Leloir glycosyltransferases and natural product glycosylation: Biocatalytic synthesis of the C-glucoside nothofagin, a major antioxidant of redbush herbal tea

Article abstract of DOI:10.1002/adsc.201300251

Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid-like C-glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin based on a one-pot coupled glycosyltransferasecatalyzed transformation that involves perfectly selective 3′-C-β-D-glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. C-Glucosyltransferase from Oryza sativa (rice) was used for phloretin C-glucosylation from uridine 5'-diphosphate (UDP)-glucose, which was supplied continuously in situ through conversion of sucrose and UDP catalyzed by sucrose synthase from Glycine max (soybean). In an evaluation of thermodynamic, kinetic, and stability parameters of the coupled enzymatic reactions, poor water solubility of the phloretin acceptor substrate was revealed as a major bottleneck of conversion efficiency. Using periodic feed of phloretin controlled by reaction progress, nothofagin concentrations (45 mM; 20 gL^-1) were obtained that vastly exceed the phloretin solubility limit (5-10 mM). The intermediate UDP-glucose was produced from catalytic amounts of UDP (1.0 mM) and was thus recycled 45 times in the process. Benchmarked against comparable glycosyltransferase-catalyzed transformations (e.g., on quercetin), the synthesis of nothofagin has achieved intensification in glycosidic product formation by up to three orders of magnitude (μM → mM range). It thus makes a strong case for the application of Leloir glycosyltransferases in biocatalytic syntheses of glycosylated natural products as fine chemicals.

Full text of DOI:10.1002/adsc.201300251

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DOI: 10.1002/adsc.201300251
Leloir Glycosyltransferases and Natural Product Glycosylation:
Biocatalytic Synthesis of the C-Glucoside Nothofagin, a Major
Antioxidant of Redbush Herbal Tea
+
a
+a
a,
Linda Bungaruang, Alexander Gutmann, and Bernd Nidetzky *
a
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010 Graz,
Austria
Fax : (+43)-316-873-8434; phone:ACTHUNGTRENNNUG
+
Received: April 2, 2013; Revised: July 6, 2013; Published online: August 20, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201300251.
ꢀ
2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article
under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use
and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no
modifications or adaptations are made.
Abstract: Nothofagin is a major antioxidant of red-
bush herbal tea and represents a class of bioactive
flavonoid-like C-glycosidic natural products. We de-
veloped an efficient enzymatic synthesis of nothofa-
gin based on a one-pot coupled glycosyltransferase-
catalyzed transformation that involves perfectly se-
lective 3’-C-b-d-glucosylation of naturally abundant
phloretin and applies sucrose as expedient glucosyl
donor. C-Glucosyltransferase from Oryza sativa
catalytic syntheses of glycosylated natural products
as fine chemicals.
Keywords: carbohydrates; C-glycosides; glycosyl-
transferases; natural products; UDP-glucose recy-
cling
Many bioactive natural products contain sugar mole-
[
1]
(
rice) was used for phloretin C-glucosylation from
cule(s) as part of their structure. Their physiological
activity, selectivity and pharmacological properties
uridine 5’-diphosphate (UDP)-glucose, which was
supplied continuously in situ through conversion of
sucrose and UDP catalyzed by sucrose synthase
from Glycine max (soybean). In an evaluation of
thermodynamic, kinetic, and stability parameters of
the coupled enzymatic reactions, poor water solubil-
ity of the phloretin acceptor substrate was revealed
as a major bottleneck of conversion efficiency.
Using periodic feed of phloretin controlled by reac-
tion progress, nothofagin concentrations (45 mM;
[2]
are often derived from the sugar component(s).
Therefore, glycosylation is often central to a natural
productꢁs efficacy in the particular application consid-
[
3]
ered. Aside from therapeutic uses, glycosylated nat-
ural products have raised interest as functional food
[
4]
additives and cosmetic ingredients. Glycosylation
pattern engineering is regarded as a highly promising
way of functional diversification of natural pro-
[
2b,3]
ducts.
This might contribute to the creation of new
ꢀ
1
20 gL ) were obtained that vastly exceed the
bioactive substances and drug leads.
phloretin solubility limit (5–10 mM). The intermedi-
ate UDP-glucose was produced from catalytic
amounts of UDP (1.0 mM) and was thus recycled
In nature, the selective modification of target com-
pounds with sugars is catalyzed by glycosyltransfer-
AHCTUNGTRENNUNGa ses (EC 2.4). These enzymes use an activated
[
5]
4
5 times in the process. Benchmarked against com-
donor substrate, typically a nucleoside diphosphate
(NDP)-sugar, for transfer of the glycosyl residue onto
a specific position of an acceptor molecule. Glycosyl-
transferases display splendid regioselectivity and ste-
reochemical control in the transformations cata-
parable glycosyltransferase-catalyzed transforma-
tions (e.g., on quercetin), the synthesis of nothofa-
gin has achieved intensification in glycosidic prod-
uct formation by up to three orders of magnitude
[
6]
(
mM!mM range). It thus makes a strong case for
lyzed, and they are therefore widely recognized as
[7]
the application of Leloir glycosyltransferases in bio-
highly valuable glycosylation catalysts. However,
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2757

Linda Bungaruang et al.
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Scheme 1. Synthesis of nothofagin is achieved by enzymatic C-glucosylation of phloretin from sucrose via UDP-glucose.
OsCGT and GmSuSy are telescoped in one pot, and the reaction proceeds in the presence of catalytic amounts of UDP.
Fructose is the only by-product formed. The overall equilibrium lies far on the C-glycoside product side, driven by the reac-
tion of OsCGT and the presence of sucrose in excess.
synthetic applications of glycosyltransferases have so reacts with uridine 5’-diphosphate (UDP)-glucose to
far been quite restricted due to complexities of the give
the
3’-C-aryl
b-d-glucoside
nothofagin
enzymes (e.g., low specific enzyme activity and stabil- (Scheme 1) as a single transfer product. Nothofagin is
[8]
ity) and the supply of donor and acceptor substrates a natural substance found in redbush herbal tea and
[
9]
for the enzymatic reactions.
represents a structural class of bioactive aryl C-glyco-
[
16]
The majority of natural product glycosylations in- sides. For efficient nothofagin synthesis, we coupled
volve O-glycosidic bonds. Glycosylations at carbon, the C-glucosyltransferase reaction to enzymatic in situ
by contrast, are relatively rare and to date only supply of the glucosyl donor substrate (Scheme 1):
a small number of natural C-glycosyltransferases have UDP-glucose is produced from sucrose and UDP
[10]
been reported.
The C-glycosidic linkage displays using recombinant sucrose synthase from soybean
outstanding resistance to chemical or enzyme-cata- (Glycine max; GmSuSy). While the applied internal
lyzed hydrolysis, surpassing that of the corresponding UDP-glucose regeneration is known in principle and
[
11]
O-glycosidic linkage by a large amount.
C-glyco- has been applied to enzymatic reactions involving dif-
[17]
sides have therefore attracted considerable attention ferent glycosyl donor substrates, a critical test of its
for functional substitution of physiologically active O- performance capability in the synthesis of natural
[
12]
[17e–h]
glycosidic compounds having low in vivo lifetimes.
product glucosides such as nothofagin
remains
Aryl glucosides derived from flavonoid-like agly- outstanding. We show the application of comprehen-
cones (Scheme 1) present a very interesting class of sive step-by-step reaction engineering to overcome
plant natural products that may involve either a C- or complexities inherent to this and similarly coupled
[
13]
an O-glycosidic linkage. They show a highly signifi- glycosyltransferase systems and report an efficient,
cant profile of biological activities that typically in- high-yielding biocatalytic production of nothofagin.
clude strong antioxidant and radical scavenger func-
We first examined reactions of OsCGT and
tions, but also comprise antiviral and cytotoxic ef- GmSuSy separately and determined their kinetic and
[14]
fects.
Because product isolation directly from the thermodynamic characteristics. Enzymes were ob-
plant is often impractical, compounds must also be tained from Escherichia coli expression cultures and
prepared by bottom-up synthesis. Despite notable purified to apparent homogeneity by Strep-tag affini-
[
15]
recent advancements,
chemical methodologies in- ty chromatography, as described in the Supporting In-
volve multiple steps and are therefore generally nei- formation (Methods, Figure S1). Their activities were
ther atom-efficient nor high-yielding. We demonstrate determined using enzymatic or HPLC-based assays
in this study that single-step glycosyltransferase-cata- (Supporting Information, Methods). The reaction of
lyzed transformation in vitro presents a powerful tool OsCGT was monitored with an HPLC assay capable
for aryl C-glycoside synthesis. We show that when of- of distinguishing between nothofagin and potential al-
fered the dihydrochalcone phloretin as acceptor, C- ternative products resulting from O-glucosyl transfer
glycosyltransferase from rice (Oryza sativa; OsCGT) at the 2’ or 4’ position of the acceptor (Supporting In-
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Adv. Synth. Catal. 2013, 355, 2757 – 2763

Leloir Glycosyltransferases and Natural Product Glycosylation
Table 1. Characterization of glycosyltransferases and their
[a]
reactions.
Parameter
GmSuSy
Cleavage
OsCGT
Synthesis
[
b]
KM sucrose [mM]
KM UDP [mM]
KM fructose [mM]
–
–
25.5ꢃ3.3
–
–
[b]
0.13ꢃ0.02
[b]
3.0ꢃ0.4
–
–
[b]
K
0.14ꢃ0.03
0.024ꢃ0.004
M UDPꢀglucose
[mM]
K
[mM]
–
–
0.009ꢃ0.003
4.4ꢃ0.3
M phloretin
ꢀ1
kcat [s ]
Spec. act.
7.5ꢃ0.4
9.3ꢃ0.3
4.8ꢃ0.2
5.9ꢃ0.2
5.1ꢃ0.3
ꢀ
1
[Umg ]
[
c,e]
[d,e]
Keq
t1/2 [h]
0.49ꢃ0.01
>400
[
f]
[g]
18.8ꢃ0.9
13.8ꢃ1.2
[a]
[b]
[c]
[d]
3
3
08C, 50 mM HEPES pH 7.5, 20% (v/v) DMSO.
08C, 20 mM HEPES, pH 7.5.
Cleavage direction (conversion of sucrose and UDP).
Glycosylation direction (conversion of phloretin and
UDP-glucose).
Figure 1. Time course analysis for individual enzymatic reac-
tions catalyzed by OsCGT and GmSuSy at pH 7.5 and 308C.
Nothofagin synthesis by OsCGT (black symbols):
ꢀ
1
8
4
0 mUmL , 5 mM phloretin (triangle, dashed line),
.75 mM UDP-glucose (circle, solid line), nothofagin
[
[
e]
f]
Calculated from data in Figure 1.
08C, 50 mM HEPES pH 7.5, 20% (v/v) DMSO, 100 mM
3
(
5
square, solid line). Reactions of GmSuSy (grey symbols):
0 mUmL , 5 mM of each substrate, UDP-glucose in su-
ꢀ
1
sucrose.
[g]
3
08C, 50 mM HEPES pH 7.5, 20% (v/v) DMSO, 5 mM
crose synthesis (squares, solid line) and cleavage (triangles,
dashed line).
phloretin.
[
18]
formation, Figure S2).
OsCGT displayed absolute ic cosolvent was essential to enhance the acceptor
selectivity (within an error limit of ꢁ0.5%) for 3’-C- substrate availability in C-glycosylations catalyzed by
glycosylation of phloretin. We determined pH-activity OsCGT. Whereas both ethanol and DMSO up to 20%
dependencies for GmSuSy (sucrose cleavage and syn- by volume caused only minor interference with
thesis) and OsCGT (nothofagin synthesis) at 308C. OsCGT activity, GmSuSy displayed a low cosolvent
The resulting pH profiles revealed suitable overlap of tolerance and its activity was almost completely (ꢂ
the enzyme activities in the pH range 6.5–8.0 (Sup- 85%) lost in the presence of 15% ethanol. DMSO was
porting Information, Figure S3). The results shown in less strongly affecting the activity of GmSuSy and
Figure 1 indicate that C-glucosylation of phloretin at around 65% of the specific enzyme activity in purely
pH 7.5 resulted in high conversion of substrates (ꢂ aqueous buffer were retained in 20% DMSO. Further-
95%). Moreover, the enzymatic reverse reaction with more, OsCGT stability and phloretin solubility were
nothofagin and UDP was not detectable under these superior in DMSO as compared to ethanol. All conver-
conditions (Supporting Information, Figure S4). We sion experiments were therefore performed in 20%
concluded, therefore, that synthesis of nothofagin by DMSO, and the maximum concentration of dissolved
OsCGT proceeds without critical thermodynamic lim- phloretin was around 10 mM under these conditions.
itations. The equilibrium for sucrose conversion is
Kinetic characterization of GmSuSy (sucrose con-
pH-dependent, and a low pH of 6 or smaller is known version) and OsCGT (nothofagin synthesis) was done
[
19]
to favor the formation of UDP-glucose.
Figure 1 at pH 7.5 and results are summarized in Table 1 along
shows that at pH 7.5, the equilibrium constant (Keq) with the relevant enzyme stability and reaction ther-
for conversion of sucrose and UDP had a value of modynamic parameters under these conditions. Both
0
.49. However, thermodynamic constraints on the enzymes showed useful specific activities (ꢂ
ꢀ1
supply of UDP-glucose at elevated pH can be elimi- 5 unitsmg
protein) and were sufficiently stable
nated effectively using sucrose in excess. We therefore under the reaction conditions with half-lives of
performed our conversion studies at pH 7.5 and 308C around 19 (GmSuSy) and 14 h (OsCGT). The Mi-
where both glycosyltransferases showed useful activity chaelis–Menten constant (K ) of GmSuSy for sucrose
M
and stability (Table 1) and quantitative transformation exceeds the corresponding K for UDP by two orders
M
of sucrose into nothofagin was feasible.
of magnitude. KM values of OsCGT are also much
The low water solubility of non-carbohydrate ac- lower than the sucrose K . Therefore, this implies
M
ceptor substrates is an important issue for carrying that relatively high sucrose concentrations should be
out natural product glycosylations in vitro. In the case used in the coupled reaction to partly saturate and
of the barely water-soluble phloretin, use of an organ- thus make optimum use of the GmSuSy activity pres-
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Linda Bungaruang et al.
COMMUNICATIONS
ent. The kinetic requirements of SuSy are therefore in creasing the UDP concentration relative to the phlor-
good accordance with the notion of using an excess of etin concentration, but this would probably have to
sucrose to drive the overall conversion. We noticed occur at the expense of a significant loss in r . It is in-
P
that the KM for UDP was 26-fold higher in our re- teresting that at the lowest UDP concentration used
combinant preparation of GmSuSy as compared to in Figure 2 (5 mM), the observed r was still 10% of
P
[
20]
the enzyme isolated from the native source.
This its maximum value at saturation with UDP.
Aside from cost-efficient supply of the UDP-glu-
large difference in apparent UDP binding affinity
might be due to effects of post-translational modifica- cose donor substrate and favorable thermodynamic
tion (e.g., covalent phosphorylation) that have been effects resulting from the use of high sucrose concen-
[
21]
described for sucrose synthases in plants and that trations, a glucosyltransferase reaction might benefit
may not occur in E. coli. The KM for UDP-glucose from its coupling to the SuSy reaction kinetically. Pro-
was also strongly elevated (12-fold) in recombinant as nounced end-product inhibition by micromolar con-
compared to native GmSuSy while, interestingly, the centrations of UDP is quite common among flavonoid
[
17e,g,h]
KM values for sucrose and fructose were not affected O-glucosyltransferases
and imposes severe re-
in the recombinant enzyme. Differences between re- strictions on the direct synthetic use of these enzymes
combinant and native GmSuSy were not further pur- which could be decreased by continuous removal of
[
17e,22]
sued in this study.
the UDP released.
We tested the influence of
We performed synthesis experiments in which the UDP inhibition on nothofagin production by compar-
ꢀ
1
initial concentration of sucrose (5–500 mM; 0.5 mM ing OsCGT (50 mUmL ) conversion of 5 mM phlor-
UDP) or UDP (0.005–1 mM; 100 mM sucrose) was etin (6 mM UDP-glucose, 100 mM sucrose) in ab-
ꢀ
1
ꢀ1
varied while enzymatic activity (10 mUmL OsCGT/ sence and presence of GmSuSy (50 mUmL ) (Fig-
GmSuSy) and phloretin concentration (5 mM) were ure 3A). Although in both reactions quantitative con-
constant. The nothofagin production rate (r ) was version (>99.5%) was achieved r showed a stronger
P
P
measured, and results are depicted in Figure 2. De- decrease above ~75% conversion (2 h) without
pendence of r on the sucrose concentration was hy- GmSuSy and final conversions (>99%) were only
P
perbolic, with a half-saturation constant (27 mM) reached after more than 10 h compared to less than
comparable to the K of GmSuSy for sucrose. There- 6 h in the presence of GmSuSy. This corresponds to
M
fore, it appears to primarily reflect saturation behav- an approximately two-fold gain in space-time yield in
ior of GmSuSy, as noted above (Table 1). The de- nothofagin production (mM product formed/time con-
pendence of r on the UDP concentration was like- sumed) resulting from the coupling of OsCGT and
P
wise hyperbolic, with a half-saturation level (51 mM) GmSuSy reactions. UDP-glucose depletion could be
in between the K values for UDP and UDP-glucose. excluded as an explanation for the reduction of r in
M
P
Figure 2B) indicates that the applied “nothofagin syn- the absence of GmSuSy due to excess of UDP-glu-
thase” activity, derived from the combined activities
of OsCGT and GmSuSy, was therefore utilized best at
a UDP concentration of 0.5 mM or higher. Using
a phloretin concentration of 5 mM, this limited the
maximum number of UDP-glucose regeneration
cycles (RCmax) to 10 (=5/0.5). It would certainly be
possible to further increase this RCmax value by de-
Figure 3. Conversions of 5 mM phloretin (square) to notho-
fagin (triangle) by OsCGT (100 mM sucrose): A) Using
ꢀ
1
Figure 2. The nothofagin production rate (r ) in a coupled-
50 mUml OsCGT and 6 mM UDP-glucose in the absence
p
ꢀ
1
ꢀ1
enzyme reaction (10 mUmL
OsCGT/GmSuSy, 5 mM
(grey) and presence (black) of 50 mUmL GmSuSy; B)
phloretin) depends on variation of A) the sucrose concen-
tration (0.5 mM UDP) and B) the UDP (100 mM sucrose)
concentration. Note: because sucrose was not fully saturat-
variation of OsCGT and GmSuSy activity in coupled conver-
ꢀ
1
sions (0.5 mM UDP): 50 mUmL OsCGT/GmSuSy (dark
ꢀ
1
ꢀ1
ꢀ1
grey); 50 mUmL
OsCGT and 250 mUmL
GmSuSy
ꢀ1
ing in B) the achieved r at high UDP is slightly lower than
(black); 250 mUmL OsCGT and 50 mUmL GmSuSy
p
in A).
(light grey, black edge).
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Leloir Glycosyltransferases and Natural Product Glycosylation
curred in the enzymatic reaction (100 mM sucrose,
ꢀ1
ꢀ1
0
.5 mM UDP, 190 mUmL OsCGT, 120 mUmL
GmSuSy). Figure 4 shows that nothofagin production
could not be upheld under conditions of insoluble ac-
ceptor being present (ꢂ10 mM) and also the final
product concentration after 24 h was strongly de-
creased in clear dependence on the phloretin concen-
tration. Using 10 mM phloretin, which was initially
dissolved completely, we noticed precipitation of the
acceptor substrate over time, limiting the maximum
amount of nothofagin obtainable in the reaction
under these conditions despite complete conversion
of all soluble phloretin (Figure 4B). Furthermore, ini-
tial rate studies of OsCGT revealed substrate inhibi-
Figure 4. Batch conversions at different phloretin concentra-
ꢀ
1
tions (100 mM sucrose, 0.5 mM UDP, 190 mUmL OsCGT,
tion (K ~5 mM) which clearly effected conversion at
i
ꢀ
1
1
20 mUmL GmSuSy); A) Nothofagin production rate de-
phloretin concentrations above 1 mM.
creases at elevated phloretin concentrations; B) final con-
version of soluble (black) and total applied (grey) phloretin
after 24 h is limited at high phloretin concentrations by sub-
strate inhibition and solubility.
To nevertheless increase the end concentration of
nothofagin in the enzymatic reaction, we changed the
operation mode from batch to fed-batch, adding fresh
phloretin to a concentration of 5 or 10 mM once the
cose (1 mM, 40-fold K ) which was monitored acceptor substrate had been depleted. Up to 8 rounds
M
throughout the conversion. Although end product in- of phloretin addition were made, using a highly con-
hibition at low millimolar UDP concentrations was centrated stock solution of 500 mM phloretin in pure
less critical for OsCGT than for O-glycosyltransferas- DMSO to minimize the resulting volume change.
es, in situ removal of UDP remains an essential fea- Table 2 presents a summary of conditions and results
ture for general application of SuSy as UDP-glucose and Figure 5 shows a reaction time course where in
recycling system for high level glycoside production.
each round fresh enzyme was supplied together with
Conversion rates were slightly lower when 5 mM 5 mM acceptor to the reaction. Feeding the phloretin
UDP-glucose were replaced with 0.5 mM UDP in acceptor was generally quite effective in enhancing
a
coupled
glycosyltransferase
conversion nothofagin production. However, the phloretin con-
ꢀ
1
(
50 mUmL OsCGT/GmSuSy) (Figure 3B; Support- version rate decreased strongly in dependence on the
ing Information, Figure S5) and complete conversion total amount of acceptor added to the reaction, so
was only reached after 10 h. Also a 5-fold excess of that without enzyme feed, the maximum concentra-
ꢀ
1
ꢀ1
GmSuSy (250 mUmL ) over OsCGT (50 mUmL ) tion of nothofagin was just around 20 mM (Table 2).
did not improve nothofagin production significantly.
On the other hand a five-fold excess of OsCGT Table 2. Nothofagin synthesis using controlled feed of phlor-
ꢀ
1
ꢀ1
[a]
(
250 mUmL ) over GmSuSy (50 mUmL ) drastical- etin and enzyme.
ly increased the conversion resulting in complete con-
version after only 4 h. Thereby C-glucosylation was
[b]
Dcphloretin [mM]
5
5
10
10
identified as a rate-limiting step at the applied condi- Enzyme feed
no
20
100
27
14.6
88
yes
45
550
135
44.1
98
no
40
100
42
19.7
63
yes
60
600
120
46.6
[c]
tions. UDP-glucose levels of roughly 0.1 mM through-
c
phloretin [mM]
[c]
ꢀ1
out all three conversions coincide with the finding Vol. act [mUmL ]
t [h]
cnothofagin [mM]
Conversion [%]
Precipitation [mM]
that the UDP-glucose supply was not critical in the
[d]
coupled conversions. Furthermore, it is worth remark-
ing that the produced nothofagin was stable in all
conversions, suggesting that the C-glucoside synthesis
is conveniently performed under thermodynamic con-
trol and in the apparent absence of chemical or
enzyme-catalyzed side reactions.
Considering that solubility of phloretin was mark-
edly enhanced (ꢂ5-fold) upon its C-glycosylation, we
raised the initial concentration of phloretin in various
steps to 30 mM, thereby exceeding the solubility limit
of the acceptor substrate by at least 3-fold. We figured
that insoluble phloretin might still be useful for the
continuous in situ supply of acceptor substrate when
gradual transformation of the dissolved phloretin oc-
[e]
90
8.1
[f]
3.5
<0.1 8.9
[g]
3
ttn (GmSuSy/OsCGT) [1·10 ] 18/16 10/9
25/21 10/8
[a]
300 mM sucrose, 1 mM UDP, 308C, 50 mM HEPES
pH 7.5, 20% DMSO.
[
[
[
[
b]
c]
d]
e]
Amount of phloretin added per feeding.
Total amount of phloretin/enzyme activity added.
Final nothofagin concentration in solution.
Based on final nothofagin and phloretin concentrations
in solution.
[f]
Difference total fed phloretin and final soluble nothofa-
gin and phloretin.
g]
[
Total turnover number (mM nothofagin/mM total
enzyme added).
Adv. Synth. Catal. 2013, 355, 2757 – 2763
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Linda Bungaruang et al.
COMMUNICATIONS
exceptions in the field of complex oligosaccharides
[
17a,d,22]
notwithstanding,
glycosyltransferase transforma-
tions have been realized almost exclusively at the ana-
[
23]
lytical or minute preparative scale.
Glycosylations
of similar poorly water-soluble natural product core
structures such as the flavonoid quercetin have fur-
nished hardly more than micromolar concentrations
[
17g,h,24]
of the desired glycosidic compound.
On com-
parison with the literature, therefore, the herein de-
scribed enzymatic process of glucosylation of phlore-
tin from sucrose stands out due to intensification, by
up to three orders of magnitude, in terms of the prod-
uct concentration that it has achieved for a coupled
glycosyltransferase-catalyzed conversion. Further-
Figure 5. Controlled feeding of phloretin is useful to en- more, with the notable exception of the recently re-
hance the nothofagin concentration in the biocatalytic syn- ported application of OsCGT for the production of
ꢀ
1
thesis. Reaction conditions: 100 mUmL OsCGT/GmSuSy,
mM phloretin, 300 mM sucrose, 1 mM UDP. After
acceptor substrate depletion, 5 mM phloretin and
nothofagin (~200 mM) and 2-hydroxynaringenin C-
5
glucoside using engineered S. cerevisiae strains for
[23c]
whole cell conversions
this is the first synthetic use
ꢀ
1
5
0 mUmL OsCGT/GmSuSy were added. Symbols: phlore-
tin added (black dashed), OsCGT/GmSuSy added (grey
dashed), nothofagin (black), phloretin (grey)
of a C-glucosyltransferase. The here reported notho-
fagin process features efficient assembly of isolated
C-glucosyltransferase in one pot with an adaptable
Co-addition of enzyme alleviated restrictions on the module for UDP-glucose supply from sucrose, which
product concentration (substrate conversion), which serves as a highly expedient glucosyl donor for the
probably resulted from the combined effect of true overall conversion. We show that systematic analysis
enzyme activity loss and product inhibition. GmSuSy of thermodynamic conditions, kinetic properties of
is inhibited by d-fructose with a reported K of the glycosyltransferases and their stabilities is the key
i
[20]
9
mM. This could also explain why lower total turn- for identifying and thus eliminating critical constraints
over numbers (ttn) were obtained with enzyme feed on the multi-component reaction system. Integration
~10,000) than without (~20,000). We also noticed of biochemical optimization with reaction engineering
(
the requirement to carefully control the phloretin was essential to overcome the restriction of acceptor
feed to keep the acceptor concentration well below substrate solubility. The number of UDP-glucose re-
its solubility limit during the reaction. Figure S6 (Sup- generation cycles was brought into a range (around
porting Information) depicts in situ precipitation of 50) where one begins to truly capitalize on the cou-
phloretin under conditions where the acceptor feeding pling with the SuSy reaction. Reported RC
values
max
[
17e 17g,17h]
rate was not matched to the enzymatic consumption in literature are by far too small
rate. Phloretin precipitation was clearly reflected in zymatic recycling of UDP-glucose. However, costs of
the mass balance in solution (Table 2). the donor substrate are expected to prohibit the
to justify en-
Applying a suitable stepwise feeding of phloretin (5 direct use of UDP-glucose for synthesis. Considering
or 10 mM) that included supplementation with fresh that unprocessed redbush tea contains nothofagin to
ꢀ
1
[25]
enzyme, it was possible to accumulate nothofagin in just about 4.31 gkg freeze-dried matter, the high-
a concentration of around 45 mM, equivalent to yielding enzymatic synthesis developed herein is ex-
2
ꢀ
1
0 gL (Figure 5, Table 2) which corresponded to pected to remove compound availability as a critical
RCmax values of approximately 45. It has to be noted bottleneck of the various medical and food-related
[
14b,16,26]
that only with addition of enzyme and a low acceptor applications of this aryl-C-glucoside.
This study
feed (5 mm) (Figure 5) could precipitation be avoided therefore makes a strong and so far missing case for
and almost quantitative conversion (~98%) was ach- the application of glycosyltransferases in biocatalytic
ieved. Nothofagin was isolated from reaction mixtures synthesis of glycosylated natural products as fine
in a single step using preparative reversed phase C-18 chemicals.
HPLC with water to methanol gradient elution. Typi-
cally more than 80% of the initially applied phloretin
(
ꢂ25 mg) could be recovered as highly pure nothofa-
Experimental Section
gin (Supporting information, Figure S8C). The isola-
tion procedure is simple and not limited in scale.
Glycosyltransferases currently underachieve to
Coupled Enzymatic Conversions
a large extent their often-quoted high potential as Unless otherwise mentioned, standard reaction mixtures
[
9]
synthetically usable glycosylation catalysts. Notable contained 5 mM phloretin, 100 mM sucrose, 0.5 mM UDP,
2762
asc.wiley-vch.de
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2013, 355, 2757 – 2763

Leloir Glycosyltransferases and Natural Product Glycosylation
1
3 mM MgCl , 50 mM KCl, 0.13% (w/v) BSA, and 20%
Hyun, J. S. Williams, A. M. Czisny, J. S. Thorson, Chem.
Biol. 2004, 11, 959–969.
2
ACHTUNGTRENNUNG( v/v) DMSO in 50 mM HEPES buffer pH 7.5. Reactions
were started by the addition of the indicated amounts of
OsCGT and GmSuSy. Enzymatic reactions were performed
in 1.5 mL Eppendorf tubes at 308C using a thermomixer
comfort for temperature control and agitation at 400 rpm.
Samples were mixed with an equal volume of acetonitrile to
stop the reaction. Precipitated protein was removed by cen-
trifugation (13,200 rpm). The supernatant was analyzed
using a reversed phase C-18 HPLC-assay. All compounds
of the reaction were analyzed. Reported conversions are
confirmed by closed mass balance.
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Wang, L. Zhu, L. Zhang, Med. Res. Rev. 2003, 23, 519–
A detailed description of all experimental procedures can
be found in the Supporting Information, which comprises:
cloning of GmSuSy; expression and purification of OsCGT
and GmSuSy; enzyme assays; details of the biocatalytic
transformations performed; product isolation and identifica-
tion; and analytical methods used. Any associated referen-
ces are also given.
5
34.
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Financial support from the Austrian Science Fund FWF is
gratefully acknowledged (DK Molecular Enzymology W901-
B05). Prof. R. Edwards (Durham, UK) is thanked for pro-
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asc.wiley-vch.de
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