In vitro precursor-directed synthesis of polyketide analogues with coenzyme a regeneration for the development of antiangiogenic agents

Article abstract of DOI:10.1021/ol901243e

Polyketide analogues are produced via in vitro reconstruction of a precursor-directed polyketide biosynthetic pathway. Malonyl-CoA synthetase (MCS) was used in conjunction with chalcone synthase (CHS), thereby allowing efficient use of synthetic starter molecules and malonate as extender. Coenzyme-A was recycled up to 50 times. The use of a simple immobilization procedure resulted in up to a 30-fold higher yield of pyrone CHS products than that obtained with the free enzyme solutions.

Full text of DOI:10.1021/ol901243e

ORGANIC
LETTERS
2009
Vol. 11, No. 17
3806-3809
In Vitro Precursor-Directed Synthesis of
Polyketide Analogues with Coenzyme A
Regeneration for the Development of
Antiangiogenic Agents
Moon Il Kim, Seok Joon Kwon, and Jonathan S. Dordick*
Department of Chemical and Biological Engineering, Center for Biotechnology &
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180
dordick@rpi.edu
Received June 3, 2009
ABSTRACT
Polyketide analogues are produced via in vitro reconstruction of a precursor-directed polyketide biosynthetic pathway. Malonyl-CoA synthetase
(MCS) was used in conjunction with chalcone synthase (CHS), thereby allowing efficient use of synthetic starter molecules and malonate as
extender. Coenzyme-A was recycled up to 50 times. The use of a simple immobilization procedure resulted in up to a 30-fold higher yield of
pyrone CHS products than that obtained with the free enzyme solutions.
Polyketides represent an exceptionally diverse class of natural
products with applications as antibiotics, anticancer com-
pounds, cholesterol-lowering drugs, and animal feed supple-
ments.1 Their often high biological activity and potential for
broad structural diversity are attractive driving forces for
exploration of polyketide analogues with tailored structures
and biological function. Along these lines, chemical synthesis
has been used to generate polyketide analogues;2 however,
the structural and chemical complexity of polyketides and
their derivatives makes this route difficult.
Alternatively, an in vivo heterologous production of
polyketides by metabolic engineering of biosynthetic path-
ways or combinatorial biosynthesis is playing an increasingly
important role in natural product production.3 For example,
precursor-directed biosynthesis using N-acetylcysteamine
thioester (SNAc) starter substrates has proven to be effective
in yielding unnatural polyketide analogues.3b However,
producing polyketides in vivo has numerous limitations, such
as limited substrate permeability into the cell, the presence
of competing pathways with complex regulatory controls,
and cellular toxicity.4
As opposed to chemical- or cell-based polyketide synthe-
sis, in vitro reconstruction of polyketide synthase (PKS)
biosynthetic pathways is an emerging route to explore novel
enzyme mechanisms, assess substrate specificity, and produce
diverse polyketides.5 However, the instability of PKS and
the burden of expensive extender (malonyl- or methylma-
lonyl-coenzyme A (CoA)) substrates have made it difficult
to achieve high-yield production of polyketide analogues and
(4) (a) Fecik, R. A. Nat. Chem. Biol. 2007, 3, 531–532. (b) Weissman,
K. J.; Bycroft, M.; Cutter, A. L.; Hanefeld, U.; Frost, E. J.; Timoney, M. C.;
Harris, R.; Handa, S.; Roddis, M.; Staunton, J.; Leadlay, P. F. Chem. Biol.
1998, 5, 743–754.
(1) Shen, B. Curr. Opin. Chem. Biol. 2003, 7, 285–295.
(2) Tatsuta, K.; Hosokawa, S. Chem. Rec. 2006, 6, 217–233.
(3) (a) Van Lanen, S. G.; Shen, B. Curr. Opin. Microbiol. 2006, 9, 252–
260. (b) Zhou, H.; Zhan, J.; Watanabe, K.; Xie, X.; Tang, Y. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 6249–6254.
(5) (a) Cheng, Q.; Xiang, L.; Izumikawa, M.; Meluzzi, D.; Moore, B. S.
Nat. Chem. Biol. 2007, 3, 557–558. (b) Roessner, C. A.; Scott, A. I. Chem.
Biol. 1996, 3, 325–330.
10.1021/ol901243e CCC: $40.75
Published on Web 08/04/2009
 2009 American Chemical Society

enable their efficient scale-up.4a Herein, we report an efficient
in vitro, enzymatic synthesis of polyketide analogues by
reconstructing a type III PKS biosynthetic pathway complete
with a CoA regeneration system that mimics in vivo
precursor-directed biosynthesis.
To demonstrate the in vitro function of a precursor-directed
biosynthetic pathway, we used CHS6a with unnatural SNAc
starter substrates (Scheme 1). A critical need for large-scale
Scheme 1
.
In Vitro Precursor-Directed Biosynthesis of
Polyketide Analogues
Figure 1. (a) Stability comparison between immobilized and free
CHS (static condition, 22 °C). (b) Comparison of benzoyl SNAC
conversion (%) among four combinations of MCS and CHS (1 day
reaction). (c) Time course of benzoyl SNAC conversion (%) using
both immobilized CHS and MCS.
porting Information). Moreover, upon covalent attachment
using the reactive amine residues of CHS, the activity and
stability were substantially lower than that of free CHS.
Similarly, CLEA formation using glutaraldehyde cross-
linking of CHS amine residues resulted in poor stability, and
this was not improved with a polymeric (dextran aldehyde)
cross-linked residue.
synthesis of novel polyketides is high enzyme activity and
stability. Unfortunately, ∼40% of CHS activity was lost
within 3 h, and after 2 days <10% activity remained for the
reaction of benzoyl-SNAc with malonyl-CoA (Figure 1a and
Figure 1S, Supporting Information).
These results can be rationalized by examining the CHS
crystal structure. As depicted in Figure 2Sa (Supporting
Information), CHS possesses numerous lysine and arginine
residues (yellow and orange balls), which are located near
the CoA binding site (green, substrate binding site). The
covalent attachment on the silica beads or cross-linking with
these reactive amine groups (lysine and arginine residues)
may block the substrate entrance of active site, resulting in
interference with starter CoA binding leading to decreased
catalytic activity. As opposed to rather random adsorption
or covalent attachment to a surface, the site-specific10
coordinated covalent attachment using the N-terminal his-
tidine (His)-tag of enzyme onto Ni2+-nitrotriacetic acid (Ni-
NTA) agarose beads enables attachment distant from the
CHS active site (cyan, Figure 2Sa, Supporting Information).
Immobilization onto Ni-NTA agarose beads resulted in ca.
50% retention of native solution activity, yet a highly stable
To improve the in vitro stability of CHS, we evaluated
several enzyme immobilization methods, including physical
adsorption onto mesoporous silica (MPS) and hydrophobic
methyltrimethoxysilane (MTMOS)-coated MPS,7 covalent
attachment onto aldehyde-functionalized MPS,8 and cross-
linked enzyme aggregate (CLEA).9 Unfortunately, physical
adsorption onto either hydrophilic or hydrophobic silica
surfaces failed to improve CHS stability Figure 1S, (Sup-
(6) (a) Ferrer, J. L.; Jez, J. M.; Bowman, M. E.; Dixon, R. A.; Noel,
J. P. Nat. Struct. Biol. 1999, 6, 775–784. (b) Katsuyama, Y.; Miyahisa, I.;
Funa, N.; Horinouchi, S. Appl. Microbiol. Biotechnol. 2007, 73, 1143–
1149.
(7) (a) Kim, S. S.; Pauly, T. R.; Pinnavaia, T. J. Chem. Commun. 2000,
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Inagaki, S. Chem. Mater. 2000, 12, 3301–3305. (c) Ying, J. I.; Mehnert,
C. P.; Wong, M. S. Angew. Chem., Int. Ed. 1999, 38, 56–77.
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Biotechnol. Bioeng. 2001, 74, 249–255.
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Kieboom, A. P.; van Rantwijk, F.; van der Wielen, L. A.; Sheldon, R. A.
Biotechnol. Bioeng. 2004, 87, 754–762.
(10) Yu, C. C.; Lin, P. C.; Lin, C. C. Chem. Commun. 2008, 1308–
1310.
Org. Lett., Vol. 11, No. 17, 2009
3807

enzyme. This stabilization was confirmed using spectroscopic
analysis. Specifically, near-UV circular dichroism (CD)
spectroscopy was used to evaluate the bulk tertiary structure
of CHS free in solution at the initial time and after 15 h
incubation in aqueous buffer (Figure 2Sb, Supporting
Information) as well as immobilized onto Ni-NTA agarose.
Secondary structural changes using far-UV CD could not
be performed using the immobilized enzyme form due to
interference by the support. A clear loss in tertiary structure
was observed for the free enzyme after 15 h incubation;
however, near-complete retention of the tertiary structure was
evident with the immobilized CHS.
CHS immobilized onto Ni-NTA agarose was extremely
stable under the operating conditions, with nearly 80%
retention of initial enzyme activity following 1 week in
aqueous buffer at room temperature (Figure 1a). This stability
enabled recycling and reuse (via gravity centrifugation) of
the immobilized CHS.
malonyl-SNAc as extender substrates and instead requires
the respective CoA esters. For this reason, we coupled
malonyl-CoA synthetase (MCS)11 with CHS, which enabled
malonyl-CoA to be used as the natural extender substrate
and also allowed recycling of malonyl-CoA upon addition
of malonate and ATP with a small amount of CoA. To that
end, glutathione S-transferase (GST)-tagged MCS was used
in either free solution or immobilized onto glutathione-
sepharose (Figure 3S, Supporting Information).11 A sequen-
tial two-step enzymatic reaction was performed with benzoyl-
SNAc (1 mM) and excess malonate (10 mM) in the presence
of CoA (1 mM) and ATP (5 mM) to produce benzoyl
pyrones (tripyrone 1a and tetrapyrone 1b; detailed reaction
conditions are described in the Supporting Information). The
conversion of benzoyl-SNAc was ∼2% when free CHS and
MCS were used, and the conversion was not increased upon
immobilization of MCS. Modest improvement in starter unit
conversion was achieved in the presence of immobilized CHS
(Figure 1b). The largest conversion (>65%) was obtained in
the two-step reaction using both immobilized CHS and MCS.
The efficient use of the CoA regeneration system enabled
us to perform larger scale biotransformations, up to 50-mL
reaction volumes, containing different concentrations of
initial CoA (0.05 to 1.0 mM) with benzoyl SNAC (1 mM),
4 mM ATP, and 0.5 mM CoASH. The time course of
benzoyl pyrone synthesis is shown in Figure 1c. As expected,
with higher concentrations of initial CoA, the reaction
proceeded faster. However, after 7 days, complete conversion
of benzoyl SNAC was achieved even with 0.05 mM of initial
CoA concentration. In the case of 0.05 mM initial CoA
concentration, ca. 50 CoA regeneration cycles were achieved
after 7 days for conversion of benzoyl-SNAc into tripyrone
(two molecules of malonyl CoA used) and tetrapyrone (three
molecules of malonyl CoA used) (Table 1S, Supporting
Information). This immobilized in vitro system, therefore,
did not require high concentrations of expensive CoA,
increasing the potential for reaction scale-up.
Following 20 cycles (2 h per cycle), ca. 50% of the initial
CHS activity remained (Figure 2Sc, Supporting Information).
Finally, the reactivity of Ni-NTA agarose immobilized CHS
was tested using several acyl-CoA starter substrates and
malonyl-CoA (Table 1). For benzoyl-CoA, the overall
Table 1. Reactivity Comparison between Free and Immobilized
CHS on Ni-NTA Agarose Using Several Acyl-CoA Substrates
as Starter and Malonyl-CoA as Extendera
immobilized CHS
free CHS
starter
substrates
tripyrone tetrapyrone tripyrone tetrapyrone
benzoyl-CoA
butyryl-CoA
acetyl-CoA
11
47
37
60
5
N.D.
22
33
1
39
N.D.
N.D.
a 100 µL reaction volume, 40 µg enzyme used in Tris-HCl buffer.
Three day reaction. Concentrations of starter substrate and malonyl CoA
are 1 and 5 mM, respectively. Conversion (%) based on starter substrate as
the limiting agent. N.D. indicates compounds were not detected.
The coupled CHS/MCS reaction system was then used to
synthesize a wide range of type III polyketides. Eight starter
SNAc compounds (1-8) were chemically synthesized and
used in 50 mL reactions. After 3-day reactions, the expected
tri- and tetrapyrones and flavonoids were detected and
analyzed by LC-MS. Selected compounds were then puri-
conversion was similar for the immobilized and free CHS,
although a larger proportion of the product of the im-
mobilized CHS was the tetrapyrone, indicative of a more
extensive reaction. However, for the intrinsically less reactive
butyryl-CoA and acetyl-CoA starter substrates, substantially
higher conversion was achieved with the immobilized
enzyme. Therefore, the combination of enzyme activity and
stability, coupled with the ease of immobilization using
Ni-NTA agarose beads (including combined protein puri-
fication and immobilization), led us to use this immobiliza-
tion strategy for this study.
CHS is capable of using SNAc thioesters as starter
substrates6b as an alternative to natural CoA esters. Although
the reactivity of CHS on SNAc substrates is lower than on
their CoA counterparts (e.g., CHS was 2-fold less active on
benzoyl-SNAc as compared to benzoyl-CoA), the ease of
synthesis and the far lower cost of the SNAc esters
sufficiently outweighs the reduced enzyme activity. CHS,
however, is incapable of using malonyl-SNAc or methyl-
1
fed by flash column chromatography and analyzed by H
NMR (Supporting Information). Overall reaction yields
ranged from 27 to 81% (Table 2) based on conversion of
the SNAc starter substrate. Several of the polyketide products
have not been reported previously, including naphthoyl-
primed pyrones (5a and 5b), indole-primed pyrones and
flavonoids (6a, 6b, and 6c), and anthracene-primed pyrones
(7a and 7b) synthesized from unnatural starter molecules
(naphthoic, indolic, and anthracenoic-SNAc esters (5, 6, and
7, respectively). The starter SNAc esters having trans-
cinnamoyl moieties (3, 4, and 6) were converted into
flavonoids as well as pyrones. These results indicated that
(11) (a) An, J. H.; Kim, Y. S. Eur. J. Biochem. 1998, 257, 395–402. (b)
An, J. H.; Lee, G. Y.; Jung, J. W.; Lee, W.; Kim, Y. S. Biochem. J. 1999,
344, 159–166.
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Org. Lett., Vol. 11, No. 17, 2009

activation or overexpression of HER-2 is associated with up-
regulation of VEGF in human breast cancer cells.13
As a result, 10 µM each of the newly synthesized
polyketides were modestly inhibitory against VEGF release
from BT-474 cells. Among them, benzoyl tri- or tetrapyrones
(1a, 1b), cinnamoyl flavonoid (3c), and naphthoyl tri- or
tetrapyrones (5a, 5b) showed ∼50% inhibition against VEGF
secretion. To compare the effectiveness of these compounds
with a known VEGF inhibitor, apigenin,14 dose-response
curves for 1b, 3c, and 5b were obtained to calculate their
IC50 (half maximal inhibitory concentration) (Figure 4S,
Supporting Information) As summarized in Table 2S (Sup-
porting Information), benzoyl tetrapyrone (1b) was the most
potent of the compounds tested against VEGF secretion.
In summary, in vitro reconstruction of a PKS biosynthetic
pathway was developed, resulting in high yields of polyketide
analogues. Main bottlenecks of in vitro PKS catalysis, e.g.,
enzyme stability and cost of malonyl-CoA, were overcome
by use of simple immobilization and CoA regeneration
catalyzed by MCS, respectively. Regeneration of ATP can
be considered as an additional approach to reduce consump-
tion of expensive cofactors.15
Eight unnatural SNAc starter substrates were successfully
used in this system, yielding the expected polyketides and
then screened for antiangiogenesis activity. Because the in
vitro reconstruction of PKS biosynthetic pathway comprised
both high enzyme stability and continuous production of
malonyl-CoA by CoA regeneration, this system may ef-
ficiently mimic in vivo PKS biosynthesis. This, in turn,
results in convenient screening of novel unnatural polyketide
analogues and may lead to new classes of compounds with
antiangiogenesis activity.
Table 2. Yields (% Based on Starter SNAc) of Polyketides
Produced by in Vitro Systema
triketides
tetraketides
yield
flavonoids
yield
yield
starter product
(%)
product
(%)
product
(%)
1
2
3
4
5
6
7
8
1a
2a
3a
4a
5a
6a
7a
8a
23
5
1b
2b
3b
4b
5b
6b
7b
8b
64
47
5
1
55
3
36
17
10
18
40
54
3c
4c
10
35
6c
6
3
24
a Percent yields based on starter SNAc ester concentration; 10 mg of
immobilized MCS and 5 mg of immobilized CHS were mixed in the reaction
buffer (50 mL Tris-HCl, pH 7.5). 10 mM sodium malonate, 1 mM starter
molecules, 10 mM MgCl2, 4 mM ATP, 10 mM dithiothreitol, and 0.5 mM
initial CoA were used for 3 day incubations. Coefficients of variation (CV)
of the results were less than 7%.
CHS has extremely broad substrate specificity for aromatic
starter SNAc esters. A total of 19 natural or unnatural
polyketides were synthesized using combined immobilized
CHS and MCS.
To test the bioactivity of isolated polyketide analogues,
an antiangiogenesis assay was performed by incubating
HER2 overexpressing breast cancer (BT-474) cells with the
compounds and determining the expression level of vascular
endothelial growth factor (VEGF). Because VEGF are crucial
regulators of blood-vessel formation (angiogenesis) as well
as vascular development (vasculogenesis), inhibition of
VEGF gene expression and blocking of VEGF function are
clinically used in cancer therapy.12
Previous reports indicate that known type III polyketides,
including genistein and apigenin, inhibit VEGF expression
and decrease tumor angiogenesis.13 Therefore, we anticipated
that unnatural polyketide analogues might also regulate
VEGF secretion and synthesis. With this rationale in mind,
we tested the polyketide analogues synthesized in vitro by
the combined action of CHS and MCS catalysis for their
ability to suppress the secretion of VEGF from BT-474 cells;
Acknowledgment. This work was supported by grants
from the National Institutes of Health (ES012619 and
GM66712), NYSTAR, and the Korea Research Foundation
funded by the Korean government (MOEHRD) (KRF-2006-
352-D00047). We thank Prof. Y. S. Kim (Yonsei University)
and Prof. J. P. Noel (Salk Institute) for kindly supplying the
genes for MCS and CHS, respectively. We thank Dr. J. H.
Kim (Rensselaer Polytechnic Institute) for NMR analysis.
Supporting Information Available: Detailed experimen-
tal methods. This material is available free of charge via the
Internet at http://pubs.acs.org.
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