-deoxytalose) differed remarkably (10-
fold) depending on the vectors used. After selection of optimal
combination of plasmid vector and E. coli strain, 98 mg of 3-O-
(6-deoxytalose)/liter was synthesized.
The biological activity of quercetin 3-O-(6-deoxytalose) re-
mains elusive. However, different quercetin 3-O-glycoses display
different biological activities. For example, quercetin 3-O-glucose
inhibits adipocyte differentiation (12), which might control obe-
sity, whereas quercetin 3-O-galactose contains neuroprotective
effects (28). Therefore, the quercetin 3-O-(6-deoxytalose) synthe-
sized here might contain a novel biological activity.
UGTs from A. thaliana, G. max, O sativa, and B. cereus were
used to nd a UGT able to use dTDP-6-deoxytalose as sugar do-
nor. Of the 50 UGTs tested, only one UGT, AtUGT78D1 was able
to use dTDP-6-deoxytalose. Most UGTs use UDP-glucose as a
sugar donor, and a few use other nucleotide sugars. UGTs from
plants are known to generally have a narrow sugar-donor range
(18). Changing the sugar selectivity of UGTs is not easy. Although
the molecular structures of several plant UGTs have been deter-
mined, there has been no report of the domain or motif critical for
recognizing the sugar donor selectivity. In silico screening of UGTs
might be alternative approach to nd an appropriate UGT. How-
ever, the number of UGTs available for in silico screening is lim-
ited. An alternate approach, site-directed mutagenesis is also
time-consuming.
In vitro and in vivo functional study of AtUGT78D1 showed
that it uses dUDP-rhamnose as a sugar donor and quercetin as a
sugar acceptor. Our results also show that AtUGT78D1 can also
use dTDP-6-deoxytalose, indicating that this enzyme is promis-
cuous. dTDP-6-deoxytalose is not present in plant. Promiscuous
enzyme activities are considered an adventitious activity without a
physiological role (1). Therefore, although plants do not utilize
dTDP-6-deoxytalose, this adventitious activity canbe usedto pro-
duce novel compounds in a heterologous expression system.
Talosin A from Kitasatospora kifunensis is genistein 7-O-(6-
deoxytalose) (27). Although talosin A displays antifungal activity,
its production is low(3.2 mg/liter after 7 days). The E. coli strain
we created in the present study could be a host for the production
of talosin A. However, the E. coli B12 strain does not produce
talosin A after biotransformation of genistein. Therefore, screen-
ing for a UGT that recognizes genistein as well as TDP-6-deoxyta-
lose is needed. Ultimately, the BgalU-rffA-rfbD string harboring
tll developed here could serve as a host to attach 6-deoxytalose to
other small molecules.
We tried to compare the nucleotide sugar proles in the wild
FIG5 Production of quercetin 3-O-(6-deoxytalose) by BgalU-rfbD-rffA har-
boring different plasmid combinations. Three independent experiments were
carried out. Error bars represent the standard deviation.
FIG6 Production of quercetin 3-O-(6-deoxytalose) by biotransformation using E. coli B12. Initially, 100 M quercetin was added, and then 50 M was added
at 8, 12, and 36 h. Therefore, the total concentration of quercetin was 250 M (75.5 mg/liter) of quercetin. Reaction product samples (500 l) were collected at
2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 h. Three independent experiments were carried out. Error bars represent the standard deviation.
Quercetin 3-O-(6-Deoxytalose) Production in E. coli
June 2012 Volume 78 Number 12 aem.asm.org 4261

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type as well as in the mutant strains; however, we could not detect
any differences. Nucleotide sugars, such as UDP-glucose, would
be in a static state because they serve as the substrates for biosyn-
thesis of various other nucleotide sugars. Therefore, the quanti-
cationof the nucleotide sugars ineachcell is difcult. Althoughwe
could not detect nucleotide sugar difference in E. coli strains, it is
likely that the pool of each nucleotide sugar signicantly changes
in each E. coli mutant since the prole of quercetin reaction prod-
ucts in each mutant was different from one another (Fig. 1). Al-
though UGTs from plants show sugar donor specicity, it is ap-
parent that UGTs can utilize other nucleotide sugars when the
appropriate proper sugar donor is not available (7).
It was assumed that the rfbDmutant did not produce quercetin
3-O-rhamnose. However, a small amount of quercetin3-O-rham-
nose was still produced in strain BrfbD. Biotransformation of
quercetin using BrfbD harboring only pG-D1 did not produce
quercetin 3-O-rhamnose. Therefore, it is likely that using dTDP-
4-dehydro-6-deoxy-L-mannose as a substrate, tll produced
dTDP-6-deoxytalose as a major product and dTDP-L-rhamnose
as a minor product. The resulting nucleotide sugars served as
sugar donors for AtUGT78D1 in production of quercetin 3-O-(6-
deoxytalose) and quercetin 3-O-rhamnose.
ACKNOWLEDGMENTS
This study was supported by a grant from the Next-Generation BioGreen
21 Program (SSAC, grant PJ007975), Rural Development Administra-
tion, Republic of Korea, andalso partially by the Priority ResearchCenters
Program through the National Research Foundation of Korea funded by
the Ministry of Education, Science, and Technology (2009-0093824).
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