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Journal ArticleDOI

Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate.

01 Apr 2015-Biotechnology Journal (Biotechnol J)-Vol. 10, Iss: 4, pp 599-609

Abstract: Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we report an engineered artificial pathway in Escherichia coli to produce natural curcuminoids through caffeic acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used to produce curcuminoids and 70 mg/L of curcumin was obtained from ferulic acid. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used. Caffeoyl-CoA 3-O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 mg/L of curcumin were produced, respectively. This is the first time DCS and CURS1 were used in vivo to produce curcuminoids and that curcumin was produced by feeding tyrosine. We have shown that curcumin can be produced using a pathway involvoing caffeic acid. This alternative pathway represents a step forward in the heterologous production of curcumin using E. coli.
Topics: Curcumin synthase (63%), Caffeic acid (62%), Tyrosine ammonia-lyase (61%), Ferulic acid (57%), Tyrosine (53%)

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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1
1 Introduction
Curcuminoids are natural phenylpropanoids from the
plant Curcuma longa Linn. Its rhizome contains a mixture
of curcuminoids, with curcumin, demethoxycurcumin,
and bisdemethoxycurcumin present in higher amounts.
These compounds present in turmeric have long been
used in traditional Asian food and medicine. Their thera-
peutic properties include anti-cancer, anti-inflammatory,
anti-oxidant, anti-Alzeimer’s, anti-HIV, and anti-Parkin-
son [1–4]. Despite their numerous benefits to human
health, curcuminoids have poor bioavailability and their
natural abundance is low, thus making their heterologous
biosynthetic production very interesting.
Recently, curcuminoids were found to be synthesized
by type III polyketide synthases (PKSs) and additional
enzymes from the phenylpropanoid pathway in plants [5].
Katsuyama et al. [6] were the first to produce curcumi-
noids using an artificial pathway in E. coli. They used
phenylalanine ammonia lyase (PAL) from Rhodotorula
rubra with tyrosine ammonia lyase (TAL) activity to con-
vert the amino acids phenylalanine and tyrosine directly
to cinnamic acid and p-coumaric acid. 4-coumarate-CoA
ligase (4CL) from Lithospermum erythrorhizon was used
to convert cinnamic acid and p-coumaric acid to cin-
namoyl-CoA and p-coumaroyl-CoA, respectively, and
Research Article
Production of curcuminoids from tyrosine by a metabolically
engineered Escherichia coli using caffeic acid as an intermediate
Joana L. Rodrigues
1,2
, Rafael G. Araújo
1
, Kristala L. J. Prather
2,3
, Leon D. Kluskens
1
, and Ligia R. Rodrigues
1,2
1
Centre of Biological Engineering, University of Minho, Braga, Portugal
2
MIT-Portugal Program, Cambridge, MA and Lisbon, Portugal
3
Department of Chemical Engineering, Synthetic Biology Engineering Research Center (SynBERC) Massachusetts Institute
of Technology, Cambridge, MA, USA
Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we report an
engineered artificial pathway in Escherichia coli to produce natural curcuminoids through caffeic
acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS)
and curcumin synthase (CURS1) were used to produce curcuminoids and 70 mg/L of curcumin
was obtained from ferulic acid. Bisdemethoxycurcumin and demethoxycurcumin were also pro-
duced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and
ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caf-
feic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and
4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used. Caffeoyl-CoA 3-O-methyl-
transferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic
acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 mg/L of curcumin were produced,
respectively. This is the first time DCS and CURS1 were used in vivo to produce curcuminoids and
that curcumin was produced by feeding tyrosine. We have shown that curcumin can be produced
using a pathway involvoing caffeic acid. This alternative pathway represents a step forward in the
heterologous production of curcumin using E. coli.
Keywords: Caffeic acid · Curcumin synthase · Curcuminoids · E. coli · Tyrosine
Correspondence: Prof. Lígia R. Rodrigues, Centre of Biological
Engineering, University of Minho, 4710 – 057 Braga, Portugal
E-mail: lrmr@deb.uminho.pt
Abbreviations: 4CL, 4-coumaroyl-CoA ligase; ACC, acetyl-CoA carboxylase;
C3H, 4-coumarate 3-hydroxylase; CCoAOMT, caffeoyl-CoA 3-O-methyltrans-
ferase; CURS, curcumin synthase; CUS, curcuminoid synthase; DCS, dike-
tide-CoA synthase; PAL, phenylalanine ammonia lyase; TAL, tyrosine
ammonia lyase
Biotechnol. J. 2015, 10 DOI 10.1002/biot.201400637
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Biotechnology
Journal
Received 15 OCT 2014
Revised 26 NOV 2014
Accepted 02 JAN 2015
Accepted
article online 08 JAN 2015
Supporting information
available online

2 © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
then to curcuminoids by curcuminoid synthase (CUS)
from Oryza sativa. Acetyl-CoA carboxylase (ACC) from
Corynebacterium glutamicum was also overexpressed to
increase the intracellular pool of malonyl-CoA. The sup-
plementation of amino acids to the medium led to the
production of bisdemethoxycurcumin and other two cur-
cuminoids, cinnamoyl-p-coumaroylmethane, and dicin-
namoylmethane. The direct supplementation of car-
boxylic acids, as ferulic acid for instance, led to other cur-
cuminoids including curcumin and demethoxycurcumin
[6]. By adding two different unnatural carboxylic acids
simultaneously (analogs of p-coumaric acid), Katsuyama
and coworkers also produced unnatural curcuminoids [7].
Moreover, Wang et al. [8] produced the curcuminoid
dicinnamoylmethane by using PALs from Trifolium
pratense, 4CL1 from Arabidopsis thaliana and CUS from
O. sativa. After CUS was discovered, Katsuyama et al. [9]
reported that in the C. longa plant, the PKSs used to pro-
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Figure 1. Proposed curcuminoid biosynthetic pathway in E. coli and the reactions catalyzed by the enzymes in this study. TAL, tyrosine ammonia lyase;
C3H, 4-coumarate 3-hydroxylase; 4CL, 4-coumarate-CoA ligase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; DCS, diketide-CoA synthase; CURS1,
curcumin synthase; CUS, curcuminoid synthase.

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3
duce curcuminoids were diketide-CoA synthase (DCS)
and curcumin synthase (CURS1). They also identified oth-
er CURS enzymes (CURS2 and CURS3) with different sub-
strate specificities [10]. It is important to bear in mind that
CUS catalyzes both steps that are catalyzed separately by
DCS and CURS [9] (Fig. 1).
In this work, we describe curcuminoid production in
E. coli using an artificial pathway (Fig. 1). We tested 4CL1
from A. thaliana (At4CL1) and different PKSs for curcum-
inoid production (CUS, DCS, and CURS1). Curcumin,
demethoxycurcumin, and bisdemethoxycurcumin were
produced by adding ferulic acid and/or p-coumaric acid
as precursors. To produce curcuminoids, including cur-
cumin, from the amino acid tyrosine, caffeic acid had to
be produced as an intermediate in the pathway. TAL from
R. glutinis and 4-coumarate 3-hydroxylase (C3H) from
S. espanaensis were selected to produce caffeic acid
based on the results obtained in our previous work
(J. Rodrigues et al., submitted). Caffeoyl-CoA was con-
verted to feruloyl-CoA by CCoAOMT from Medicago sati-
va. This alternative pathway through caffeic acid allowed,
for the first time, the production of curcumin, the most
studied curcuminoid for therapeutic purposes and con-
sidered in many studies as the most potent and active
[3, 4], from the amino acid tyrosine, thus representing an
advance in the heterologous production of curcumin by
E. coli.
2 Materials and methods
2.1 Bacterial strains, plasmids, and chemicals
E. coli NZY5α competent cells (NZYTech, Lisbon, Portu-
gal) were used for molecular cloning and vector propaga-
tion and E. coli K-12 MG1655(DE3) [11] was used as the
host for the expression of genes under T7 promoter con-
trol. The characteristics of all the strains and plasmids
used in this study are described in Table 1. TAL, C3H,
DCS, CURS1, CUS, and CCoAOMT genes were codon-
optimized for E. coli, synthesized and cloned in the plas-
mid vector pUC57 by GenScript (Piscataway, NJ, USA) or
NZYTech (Lisbon, Portugal). The DNA sequences of the
codon-optimized genes are provided in Supporting Infor-
mation, Table S1. pAC_At4CL1 was purchased from
Addgene (Cambridge, MA, USA).
Restriction and ligation enzymes (NEB, Ipswich, MA,
USA), KAPA HiFi DNA polymerase enzyme (Kapa Biosys-
tems, Wilmington, MA, USA), NucleoSpin
®
Plasmid
Miniprep Kit (Macherey-Nagel, Düren, Germany) and
DNA Clean and Concentrator and Gel DNA Recovery Kits
(Zymo Research, Orange, CA, USA) were used according
to the instructions provided by the manufacturers.
L-tyrosine, p-coumaric, caffeic acid, demethoxycur-
cumin, and bisdemethoxycurcumin were purchased from
Sigma–Aldrich (Steinheim, Germany), ferulic acid from
Acros (Geel, Belgium), curcumin from Fisher Scientific
(Loughborough, UK), isopropyl β-
D-thiogalactopyranoside
(IPTG) and Luria-Bertani (LB) medium from NZYTech
(Lisbon, Portugal) and anhydrotetracycline (aTc) from
Acros. Glucose (Acros), Na
2
HPO
4
(Scharlau, Sentmenat,
Spain), MgSO
4
, KH
2
PO
4
(Riel-deHaën, Seelze, Germany),
NH
4
Cl, NaCl, CaCO
3
(Panreac, Barcelona, Spain), and
thiamine (Fisher Scientific, Loughborough, UK) were
used to prepare the M9 modified salt medium. The fol-
lowing mineral traces and vitamins were supplemented
to the M9 medium: FeCl
3
, ZnCl
2
, CoCl
2
, CuCl
2
, nicotinic
acid (Riedel-deHaën), NaMoO
4
, H
2
BO
3
, pyridoxine,
biotin, folic acid (Merck), riboflavin, and pantothenic acid
(Sigma–Aldrich). Ampicillin (Applichem, Darmstadt, Ger-
many), chloramphenicol, kanamycin (NZYTech), and
spectinomycin (Panreac) were used when necessary.
2.2 Construction of plasmids
The genes encoding CUS, CURS1, DCS, CCoAOMT, TAL,
and C3H were expressed in E. coli cells using the
pETDuet-1, pCDFDuet-1, pRSFDuet-1 and pKVS45 vec-
tors (Table 1) and were cloned using the appropriate
restriction enzymes (Supporting information, Table S2).
All constructed plasmids herein described were veri-
fied by colony PCR or digestion and confirmed by
sequencing (Macrogen, Amsterdam, The Netherlands).
2.3 Curcuminoid production
E. coli cells for gene cloning, plasmid propagation, and
inoculum preparation were grown in LB medium at 37°C
and 200 rpm.
For the production of curcuminoids, the cultures were
grown at 37°C in LB (50 mL) to an optical density at
600 nm of 0.3–0.4. IPTG and/or aTc were added (final con-
centration of 1mM and 100ng/mL, respectively) to induce
protein expression. The culture was then incubated for
5h at 26°C. Next, the cells were harvested by centrifuga-
tion, suspended, and incubated at 26°C for 63h in modi-
fied M9 minimal salt medium (50 mL) containing (per
liter): glucose (40g), Na
2
HPO
4
(6g), KH
2
PO
4
(3g), NH
4
Cl
(1g), NaCl (0.5g), CaCl
2
(17mg), MgSO
4
(58mg), thiamine
(340mg), and CaCO
3
(5g) (to control the pH). Trace ele-
ments [FeCl
3
(54mg), ZnCl
2
(4mg), CoCl
2
(4mg), NaMoO
4
(4 mg), CuCl
2
(2 mg), and H
2
BO
3
(1 mg)] and vitamins
[riboflavin (0.84mg), folic acid (0.084mg), nicotinic acid
(12.2mg), pyridoxine (2.8mg), biotin (0.12mg), and pan-
tothenic acid (10.8mg)] were also supplemented to the
M9 medium. Depending on the plasmid(s) present in the
strain, ampicillin (100μg/mL), spectinomycin (100μg/mL),
chloramphenicol (30μg/mL), and/or kanamycin (50μg/mL)
were added. aTc and IPTG were added at the same time.
Substrates were added at time 0 of induction in M9 medi-
um (unless otherwise stated): tyrosine (3mM), p-coumar-
ic acid (2 mM), caffeic acid (1 mM) and ferulic acid
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4 © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
(2mM). All the experiments were performed in triplicate
unless otherwise specified.
2.4 Curcuminoid extraction
For posterior analysis, 2mL of culture broth were taken at
several points during the fermentation and adjusted to
pH 3.0 with HCl (6M). Then, the curcuminoids present in
the samples were extracted with an equal volume of eth-
yl acetate. The extraction procedure was performed more
than once for the cases in which the presence of curcum-
inoids inside the cells after the first extraction was still
visible to the naked eye (yellow coloration). The extracts
were concentrated by solvent evaporation in a fume hood,
suspended in at least 200μL of acetonitrile and then sub-
jected to product analysis by high-performance liquid
chromatography (HPLC).
2.5 HPLC analysis of hydroxycinnamic acids
and curcuminoids
HPLC analysis was used to quantify p-coumaric acid, caf-
feic acid, ferulic acid, curcumin, demethoxycurcumin,
and bisdemethoxycurcumin using a system from Jasco
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Table 1. Bacterial strains and plasmids used in this study
Strains Relevant Genotype Source
E. coli NZY5a fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 NZYTech
Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17
E. coli K-12 MG1655 DE3 F
-
λ
-
ilvG
-
rfb
-
50 rph
-
1 λ(DE3) [11]
Plasmids Construct Source
pETDuet-1 ColE1(pBR322) ori, lacI, double T7lac, Amp
R
Novagen
pCDFDuet-1 CloDF13 ori, lacI, double T7lac, Strep
R
Novagen
pRSFDuet-1 RSF ori, lacI, double T7lac, Kan
R
Novagen
pKVS45 p15A ori, tetR, P
tet
, Amp
R
[24]
pUC57_CCoAOMT pUC57 carrying codon-optimized CCoAOMT from M. sativa GenScript
pUC57_CUS pUC57 carrying codon-optimized CUS from O. sativa NZYTech
pUC57_DCS pUC57 carrying codon-optimized DCS from C. longa NZYTech
pUC57_CURS1 pUC57 carrying codon-optimized CURS1 from C. longa NZYTech
pAC_At4CL1 pAC carrying not codon-optimized 4CL1 from A. thaliana Addgene (35947)
pETDuet_TAL pETduet-1 carrying codon-optimized TAL from R. glutinis unpublished data
pETDuet_C3H pETduet-1 carrying codon-optimized C3H from S. espanaensis unpublished data
pETDuet_TAL_C3H pETduet-1 carrying codon-optimized TAL from R. glutinis and C3H from unpublished data
S. espanaensis
pKVS45_C3H pKVS45 carrying codon-optimized C3H from S. espanaensis unpublished data
pCDFDuet_TAL pCDFDuet-1 carrying codon-optimized TAL from R. glutinis unpublished data
pRSFDuet_CUS pRSFDuet-1 carrying codon-optimized CUS from O. sativa this study
pRSFDuet_CURS1 pRSFDuet-1 carrying codon-optimized CURS1 from C. longa this study
pCDFDuet_DCS pCDFDuet-1 carrying codon-optimized DCS from C. longa this study
pRSFDuet_CCoAOMT pRSFDuet-1 carrying codon-optimized CCoAOMT from M. sativa this study
pRSFDuet_At4CL1_CUS pRSFDuet_At4CL1 carrying codon-optimized CUS from O. sativa this study
pCDFDuet_DCS_CCoAOMT pCDFDuet_DCS carrying codon-optimized CCoAOMT from M. sativa this study
pRSFDuet_CURS1_CCoAOMT pRSFDuet_CURS carrying codon-optimized CCoAOMT from M. sativa this study
pCDFDuet_DCS_TAL pCDFDuet_DCS carrying codon-optimized TAL from R. glutinis this study
pCDFDuet_DCS_At4CL1 pCDFDuet_DCS carrying 4CL1 from A. thaliana this study

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5
(Easton, MD, USA) (PU-2080 Plus Pump unit, LG-2080-02
Ternary Gradient unit, a DG-2080-53 3-Line Degasser
unit, a UV-2075 Plus Intelligent UV/VIS Detector unit, and
AS-2057 Plus Intelligent Sampler unit) and a Grace All-
tech Platinum EPS C18 column (3μm, 150mm×4.6mm)
(Grace, Columbia, MD, USA). Mobile phases A and B were
composed of water (0.1% trifluoroacetic acid) and ace-
tonitrile, respectively. For the hydroxycinnamic acids
quantification, the following gradient was used at a con-
stant flow rate (1 mL/min): 10–20% acetonitrile (mobile
phase B) for 16min. Quantification was based on the peak
areas obtained at 310nm for p-coumaric acid, caffeic acid
and ferulic acid. The retention times of p-coumaric acid,
caffeic acid and ferulic acid were 8.0, 11.8, and 13.8min,
respectively. For the curcuminoid quantification, a gradi-
ent of 40–43% acetonitrile (mobile phase B) for 15min and
43% acetonitrile for an additional 5 min was used. The
curcuminoids were detected at 425 nm and the retention
times of bisdemethoxycurcumin, demethoxycurcumin
and curcumin were 12.4, 13.5, and 14.5min, respectively.
2.6 Protein analysis
(CUS, CURS1, DCS, and CCoAOMT)
E. coli K-12 MG1655(DE3) cells harboring pRSFDuet_CUS,
pRSFDuet_CURS1, pRSFDuet_DCS and pRSFDuet_
CCoAOMT were grown in LB at 37°C to an optical densi-
ty at 600nm of 0.6. IPTG was added (at a final concentra-
tion of 1mM) to induce protein expression, and the cul-
ture was incubated for 5h. Samples (10mL culture medi-
um) were taken at time 0 and 5h of induction. Samples
were centrifuged and the cells were suspended in phos-
phate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl,
10mM Na
2
PO
4
, 1.8mM KH
2
PO
4
, pH 7.4) and disrupted by
sonication on ice for 3min. After centrifugation, the pro-
tein concentration from the resulting supernatant was
determined using Protein Assay Dye Reagent Concen-
trate (BioRad, Hercules, CA, USA) and bovine gamma
globulin (BSA) (NEB) as a standard. The expression levels
of the enzymes were examined using sodium dodecyl sul-
fate polyacrylamide gel electrophoretic (SDS–PAGE)
analysis. Fifteen to 20μg of total protein were loaded in
4–20% Mini-PROTEAN
®
TGX™ Precast Gels (BioRad).
The protein marker used was Precision Plus Protein™
Unstained (BioRad) and/or ColorPlus™ Prestained Protein
Ladder Broad Range (NEB). For gel staining, Bio-Safe
Coomassie Stain (BioRad) was used.
3 Results
3.1 The 4-coumarate-CoA ligase (4CL) limiting step
4CL enzymes convert the hydroxycinnamic acids
(p-coumaric acid, caffeic acid, and ferulic acid) to their
corresponding CoA esters (p-coumaroyl-CoA, caffeoyl-
CoA, and feruloyl-CoA) (Fig. 1) with different substrate
preferences and specificities. We studied three different
4CL enzymes (LeAt4CL1, At4CL2, and At4CL1) and only
At4CL1 was found to be functionally expressed. Recently,
this enzyme was successfully used in the production of
curcuminoids [8] and in stilbene and flavonoid biosynthe-
sis [12, 13]. In the current work, this enzyme was tested in
vivo with CUS and CURS1/DCS. Since the results
obtained regarding the production of curcuminoids were
positive, all the further optimization tests were performed
using this 4CL enzyme.
3.2 Very low production of curcuminoids
using CUS from O. sativa
Codon-optimized CUS was cloned in pRSFDuet-1 and its
expression was confirmed on a protein gel (44.3 kDa)
(Supporting information, Fig. S1). After that, CUS was
tested in E. coli with At4CL1 (pRSFDuet_CUS +
pAC_At4CL1) (Fig. 2). These enzymes produced bis -
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Figure 2. Curcuminoid production from
p-coumaric acid and ferulic acid using
4-coumarate-CoA ligase (4CL1) from
A. thaliana and curcuminoid synthase
(CUS) from O. sativa. Bisdemethoxycur-
cumin was also produced from tyrosine
using TAL from R. glutinis. Error bars are
standard deviations from triplicate
experiments.

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Claire M. Palmer1, Hal S. Alper1Institutions (1)
TL;DR: In this review, the recent advances in the engineering of microbial hosts for the production of type III PKS‐derived polyketides are highlighted and tools and strategies that can be leveraged to realize the potential of microbial production and diversification of these molecules are highlighted.
Abstract: Polyketides are a unique class of molecules with attractive bioactive and chemical properties. As a result, biorenewable production is being explored with these molecules as potential pharmaceutical, fuel, and material precursors. In particular, type III polyketide synthases enable access to a diverse class of chemicals using a relatively simple biochemical synthesis pathway. In this review, the recent advances in the engineering of microbial hosts for the production of type III PKS-derived polyketides are highlighted. In particular, the field has moved beyond simple proof-of-concept and has been exploring engineering efforts that have led to improved production scales. This review details engineering progress for the production of acetyl-CoA- and malonyl-CoA-derived polyketides including the products triacetic acid lactone and phloroglucinol as well as polyphenolic, phenylpropanoid-derived compounds including flavonoids, stilbenoids, and curcuminoids. Specifically, the authors focus on enumerating the metabolic engineering strategies employed and product titers achieved for these molecules. Finally, the authors highlight tools and strategies that can be leveraged to realize the potential of microbial production and diversification of these molecules.

21 citations


Journal ArticleDOI
TL;DR: It was demonstrated that the biosynthetic pathway of p-coumaric acid, caffeic acid and curcumin in E. coli can be triggered by using heat shock promoters, suggesting its potential for the development of new industrial bioprocesses or even new bacterial therapies.
Abstract: Hydroxycinnamic acids and curcumin are compounds with great therapeutic potential, including anticancer properties. In this study, p-coumaric acid, caffeic acid and curcumin were produced in Escherichia coli. Their production was induced by heat using the dnaK and ibpA heat shock promoters. The ribosome binding site (RBS) used was tested and further optimized for each gene to assure an efficient translation. p-Coumaric acid was successfully produced from tyrosine and caffeic acid was produced either from tyrosine or p-coumaric acid using tyrosine ammonia lyase (TAL) from Rhodotorula glutinis, 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris. The highest p-coumaric acid production obtained was 2.5 mM; caffeic acid production reached 370 μM. Regarding curcumin, 17 μM was produced using 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana, diketide-CoA synthase (DCS) and curcumin synthase 1 (CURS1) from Curcuma longa. Stronger RBSs and/or different induction conditions should be further evaluated to optimize those production levels. Herein it was demonstrated that the biosynthetic pathway of p-coumaric acid, caffeic acid and curcumin in E. coli can be triggered by using heat shock promoters, suggesting its potential for the development of new industrial bioprocesses or even new bacterial therapies.

20 citations


References
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Journal ArticleDOI
TL;DR: Curcumin, a spice once relegated to the kitchen shelf, has moved into the clinic and may prove to be "Curecumin", a therapeutic agent in wound healing, diabetes, Alzheimer disease, Parkinson disease, cardiovascular disease, pulmonary disease, and arthritis.
Abstract: Although turmeric (Curcuma longa; an Indian spice) has been described in Ayurveda, as a treatment for inflammatory diseases and is referred by different names in different cultures, the active principle called curcumin or diferuloylmethane, a yellow pigment present in turmeric (curry powder) has been shown to exhibit numerous activities. Extensive research over the last half century has revealed several important functions of curcumin. It binds to a variety of proteins and inhibits the activity of various kinases. By modulating the activation of various transcription factors, curcumin regulates the expression of inflammatory enzymes, cytokines, adhesion molecules, and cell survival proteins. Curcumin also downregulates cyclin D1, cyclin E and MDM2; and upregulates p21, p27, and p53. Various preclinical cell culture and animal studies suggest that curcumin has potential as an antiproliferative, anti-invasive, and antiangiogenic agent; as a mediator of chemoresistance and radioresistance; as a chemopreventive agent; and as a therapeutic agent in wound healing, diabetes, Alzheimer disease, Parkinson disease, cardiovascular disease, pulmonary disease, and arthritis. Pilot phase I clinical trials have shown curcumin to be safe even when consumed at a daily dose of 12g for 3 months. Other clinical trials suggest a potential therapeutic role for curcumin in diseases such as familial adenomatous polyposis, inflammatory bowel disease, ulcerative colitis, colon cancer, pancreatic cancer, hypercholesteremia, atherosclerosis, pancreatitis, psoriasis, chronic anterior uveitis and arthritis. Thus, curcumin, a spice once relegated to the kitchen shelf, has moved into the clinic and may prove to be "Curecumin".

1,714 citations


Journal ArticleDOI
Bharat B. Aggarwal1, Bokyung Sung1Institutions (1)
TL;DR: Because of the crucial role of inflammation in most chronic diseases, the potential of curcumin has been examined in neoplastic, neurological, cardiovascular, pulmonary and metabolic diseases.
Abstract: Curcumin (diferuloylmethane), a yellow pigment in the spice turmeric (also called curry powder), has been used for centuries as a treatment for inflammatory diseases. Extensive research within the past two decades has shown that curcumin mediates its anti-inflammatory effects through the downregulation of inflammatory transcription factors (such as nuclear factor κB), enzymes (such as cyclooxygenase 2 and 5 lipoxygenase) and cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6). Because of the crucial role of inflammation in most chronic diseases, the potential of curcumin has been examined in neoplastic, neurological, cardiovascular, pulmonary and metabolic diseases. The pharmacodynamics and pharmacokinetics of curcumin have been examined in animals and in humans. Various pharmacological aspects of curcumin in vitro and in vivo are discussed in detail here.

865 citations


Journal ArticleDOI
TL;DR: An overview of the extensive published literature on the use of curcumin as a therapy for malignant and inflammatory diseases and its potential use in the treatment of degenerative neurologic diseases, cystic fibrosis, and cardiovascular diseases is provided.
Abstract: Curcumin is a natural polyphenol used in ancient Asian medicine. Since the first article referring to the use of curcumin to treat human disease was published in The Lancet in 1937, >2,600 research studies using curcumin or turmeric have been published in English language journals. The mechanisms implicated in the inhibition of tumorigenesis by curcumin are diverse and appear to involve a combination of antiinflammatory, antioxidant, immunomodulatory, proapoptotic, and antiangiogenic properties via pleiotropic effects on genes and cell-signaling pathways at multiple levels. The potentially adverse sequelae of curcumin's effects on proapoptotic genes, particularly p53, represent a cause for current debate. When curcumin is combined with some cytotoxic drugs or certain other diet-derived polyphenols, synergistic effects have been demonstrated. Although curcumin's low systemic bioavailability after oral dosing may limit access of sufficient concentrations for pharmacologic effects in tissues outside the gastrointestinal tract, chemical analogues and novel delivery methods are in preclinical development to overcome this barrier. This article provides an overview of the extensive published literature on the use of curcumin as a therapy for malignant and inflammatory diseases and its potential use in the treatment of degenerative neurologic diseases, cystic fibrosis, and cardiovascular diseases. Despite the breadth of the coverage, particular emphasis is placed on the prevention and treatment of human cancers.

536 citations


Journal ArticleDOI
TL;DR: The current review provides an overview of the history, chemistry, analogs, and mechanism of action of curcumin.
Abstract: Although the history of the golden spice turmeric (Curcuma longa) goes back thousands of years, it is only within the past century that we learned about the chemistry of its active component, curcumin. More than 6000 articles published within the past two decades have discussed the molecular basis for the antioxidant, anti-inflammatory, antibacterial, antiviral, antifungal, and anticancer activities assigned to this nutraceutical. Over sixty five clinical trials conducted on this molecules, have shed light on the role of curcumin in various chronic conditions, including autoimmune, cardiovascular, neurological, and psychological diseases, as well as diabetes and cancer. The current review provides an overview of the history, chemistry, analogs, and mechanism of action of curcumin.

490 citations


Journal ArticleDOI
Jürgen Ehlting1, Daniela Büttner1, Qing Wang2, Carl J. Douglas2  +2 moreInstitutions (2)
TL;DR: Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At 4CL2 isoforms belonging to class Iand At4 CL3 to class II.
Abstract: Summary The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and specificities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to flavonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than flavonoids.

382 citations


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No. of citations received by the Paper in previous years
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20218
20208
20195
20184
20176
20164