Identification of a new curcumin synthase from ginger and construction of a curcuminoid-producing unnatural fusion protein diketide-CoA synthase::curcumin synthase

Article abstract of DOI:10.1039/c5ra23401h

The biosynthesis and metabolic engineering of curcuminoids received considerable attention for their important pharmaceutical properties. In the present study, a new curcumin synthase (ZoCURS) was identified from ginger (Zingiber officinale). Notably, ZoCURS efficiently accepts 3-(4-hydroxyphenyl)propionyl-CoA to produce tetrahydrobisdemethoxycurcumin, which would be meaningful to further understand the biosynthesis of curcuminoids in ginger. In addition, a curcuminoid-producing unnatural fusion protein diketide-CoA synthase::curcumin synthase (DCS::CURS) was constructed. Comparing to ZoCURS, DCS::CURS indicated similar substrate specificities and catalytic potentials to catalyze the formation of various curcuminoids, however, the yield of curcuminoids produced by DCS::CURS was obviously increased, which would be useful for metabolic engineering of pharmaceutically important curcuminoid analogs, particularly including asymmetric dihydrocurcuminoids such as 3-(4-hydroxyphenyl)propionyl-feruloylmethane, 3-(4-hydroxyphenyl)propionyl-p-coumaroylmethane, and 3-(4-hydroxyphenyl)propionyl-cinnamoylmethane.

Full text of DOI:10.1039/c5ra23401h

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: L. Zhang, B. gao,
X. Wang, Z. Zhang, X. liu, J. Wang, T. mo, Y. liu, S. Shi and P. Tu, RSC Adv., 2016, DOI:
10.1039/C5RA23401H.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
Identification of a new curcumin synthase from ginger and construction of a
curcuminoid-producing unnatural fusion protein diketide-CoA
synthase::curcumin synthase
Le Zhang a,b, Bowen Gao a,b,c, Xiaohui Wang a, Zhongxiu Zhang a,b, Xiao Liu a, Juan
Wang a, Ting Mo a, Yuyu Liu a, Shepo Shi a,*, Pengfei Tu a,*
a Modern Research Center for Traditional Chinese Medicine, Beijing University of
Chinese Medicine, Beijing 100029, People’s Republic of China;
b School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing
100102, People’s Republic of China;
c Baotou Medical College, Baotou 014060, China
*To whom correspondence should be addressed: Tel/Fax: 86-10-64286350, E-mail:
shishepo@163.com (S.-P. Shi); pengfeitu@163.com (P.-F. Tu).
1

RSC Advances
Page 2 of 21
View Article Online
DOI: 10.1039/C5RA23401H
Abstract：The biosynthesis and metabolic engineering of curcuminoids received
considerable attention for their important pharmaceutical properties. In the present
study, a new curcumin synthase (ZoCURS) was identified from ginger (Zingiber
officinale). Notably, ZoCURS efficiently accepts 3-(4-hydroxyphenyl) propionyl-CoA
to produce tetrahydrobisdemethoxycurcumin, which would be meaningful to further
understand the biosynthesis of curcuminoids in ginger. In addition, a
curcuminoid-producing unnatural fusion protein diketide-CoA synthase::curcumin
synthase (DCS::CURS) was constructed. Comparing to ZoCURS, DCS::CURS
indicated similar substrates specificities and catalytic potentials to catalyze the
formation of various curcuminoids, However, the yield of curcuminoids produced by
DCS::CURS was obviously increased, which would be useful for metabolic
engineering of pharmaceutically important curcuminoid analogs, particularly
including asymmetric dihydrocurcuminoids such as 3-(4-hydroxyphenyl)
propionyl-feruloylmethane, 3-(4-hydroxyphenyl) propionyl-p-coumaroylmethane, and
3-(4-hydroxyphenyl) propionyl-cinnamoylmethane.
Keywords: Diketide-CoA synthase; Curcumin synthase; Unnatural fusion protein;
Curcuminoids; Zingiber officinale.
2

Page 3 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
1. Introduction
Curcumin, the major component of Curcuma longa, is widely used as traditional
medicine and food additive for their promising bioactivities, unique coloring and
aromatic properties.1,2 Due to its important pharmaceutical properties such as
anti-inflammatory, antitumor, and anti-oxidation activities, the biosynthesis of
curcumin has attracted much attention.3-6 The recent characterization of two novel
type Ⅲ polyketide synthases from C. longa termed as diketide-CoA synthase (DCS)
and curcumin synthase (CURS) well established the biosynthesis pathway of
curcumin in C. longa.7,8 Firstly, DCS catalyzes the condensation of feruloyl-CoA and
malonyl-CoA to produce feruloyldiketide-CoA. And then, CURS catalyzes the
conversion of feruloyldiketide-CoA to β-keto acid and sequentially condenses with
another molecule of feruloyl-CoA to produce curcumin (Scheme 1A).7,8 Besides, a
novel type Ⅲ polyketide synthase (curcuminoid synthase, CUS) from rice plant
Oryza sativa catalyzes one-pot formation of bisdemethoxycurcumin from the
condensation of two molecules of p-coumaroyl-CoA and one molecule of
malonyl-CoA (Scheme 1B).9-11 Although curcuminoids have never been isolated from
O. sativa, the identification of CUS in O. sativa is still interesting and its adaptive
function is expecting to be found. In contrast, ginger (Zingiber officinale.) as another
major source of curcuminoids, enzymes involved in the biosynthesis of curcuminoids
in ginger have not been characterized. Thus, characterization of functionally related
enzymes from ginger would be advantage to further understand the biosynthesis of
curcuminoids in this plant.
3

RSC Advances
Page 4 of 21
View Article Online
DOI: 10.1039/C5RA23401H
On the other hand, advances in biogenetic engineering technologies facilitate to
manipulate and partially/entirely reconstruct a biosynthesis pathway in heterologous
microorganisms for the production of specific metabolites with high-yields for
medicine.12-15 Among of the approaches used in the biosynthetic-process manipulation
and reconstruction, generation of unnatural fusion protein by insertion of artificial
linkers between sequential biosynthetic enzymes, which mimics the natural
colocalization of proteins in cells and/or the physical channeling of reactive
intermediates between enzyme active sites, is of great promising to simplify the
biosynthetic process and increase the metabolic efficiency.16-18 Given the important
pharmaceutical properties of curcuminoids, reconstruction of the biosynthesis
pathway in heterologous microorganisms to produce curcuminoids is getting
increasingly attractive. However, comparing to DCS and CURS synergically
catalyzed the formation of curcuminoids, CUS from O. sativa possessing the
remarkable function of one-pot formation of curcuminoids is more convenient and
thus preferred to be used in the construction of metabolically engineered
microorganisms for curcuminoids production.19-22 We propose that construction of an
unnatural fusion protein DCS::CURS possibly achieve the synergistic function of
DCS and CURS, and the fusion protein would be convenient to be used in metabolic
engineering curcuminoids production in microorganisms.
Herein, we report the identification of a new curcumin synthase (ZoCURS) from
ginger, and functional analysis of DCS::CURS, a curcuminoid-producing unnatural
fusion protein constructed by inserting of an artificial linker between a known
4

Page 5 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
diketide-CoA synthase (DCS) from C. longa and the newly identified ZoCURS.
2. Materials and methods
2.1. Chemicals
Cinnamoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and 3-(4-hydroxyphenyl)
propionyl-CoA were enzymatically synthesized according to reported methods.23
Malonyl-CoA, curcumin (1a), bisdemethoxycurcumin (2a),
3-methoxy-4-hydroxybenzalacetone (dehydrozingerone) (1b),
4-hydroxybenzalacetone (2b), 4-hydroxybenzylacetone (raspberry ketone) (4b), and
were purchased from Sigama (St Louis, MO, USA). Dicinnamoylmethane (3a),
tetrahydrobisdemethoxycurcumin (THBDC) (4a) were synthesized according to
reported methods.24,25
2.2. cDNA cloning and expression
Total RNA samples were prepared from 100 mg of young fresh rhizomes of C.
longa and leaves of Z. officinale, respectively, using Plant RNA kit (Omega) and
reverse-transcribed using Reverscript (Wakon) and oligo-dT primer (RACE 32 = 5'
-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3'). The
full-length cDNA of DCS and ZoCURS were amplified by PCR using N- and
C-terminal primers synthesized according to the sequence registered (NCBI accession
numbers: AB495006 for DCS, and DQ486012 for ZoCURS) (primer sequences could
be found in SI, S1). The amplified DNA was cloned between the BamHI and HindIII
sites of pET28a (Novagen) to obtain the constructed plasmids pET28a-DCS and
pET28a-ZoCURS. To construct the DCS::CURS fusion gene, the stop codon of DCS
5

RSC Advances
Page 6 of 21
View Article Online
DOI: 10.1039/C5RA23401H
was removed and a three amino acid linker (Gly-Ser-Gly) was introduced between the
open reading frames of DCS and ZoCURS by site directed-mutagenesis through two
rounds of PCR (primer sequences could be found in SI, S1). It resulted in a 2.349 kb
fused gene encoding DCS, the tripeptide linker, and ZoCURS. Finally, the fusion gene
was inserted into the BamHI ⁄ HindIII site of pET28a (Novagen) to obtain the
constructed plasmid pET28a-DCS::CURS. After confirmation of the sequences, the
plasmid of pET28a-DCS, pET28a-ZoCURS and pET28a-DCS::CURS was
transformed into E. coli BL21(DE3), respectively. The cells harboring the
corresponding plasmid were cultured to an A600 of 0.6 in LB medium containing 50
g/ml Kanamycin and induced by 0.6 mM IPTG at 23 °C for 16 h.
2.3. Enzyme purification
The E. coli cells were harvested by centrifugation and resuspended in 40 mM
potassium phosphate buffer (KPB) (pH = 7.9) containing 100 mM NaCl, 5 mM
imidazole). Cell lysis was carried out by sonication and centrifuged at 12 000 g at
4 °C for 30 min. The supernatant containing crude recombinant protein was collected
and passed through a column of Ni-NTA His-Bind™ Resin (CWBIO, Beijing, China)
containing Ni2+ as an affinity ligand. After washing with 20 mM KPB (pH = 7.9)
containing 500 mM NaCl and 40 mM imidazole, the recombinant enzyme was finally
eluted with 15 mM KPB (pH = 7.8) containing 10% glycerol and 500 mM imidazole.
After desalted and concentrated by Amicon Ultra-15 centrifugal concentrators
(Millipore, MI, USA), the recombinant protein was finally kept in 20 mM potassium
phosphate buffer (pH = 8.0) containing 1 mM EDTA and 10% glycerol. The
6

Page 7 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
determination of protein concentration was carried out according to BCA method
(Protein Assay, BIOMIGA) using bovine serum albumin as standard. The expression
yield of DCS, ZoCURS, and DCS::CURS is 1.0 mg/L, 1.6 mg/L, and 1.5 mg/L,
respectively.
2.4. Enzyme reaction
The standard reaction mixture contained 100 µM starter substrate
(cinnamoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and 3-(4-hydroxyphenyl)
propionyl-CoA), 100 µM extender substrate (malonyl-CoA), 100 mM KPB (pH = 7.5),
and 166 nM of purified recombinant enzyme in a total volume of 500 µl. And
reactions were incubated at 37°C for 8 h before being quenched with 40 µl 20% HCl.
The products were extracted twice with 800 µl ethyl acetate, and the organic layer was
evaporated to dryness. The residual materials were dissolved in 100 µl of methanol
for LCMS-IT-TOF (Shimadzu, Japan) analysis. The HPLC was equipped with an
Agilent Eclipse XDB C18 Column (250 × 4.6 mm I.D. 5 µm) and eluted with a linear
acetonitrile gradient (30 – 100% over 30 min) in water at a flow rate of 1.0 mL min-1.
The enzymatic products were identified and quantified using corresponding authentic
compounds.
2.5. Time course of DCS::CURS catalyzing the production of curcumin (1a) and
3ꢀmethoxyꢀ4ꢀhydroxybenzalactone (1b)
100 µM feruloyl-CoA, 100 µM malonyl-CoA, 100 mM KPB (pH = 7.5), and 83.4
nM of purified recombinant enzyme in a total volume of 500 µl was incubated at
30 °C for 5 –360 min. The reaction mixture was processed according to the method
7

RSC Advances
Page 8 of 21
View Article Online
DOI: 10.1039/C5RA23401H
mentioned in section 2.4, and the enzymatic products were quantified using standard
compounds.
3. Results and Discussion
3.1. cDNA cloning and enzyme purification
The full length cDNA of DCS was directly amplified from C. longa according to
the known sequence of DCS (NCBI accession numbers AB495006). Considering the
extensive reports of curcumin synthases from C. longa (CURS1, CURS2, and
CURS3),7, 26 we have no attempt to screen and clone CURS from C. longa. However,
enzymes involved in the biosynthesis of curcuminoids in ginger have not been
characterized before. Although a type Ⅲ polyketide synthase named ZoPKS has
been proposed to be involved in the biosynthesis of phenylbutanone derivatives in
ginger. Its unambiguous activity is currently unknown.27 Therefore, we cloned and
expressed ZoPKS (NCBI accession numbers DQ486012) based on its known full
length. With a series of in vitro functional analysis, recombinant protein ZoPKS was
identified to be a new curcumin synthase, named ZoCURS. As the construction of the
fusion protein DCS::CURS, the stop codon of DCS was removed and a three amino
acid linker (Gly-Ser-Gly) was introduced between the open reading frames of DCS
and ZoCURS by site directed-mutagenesis through two rounds of PCR.
DCS, ZoCURS, and the unnatural fusion protein DCS::CURS His-tagged at their
N terminus were produced in E. coli BL21 (DE3) and purified by a nickel chelated
affinity column to obtain three recombinant proteins of 42.04 KDa, 43.24 KDa, and
85.47 KDa, respectively (SDS-page could be found in SI, S2).
8

Page 9 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
3.2. Analysis of the reactions catalyzed by ZoCURS
In the absence of DCS, only a trace amount of curcumin (1a) was detectable by
LC-MS in the reaction of ZoCURS with feruloyl-CoA (1) and malonyl-CoA.
However, the yield of curcumin (1a) was dramatically increased by co-incubation
DCS and ZoCURS with feruloyl-CoA and malonyl-CoA (Scheme 2A and Fig. 1A).
Further analysis of the substrate specificity revealed that ZoCURS, in the presence of
DCS, could also accept p-coumaroyl-CoA (2) and cinnamoyl-CoA (3) to produce
bidemethoxycurcumin (2a) and dicinnamoylmethane (3a), respectively (Scheme 2A
and Fig. 1B-C). In addition, C6-C4 scaffolds of 3-methoxy-4-hydroxybenzalacetone
(dehydrozingerone) (1b) and 4-hydroxybenzalacetone (2b) could also be produced by
ZoCURS (co-incubated with DCS) as by-product from feruloyl-CoA and
p-coumaroyl-CoA, respectively. Very interestingly, when DCS and ZoCURS were
co-incubated in the presence of 3-(4-hydroxyphenyl) propionyl-CoA (4) and
malonyl-CoA, tetrahydrobisdemethoxycurcumin (THBDC) (4a) and
4-hydroxybenzylacetone (4b) were produced (Scheme 2B and Fig. 1D). All structures
of the enzymatic products were unambiguously characterized by comparing their UV
and MS data as well as retention time (Rt) on HPLC chromatogram with those of the
authentic compounds (Fig 1, and S3-S4). It is generally accepted that the biosynthesis
of THBDC (4a) and gingerol-related compounds in ginger involved in a
NAD(P)H-dependent reductase to selectively reduce the unsaturated double bonds.28
However, ZoCURS from ginger efficiently accepts 3-(4-hydroxyphenyl)
propionyl-CoA as a starter to produce diarylheptanoid scaffold of THBDC (4a),
9

RSC Advances
Page 10 of 21
View Article Online
DOI: 10.1039/C5RA23401H
which suggests that THBDC (4a) and gingerol-related compounds in Z. officinale are
possible to be directly synthesized from 3-(4-hydroxyphenyl) propionic acid,
excepting for the possibility of that from p-coumaric acid. In addition, it should be
notable that 4-hydroxybenzylacetone (raspberry ketone) (4b) was previously reported
to be biogenetically synthesized from 4-hydroxybenzalacetone which was formed by
benzalactone synthase (BAS) from the condensation of p-coumaroyl-CoA and one
molecule of malonyl-CoA.29-32 Enzymes directly involved in the synthesis of
4-hydroxybenzylacetone (4b) from 3-(4-hydroxyphenyl) propionyl-CoA has never
been reported. The C6-C4 scaffolds of 1b, 2b, and 4b should be nonenzymatic
hydrolysis and subsequent decarboxylation of diketide-CoA intermediates produced
by DCS.
Although curcuminoids have been extensively reported from ginger, ZoCURS is
the first polyketide synthase functionally identified to be involved in the biosynthesis
of curcuminoids in ginger. Particularly, enzymatic formation of THBDC (4a) from
3-(4-hydroxyphenyl) propionyl-CoA by wild plant polyketide synthases has not been
previously reported. Sequence alignment reveals that ZoCURS shares 81.03% amino
acid sequence identity with CURS1 from C. longa, the highly conserved Cys-His-Asn
catalytic triad in other plant polyketide synthases is also observed in ZoCURS.
Additionally, ZoCURS shares almost the same amino residues lining around the
catalytic cavity in CURS1, including Phe215, Phe265, Phe267, Gln338, and Met137.
It was proposed that a hydrophobic cavity surrounded by the side chains of Phe215,
Phe265, Phe267, and Gly211 in CURS1 was very important for CURS1 to use a
10

Page 11 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
β-keto acid as an extender substrate.8 The observation of Phe215, Phe265, and Phe267
in ZoCURS suggests that ZoCURS probably utilizes similar catalytic machinery to
that of CURS1.
3.3. Analysis of the reactions catalyzed by DCS::CURS
Comprehensive investigations on catalytic functions of the fusion protein
DCS::CURS revealed that DCS::CURS showed similar substrate specificities and
catalytic potentials to those of ZoCURS (co-incubate with DCS). The fusion protein
DCS::CURS could also efficiently accept feruloyl-CoA, p-coumaroyl-CoA,
cinnamoyl-CoA, and 3-(4-hydroxyphenyl) propionyl-CoA to produce various
diarylheptanoid scaffolds of curcumin (1a), bisdemethoxyCurcumin (2a),
dicinnamoylmethane (3a), and THBDC (4a), respectively (Fig. 1). Comparing to the
reactions catalyzed by DCS and ZoCURS co-incubation, the yield of curcumin,
bisdemethoxycurcumin, dicinnamoylmethane, and THBDC produced by DCS::CURS
was dramatically increased (Fig. 2). In contrast, the yields of the by-products of 1b,
2b, and 4b decreased (Fig. 2). The fusion protein DCS::CURS showed the optimal pH
and temperature at 7.5 and 30 °C, respectively (SI, S5). Investigation on the time
course of the unnatural fusion protein DCS::CURS using feruloyl-CoA and
malonyl-CoA as substrates revealed that the production rate of the major product
curcumin (1a) and the minor product 3-methoxy-4-hydroxy benzalactone (1b) varied
at different time points. In the first 60 min after the reaction initiation, the velocity of
curcumin synthesis was very slow, and sharply increased in the following 3 h. In
contrast, the accumulation of the minor product 3-methoxy-4-hydroxybenzalacetone
11

RSC Advances
Page 12 of 21
View Article Online
DOI: 10.1039/C5RA23401H
(1b) in the reaction mixture kept increasing in the first 60 min, and then reached
saturation (SI, S6). The results allowed us to propose that DCS::CURS catalyzing the
formation of curcumin (1a) most likely involved in the formation and release of an
intermediate diketide-CoA. At the initial stage, the low concentration of diketide-CoA
in the reaction mixture result in the formation of curcumin (1a) in low velocity, with
the concentration of diketide-CoA rising up, the velocity of curcumin (1a) formation
increased. However, long time incubation would cause degradation of curcumin (1a).
Notably, when a 3:1 mixture of feruloyl-CoA and malonyl-CoA was incubated with
DCS::CURS, the reaction exclusively produced a single product of curcumin (1a).
Presumably, the excess feruloyl-CoA completely exhausts the intermediate
diketide-CoA to avoid the formation of the by-product 3-methoxy-4-hydroxy
3-methoxy-4-hydroxy benzalactone (1b). In order to further probe the catalytic
potentials of the fusion protein, a mixture of feruloyl-CoA and p-coumaroyl-CoA was
incubated with malonyl-CoA and the fusion protein. An asymmetric diarylheptanoid
of p-coumaroyl-feruloylmethane was unexpectedly produced as major product,
excepting the formation of curcumin and a minor product bidemethoxycurcumin (2a)
(HPLC chromatograms could be found in SI, S7A). Likewise, various asymmetric
curcuminoids such as p-coumaroyl-cinnamoylmethane, feruloyl-cinnamoylmethane
could also be formed by DCS::CURS (HPLC chromatograms could be found in SI,
S7B and S7C). Notably, DCS::CURS catalyzed the formation of asymmetric
curcuminoids such as 3-(4-hydroxyphenyl) propionyl-cinnamoylmethane,
3-(4-hydroxyphenyl) propionyl-feruloylmethane, and 3-(4-hydroxyphenyl)
12

Page 13 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
propionyl-p-coumaroylmethane would be useful to regioselectively synthesize this
type of dihydrocurcuminoids (HPLC chromatograms could be found in SI, S8). In
contrast, although CUS from O. sativa is preferred to be used for heterologous
production of curcuminoids, wild CUS could not accept 3-(4-hydroxyphenyl)
propionyl-CoA as a substrate preventing the smoothly utilization of CUS in
heterologous production of dihydrocurcuminoids.10 The unnatural fusion protein
DCS::CURS with broader substrate tolerance would thus be meaningful for synthesis
of structurally more diverse curcuminoids.
In summary, we identified a new curcumin synthase (ZoCURS) from ginger and
constructed a curcuminoid-producing unnatural fusion protein DCS::CURS. These
would be not only contributable to further understand the biosynthesis of
curcuminoids in ginger but also be advantage to further manipulate the biosynthesis of
curcuminoid analogs, particularly including THBDC and various dihydrocurcuminoid
derivatives in microorganisms.
Acknowledgments
We are thankful to the financial supports by Beijing Natural Science Foundation (No.
5132022) and the Program for New Century Excellent Talents in University (No.
NCET-11-0604) and graduate independent project in Beijing University of Chinese
Medicine (No. 2015-JYB-XS060).
References
13

RSC Advances
Page 14 of 21
View Article Online
DOI: 10.1039/C5RA23401H
[1] R. A. Sharma, A. J. Gescher and W. P. Steward, Eur. J. Cancer., 2005, 41, 1955.
[2] R. F. Tayyem, D. D. Heath, W. K. Al-Delaimy and C. L. Rock, Nutr. Cancer., 2013
55, 126.
[3] P. J. Roughley and D. A. Whiting, J. Chem. Soc., Perkin Trans., 1973, 1, 2379.
[4] P. Denniff, l. Macleod and D. A. Whiting, J. Chem. Soc., Perkin Trans., 1980, 1,
2637.
[5] P. Denniff and D. A. Whiting, J. Chem. Soc., Chem. Commun., 1976, 18, 711.
[6] M. del. C. Ramirez-Ahumada, B. N. Timmermann and D. R. Gang,
Phytochemistry, 2006, 67, 2017.
[7] Y. Katsuyama, T. Kita, N. Funa and S. Horinouchi, J. Biol. Chem., 2009, 284,
11160.
[8] Y. Katsuyama, K. Miyazono, M. Tanokura, Y. Ohnishi and S. Horinouchi, J. Biol.
Chem., 2011, 286, 6659.
[9] Y. Katsuyama, M. Matsuzawa, N. Funa and S. Horinouchi, J. Biol. Chem., 2007,
282, 37702.
[10] H. Morita, K. Wanibuchi, H. Nii, R. Kato, S. Sugio and I. Abe, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 19778.
[11] I. Abe and H. Morita, Nat. Prod. Rep., 2010, 27, 809.
[12] W. Runguphan, X. Qu and S. E. O’Connor, Nature, 2010, 468, 461.
[13] P. K. Ajikumar, W. H. Xiao, K. E. J. Tyo, Y. Wang, F. Simeon, E. Leonard, O.
Mucha, T. H. Phon, B. Pfeifer and G. Stephanopoulos, Science, 2010, 330, 70.
[14] J. D. Newman, J. Marshall, M. Chang, F. Nowroozi, E. Paradise, D. Pitera, K. L.
Newman and J. D. Keasling, Biotechnol. Bioeng., 2006, 95, 684.
[15] S. Galanie, K. Thodey, I. J. Trenchard, M. F. Interrante and C. D. Smolke,
Science, 2015, 349, 1095.
[16] Y. Li and P. C. Cirino, Biotechnol. Bioeng., 2014, 111, 1273.
[17] Y. S. Zhang, S. Z. Li, J. Li, X. Q. Pan, R. E. Cahoon, J. G. Jaworski, X. M. Wang,
J. M. Jez, F. Chen and Y. Oliver, J. Am. Chem. Soc., 2006, 128, 13030.
[18] Y. Wang, H. Yi, M. Wang, O. Yu and J. M. Jez, J. Am. Chem. Soc., 2011, 133,
14

Page 15 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
20684.
[19] Y. Katsuyama, M. Matsuzawa, N. Funa and S. Horinouchi, Microbiol., 2008, 154,
2620.
[20] Y. Katsuyama, Y. Hirose, N. Funa, Y. Ohnishi and S. Horinouchi, Biosci.
Biotech. Bioch., 2010, 74, 641.
[21] Y. Katsuyama, Y. Ohnishi and S. Horinouchi, ChemBioChem, 2010, 11, 2034.
[22] J. L. Rodrigues, R. G. Araújo, K. L. Prather, L. D. Kluskens and L. R. Rodrigues,
Biotechnol. J., 2015, 10, 599.
[23] T. Mo, J. Wang, B. W. Gao, L. Zhang, X. Liu, X. H. Wang, P. F. Tu and S. P.
Shi, Chinese J. Org. Chem., 2015, 35, 1052.
[24] A. Nurfina, M. Reksohadiprodjo, H. Timmerman, U. Jenie, D. Sugiyanto and H.
Van der Goot, Eur. J. Med. Chem., 1997, 32, 321.
[25] W. M. Weber, L. A. Hunsaker, S. F. Abcouwer, L. M. Deck and D. L. Vander
Jagt, Bioorg. Med. Chem. Lett., 2005, 13, 3811.
[26] Y. Katsuyama, T. Kita and S. Horinouchi, FEBS Lett., 2009, 583, 2799-2803.
[27] E. Radhakrishnan, K. Sivakumar and E. Soniya, J. Proteom. Bioinform., 2009, 2,
310.
[28] H. J. Koo, E. T. McDowell, X. Q. Ma, K. A. Greer, J. Kapteyn, Z. Z. Xie, A.
Descour, H. Kim, Y. Yu, D. Kudrna, R. A. Wing, C. A. Soderlund and D. R.
Gang, BMC plant Biol., 2013,13, 27.
[29] S. Pedapudi, C. K. Chin and H. Pedersen, Biotechnol. Progr., 2000, 16, 346-349.
[30] T. Abe, H. Morita, H. Noma, T. Kohno, H. Noguchi and I. Abe, Bioorg. Med.
Chem. Lett., 2007, 17, 3161.
[31] I. Abe, Y. Sano, Y. Takahashi and H. Noguchi, J. Biol. Chem., 2003, 278, 25218.
[32] H. Morita, Y. Shimokawa, M. Tanio, R. Kato, H. Noguchi, S. Sugio, T. Kohno
and I. Abe, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 669.
15

RSC Advances
Page 16 of 21
View Article Online
DOI: 10.1039/C5RA23401H
Figures and legends
Scheme 1 Proposed mechanisms of Curcuminoids formation. (A) Curcumin from two
molecules of feruloyl-CoA and one molecule of malonyl-CoA by DCS and CURS; (B)
Bisdemethoxycurcumin from two molecules of 4-coumaroyl-CoA and one molecule
of malonyl-CoA by CUS.
Scheme 2 Proposed mechanisms of curcuminoids formation catalyzed by
DCS::CURS.
Figure 1 HPLC chramatograms of the reaction products produced by ZoCURS and
DCS::CURS from malonyl-CoA and varied substrates (A) feruloyl-CoA (A360 nm), (B)
4-coumaroyl-CoA (A360 nm), (C) cinnamoyl-CoA (A360 nm), and (D)
3-(4-hydroxyphenyl) propionyl-CoA (A280 nm).
Figure 2 Increased production of curcuminoids (1a – 4a) and decreased formation of
benzalactones/benzylactone (1b, 2b, and 4b) by DCS::CURS. Results are presented
as mean ± S.E. (n = 3).
16

Page 17 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
Scheme 1
17

RSC Advances
Page 18 of 21
View Article Online
DOI: 10.1039/C5RA23401H
Scheme 2
(A)
R1
R2
R1
R2
R1
R2
H2O
CoAS
O
CoAS
OH
EnzS
H3C
DCS
+
1
x
O
O
O
O
O
1: R1 = OH, R2 = OCH3;
2: R1 = OH, R2= H;
3: R1 = R2 = H
1b
2b
: R1 = OH, R2 = OCH3;
: R1 = OH, R2 = H
CO2
H2O
R1
R2
R1
R2
R1
R2
R1
R2
ZoCURS
CoAS
HO
+
O
O
O
O
O
1: R1 = OH, R2 = OCH3;
2: R1 = OH, R2= H;
R1 = OH, R2 = OCH3;
R1 = OH, R2= H;
R1 = R2 = H
1a: R1 = OH, R2 = OCH3;
2a: R1 = OH, R2= H;
3a:
R1 = R2 = H
3:
R1 = R2 = H
(B)
OH
OH
OH
H2O
OH
CoAS
CoAS
EnzS
H3
C
DCS
+
1
x
O
O
O
O
O
O
CO2
3-(4-hydroxyphenyl)
propionyl-CoA (4)
4-hydroxybenzylacetone (4b)
H2O
OH
OH
OH
HO
ZoCURS
HO
CoAS
+
O
O
O
O
O
tetrahydrobisdemethoxycurcumin (4a)
18

Page 19 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
Fig. 1
19

RSC Advances
Page 20 of 21
View Article Online
DOI: 10.1039/C5RA23401H
Fig. 2
20

Page 21 of 21
RSC Advances
View Article Online
DOI: 10.1039/C5RA23401H
Graphical Abstract
The new curcumin synthase and the unnatural fusion protein reported here are useful for metabolic
engineering of pharmaceutically important curcuminoids.