Synthesis of Diverse Hydroxycinnamoyl Phenylethanoid Esters Using Escherichia coli

Article abstract of DOI:10.1021/acs.jafc.9b00041

Caffeic acid phenethyl ester (CAPE) is an ester of a hydroxycinnamic acid (phenylpropanoid) and a phenylethanoid (2-phenylethanol; 2-PE), which has long been used in traditional medicine. Here, we synthesized 54 hydroxycinnamic acid-phenylethanoid esters by feeding 64 combinations of hydroxycinnamic acids and phenylethanols to Escherichia coli harboring the rice genes OsPMT and Os4CL. The same approach was applied for ester synthesis with caffeic acid and eight different phenyl alcohols. Two hydroxycinnamoyl phenethyl esters, p-coumaroyl tyrosol and CAPE, were also synthesized from glucose using engineered E. coli by introducing genes for the synthesis of substrates. Consequently, we synthesized approximately 393.4 mg/L p-coumaroyl tyrosol and 23.8 mg/L CAPE with this approach. Overall, these findings demonstrate that the rice PMT and 4CL proteins can be used for the synthesis of diverse hydroxycinnamoyl phenylethanoid esters owing to their promiscuity and that further exploration of the biological activities of these compounds is warranted.

Full text of DOI:10.1021/acs.jafc.9b00041

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Biotechnology and Biological Transformations
Synthesis of diverse hydroxycinnamoyl
phenylethanoid esters using Escherichia coli
Min Kyung Song, A Ra Cho, Geun Young Sim, and Joong-Hoon Ahn
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00041 • Publication Date (Web): 30 Jan 2019
Downloaded from http://pubs.acs.org on February 2, 2019
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Synthesis of diverse hydroxycinnamoyl phenylethanoid esters using Escherichia coli
Min Kyung Song, A Ra Cho, Geun Young Sim, Joong-Hoon Ahn*
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk
University, Seoul 05029, Republic of Korea
*Corresponding author
Phone: +82-2-45-3764; Fax: +82-2-3437-6106
E-mail: jhahn@konkuk.ac.kr
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ABSTRACT
Caffeic acid phenethyl ester (CAPE) is an ester of a hydroxycinnamic acid
(phenylpropanoid) and a phenylethanoid (2-phenylethanol; 2-PE), which has long been used
in traditional medicine. Here, we synthesized 54 hydroxycinnamic acid-phenylethanoid esters
by feeding 64 combinations of hydroxycinnamic acids and phenylethanols to Escherichia coli
harboring the rice genes OsPMT and Os4CL. The same approach was applied for ester
synthesis with caffeic acid and eight different phenyl alcohols. Two hydroxycinnamoyl
phenethyl esters, p-coumaroyl tyrosol and CAPE, were also synthesized from glucose using
engineered E. coli by introducing genes for the synthesis of substrates. Consequently, we
synthesized approximately 393.4 mg/L p-coumaroyl tyrosol and 23.8 mg/L CAPE with this
approach. Overall, these findings demonstrate that the rice PMT and 4CL proteins can be used
for the synthesis of diverse hydroxycinnamoyl phenylethanoid esters owing to their
promiscuity and that further exploration of the biological activities of these compounds is
warranted.
KEYWORDS: Caffeic acid phenethyl ester, Hydroxycinnamic acids, Metabolic engineering,
Phenylethanoids
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INTRODUCTION
Diverse compounds with beneficial biological activities are readily available in nature,
which can be exploited for medicinal and industrial purposes. In particular, the secondary
metabolites of plants, derived from certain core structures, show extensive structural diversity.
For example, polyphenols synthesized from either phenylalanine or tyrosine are among the
most abundant natural products found in plants,1 comprising at least four groups of compounds
based on the carbon skeleton2: hydroxybenzoic acids, hydroxycinnamic acids, stilbenes, and
flavonoids. Hydroxycinnamic acids with a C6-C3 carbon skeleton include p-coumaric acid,
caffeic acid, ferulic acid, and others, which exhibits diverse biological activities.
Phenylethanoids are a recently discovered group of polyphenols3 with a C6-C2 carbon skeleton;
typical members of this group include tyrosol and hydroxytyrosol.4, 5 Thus, the development
of engineering strategies for the enhanced synthesis of polyphenols and their derivatives could
enable the production of diverse bioactive products.
Ester formation between carboxylic acids and alcohols is a well-established chemical
reaction. Likewise, the formation of esters between hydroxycinnamic acids and
phenylethanoids extends the diversity of natural products. Caffeic acid phenethyl ester (CAPE)
is one such ester of caffeic acid and 2-phenylethanol (2-PE) and is a component of propolis,
derived from the honeybee.6 CAPE has also been used as a traditional medicine given its
diverse biological activities, including antioxidant, antimicrobial, anti-inflammatory,
anticancer, neuroprotective, and immunomodulatory activities.7-9 In addition, CAPE has
emerged as a candidate drug for the prevention of neurodegenerative diseases, such as
Alzheimer’s disease and Parkinson’s disease.10-12
Despite the application potential of CAPE, its biological synthetic pathway has not yet
been elucidated, although the chemical synthesis of its derivatives has been attempted.11, 13
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Determination of its biosynthetic pathway would allow for the rational design of a complete
CAPE pathway via assembly of relevant genes from various pathways or organisms. Based on
other known biological ester formation reactions, it can be speculated that acyl-coenzyme A
(CoA), which stores a large amount of energy in its thioester bond, may also serve as an acyl
donor for the formation of CAPE.14 In this scenario, caffeic acid would be activated by CoA
attachment, followed by conjugation with 2-PE. CoA is known to attach to hydroxycinnamic
acid via 4-cinnamate CoA ligase (4CL).15 Although the CAPE synthetic pathway is unknown,
those of its two substrates, caffeic acid and 2-PE, have been elucidated, and their biological
synthesis was successfully achieved using genes from various sources engineered in a
microbial system. Caffeic acid has been synthesized in Escherichia coli from tyrosine using
tyrosine ammonia lyase (TAL) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC),16 and
2-PE has been synthesized in both Kluyveromyces marxianus and E. coli from phenylpyruvate
18
using phenylpyruvate decarboxylase (encoded by aro10).17,
In plants, 2-PE can be
synthesized from phenylalanine by aromatic amino acid decarboxylases (AAADs).19 However,
the enzyme involved in the formation of esters from hydroxycinnamic acids and 2-PE remains
unknown, although a yeast gene was used to achieve the esterification of caffeic acid and 2-
PE.20 Therefore, to effectively synthesize CAPE and other phenylpropanoid hydroxycinnamate
esters (PHEs), it is necessary to comprehensively explore and identify the gene(s) responsible
for the esterification reaction. Accordingly, we focused on BAHD acyltransferase,21 also
known as HxxxD acyltransferase, as an enzyme that may potentially catalyze this reaction. We
cloned the BAHD transferase (PMT) gene and an E. coli strain harboring PMT and 4CL from
Oryza sativa was used to synthesize diverse PHEs. Furthermore, two PHEs (p-coumaroyl
tyrosol and CAPE) were synthesized from glucose using engineered E. coli.
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MATERIALS AND METHODS
Chemicals
Hydroxycinnamic acid derivatives (p-coumaric acid, caffeic acid, ferulic acid, 3-
methoxycinnamic acid, cinnamic acid, o-coumaric acid, m-coumaric acid, and 4-
methoxycinnamic acid) and benzyl alcohol derivatives (benzyl alcohol, 2-PE, 3-phenyl-1-
propanol, 4-phenyl-1-butanol, 5-phenyl-1-pentanol, 7-phenyl-1-heptanol, 8-phenyl-1-octanol,
and 9-phenyl-1-nonanol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tyrosol,
hydroxytyrosol,
2-hydroxyphenethyl
alcohol,
3-hydroxyphenethyl
alcohol,
2-
methoxyphenethyl alcohol, 3-methoxyphenethyl alcohol, and 4-methoxyphenethyl alcohol
were also purchased from Sigma-Aldrich. CAPE was purchased from Tokyo Chemical
Industry (Tokyo, Japan).
Plasmid construction
The p-coumarate monolignol transferase (PMT) genes from rice (O. sativa;
XM_015765814) and corn (Zea mays; ZM_BFc0019F21) were cloned using reverse
transcription-polymerase chain reaction (RT-PCR). cDNA was synthesized from the total RNA
of 3-week-old rice or corn using the Omniscript reverse transcriptase (Qiagen, Hilden,
Germany)
aagaattcaATGGGGTTCGCGGTGGT-3′
aagcggccggTTACTTGTCGAAGGCCTTGATCTC-3′;
with
the
following
primers:
O.
sativa
PMT,
5′-
5′-
5′-
and
Z.
mays
PMT,
aagaattcaATGGGCACCATCGTCGAC-3′ and 5′-aagcggccgcCTAGGCGGCCTGCTGGC-3′
(lowercase letters indicate EcoRI and NotI sites, respectively). The resulting PCR products
were sequenced and subcloned into the EcoRI/NotI sites of the pGEX 5X-3 or pET Duet1
plasmids (GE Healthcare, Oxford, UK) to obtain pG-OsPMT, pE-OsPMT, and pG-ZmPMT.
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E. coli cells harboring both an empty pGEX vector and 4CL were used as a control. The 4CL
and AAS genes were cloned as previously described,15, 22 and the pC-AAS-4CL plasmid was
constructed by subcloning AAS into the EcoRI/NotI site of pCDF-Duet1 and subcloning 4CL
into the NdeI/XhoI site of pCDF-Duet 1. pA-SeTAL, pA-SeTAL-aroG-TyrA, and pA-SeTAL-
aroGfbr-TyrAfbr were constructed as previously described.23 HpaBC was cloned as previously
described16 and subcloned into the NdeI/XhoI site of pE-OsPMT.
Production of CAPE derivatives
To verify the production of hydroxycinnamic acid–phenylethanoid conjugates, E. coli
B-CP1 cells harboring both pG-OsPMT and pC-4CL were grown in Luria-Bertani (LB)
medium containing 50 μg/mL ampicillin and spectinomycin for 18 h at 37 C. A 1/50 volume
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of the culture was then inoculated into fresh medium and incubated at 37 C until the optical
density at 600 nm (OD600) reached 0.8. Isopropyl--D-1-thiogalactopyranoside (IPTG) was
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added at a final concentration of 1 mM, and the culture was further incubated at 18 C for 20
h. The cells were harvested and resuspended at OD600 = 3.0 in M9 medium containing 100 μM
of each substrate. The mixture was incubated at 30 C for 24 h, and the culture supernatant was
extracted with ethyl acetate. The reaction product was analyzed with high-performance liquid
chromatography (HPLC).
To synthesize p-coumaroyl tyrosol and CAPE from glucose, the E. coli strain was
grown in LB containing appropriate antibiotics at 37 C for 18 h, and then the culture was
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inoculated into fresh medium and further incubated at 37 C for 4 h with shaking. The cells
were harvested and resuspended in M9 medium containing 1% yeast extract, antibiotics, and 1
mM IPTG. Dimethyl sulfoxide (DMSO) was added to the medium at a final concentration of
10%, and the culture was incubated at 30 C.
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Analysis of reaction products
The culture, including cells, was extracted with ethyl acetate twice. The organic layer
was recovered, dried, and dissolved in methanol or DMSO, and then HPLC analysis was
carried out as described previously,16 except that ultraviolet detection was carried out at 270
nm or 320 nm. For matrix-assisted laser desorption/ionization-time-of-flight mass
spectrometry (MALDI-TOF MS), the sample was analyzed in methanol using gold
nanoparticles as a matrix. Equal volumes (1 μL) of the sample and matrix were pipetted into
the wells of a stainless steel 384-well target plate (Bruker Daltonics, Bremen, Germany), dried
in air at room temperature, and analyzed directly by mass spectrometry (MS) using an Autoflex
III MALDI-TOF mass spectrometer (Bruker Daltonics) equipped with a smart beam laser as
an ionization source. All spectra were acquired with a 19 kV accelerating voltage, 100 Hz
repetition rate, and an average of ~500 shots. High-resolution MS (Q Exactive mass
spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) was carried out as described
previously.24 In brief, the data-dependent top method was used to obtain mass spectra using an
electrospray ionization source in positive-ionization mode. The mass spectra were recorded in
the range of m/z 300–2,000 Da.
Nuclear magnetic resonance (NMR) spectroscopy was carried out as described by Yoon et
al.25 The NMR data were as follows: CAPE: 1H NMR (400 MHz, acetone) δ (ppm): 2.99 (2H,
t, J=7.0 Hz), 4.35 (2H, t, J=7.0 Hz), 6.26 (1H, d, J=16.0 Hz), 6.86 (1H, d, J=8.2 Hz), 7.02 (1H,
dd, J=8.2, 2.0 Hz), 7.14 (1H, d, J=2.0 Hz), 7.22 (1H, m), 7.31 (4H, m), 7.52 (1H, d, J=16.0
Hz).
p-coumaroyl tyrosol: 1H NMR (acetone-d6, 400 MHz) δ: 2.88 (2H, t, J = 6.9 Hz, H-
α), 4.28 (2H, t, J = 6.9 Hz, H-β), 6.28 (1H, d, J = 15.7 Hz, H-γ), 6.78 (2H, d, J = 8.4 Hz, H-3),
6.87 (2H, d, J = 8.5 Hz, H-3'), 7.12 (2H, d, J = 8.4 Hz, H-2), 7.50 (2H, d, J = 8.5 Hz, H-2'),
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7.58 (1H, d, J = 15.7 Hz, H-δ).
RESULTS
Screening of enzymes responsible for esterification of hydroxycinnamoyl-CoA and 2-PE
The esterification reaction that produces CAPE from caffeic acid and 2-PE requires a large
amount of energy,26 which was supplied by the attachment of CoA to caffeic acid. Accordingly,
we focused on BAHD acyltransferase,21 also known as HxxxD acyltransferase (PMT), as a
potential enzyme for catalyzing this reaction. PMT from rice has been shown to catalyze ester
formation between monolignols (p-coumaroyl alcohol, caffeoyl alcohol, coniferyl alcohol, and
sinapyl alcohol) and hydroxycinnamoyl-CoA.27 Given the structural similarity between
monolignols and phenylethanoids, we selected PMT as a candidate for the esterification
between caffeoyl-CoA and 2-PE. We tested the PMTs from rice (Oryza sativa) and corn (Zea
mays) for their ability to synthesize CAPE from 2-PE and caffeic acid. The genes encoding
these proteins were transformed into E. coli along with the 4CL gene (strains B-CP1 and B-
CP2). p-Coumaric acid, o-coumaric acid, m-coumaric acid, and caffeic acid were all tested as
hydroxycinnamic acids, and 2-PE was used as the phenylethanoid in all assessments. E. coli
B-CP1 produced a new product peak in the presence of p-coumaric acid, m-coumaric acid, and
caffeic acid (Fig. 1), while strain B-CP2 did not synthesize any product with any of the
substrates tested. In addition, as a control, E. coli harboring an empty pGEX vector and 4CL
did not produce any new compound. MS analysis demonstrated that the molecular masses of
the reaction products from B-CP1 corresponded with those of p-coumaric acid phenyl ester, m-
coumaric acid phenyl ester, and CAPE, respectively. Furthermore, the structure of the product
produced from caffeic acid and 2-PE using B-CP1 was confirmed to be CAPE using proton
NMR. These results indicated that OsPMT could be used to synthesize diverse PHEs. We used
OsPMT for subsequent analysis.
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Synthesis of diverse CAPE derivatives using strain B-CP1
The engineered E. coli strain B-CP1 expressing OsPMT and 4CL was used as a
biocatalyst to synthesize hydroxycinnamoyl phenethyl esters. Initially, we tested eight
hydroxycinnamic acid derivatives (cinnamic acid, o-coumaric acid, m-coumaric acid, p-
coumaric acid, caffeic acid, ferulic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic
acid) with 2-PE. Among them, all of the combinations except for 3-methoxycinnamic acid and
2-PE resulted in a new peak, as observed by HPLC, which was not observed in the control E.
coli strain harboring only 4CL. The MS analysis of all combinations showed the expected
molecular mass (Table 2 and Supporting Information), demonstrating that the product from
each combination was the expected ester formed between the hydroxycinnamic acid and 2-PE.
Since Sim et al28 showed that 4CL can convert eight hydroxycinnamic acids into the
corresponding hydroxycinnamic acid-CoAs as the first step of the reaction, thereby facilitating
ester formation, we extended the substrates further to 2-PE derivatives, including tyrosol,
hydroxytyrosol,
2-hydroxyphenethyl
alcohol,
2-(3-hydroxyphenyl)
alcohol,
2-
methoxyphenethyl alcohol, 3-methoxyphenethyl alcohol, and 4-methoxyphenethyl alcohol.
Overall, 64 combinations of the eight phenylethanoids and eight hydroxycinnamic acids were
tested using the BCP-1 strain, resulting in the formation of 54 hydroxycinnamoyl phenethyl
esters (Table 2 and Supporting Information), as confirmed by MS analysis (Supporting
Information). p-Coumaric acid, m-coumaric acid, and caffeic acid formed esters with all of the
2-PE derivatives tested. 3-Methoxycinnamic acid, 4-methoxycinnamic acid, and cinnamic acid
formed esters with seven of the 2-PE derivatives, while ferulic acid formed esters with six of
the 2-PE derivatives. o-Coumaric acid was the poorest acyl group donor and formed a product
with only three of the 2-PE derivatives. Overall, these results suggested that OsPMT used
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diverse substrates and can form esters. The observed differences in ester formation were related
to variations in the substrate specificity of OsPMT and not that of Os4CL, as all
hydroxycinnamic acid derivatives that were converted into hydroxycinnamic acid-CoA by
Os4CL formed an ester with at least one 2-PE derivative in all cases.
Moreover, we tested various phenyl alcohols (benzyl alcohol, 3-phenyl-1-propanol, 4-
phenyl-1-butanol, 5-phenyl-1-pentanol, 7-phenyl-1-heptanol, 8-phenyl-1-octanol, and 9-
phenyl-1-nonanol) as substrates along with caffeic acid in strain B-CP1. HPLC analysis of the
culture medium after ethyl acetate extraction from each combination of substrates showed a
new peak (Fig. 2), and the MS analysis of each product corresponded with the expected
molecular mass (Table 3 and Supporting Information). This indicated that OsPMT could also
use diverse phenyl alcohols as hydroxycinnamic acid acceptors, further demonstrating its broad
substrate range.
Synthesis of p-coumaroyl tyrosol and CAPE from glucose in engineered E. coli
We attempted to synthesize p-coumaroyl tyrosol without feeding any substrates. p-
Coumaroyl tyrosol is synthesized from p-coumaric acid and tyrosol, both of which were
derived from tyrosine: AAS synthesizes tyrosol from tyrosine,22 and TAL produces p-coumaric
acid from tyrosine. Thus, genes encoding these two proteins, along with 4CL and PMT, were
used to synthesize p-coumaroyl tyrosol from glucose. The resulting strain (B-CT1) was
examined for its ability to produce p-coumaroyl tyrosol. The strain B-CT1 produced a new
peak, and this product was determined to be p-coumaroyl tyrosol by NMR. We also observed
the synthesis of p-coumaroyl 2-PE, as AAS also converted phenylalanine into 2-PE.19
As both p-coumaric acid and tyrosol, the precursors for p-coumaroyl tyrosol, were
synthesized from tyrosine, we overexpressed genes (aroG and tyrA) that were known to
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increase the tyrosine content in E. coli.29 Because AroG and TyrA are inhibited by tyrosine,14
feedback inhibition-free versions of aroG and tyrA (aroGfbr and tyrAfbr, respectively) were also
used. The combination of aroG, tyrA, aroGfbr, and tyrAfbr was tested (Table 1) for comparison
of the production of p-coumaroyl tyrosol in three strains (B-CT1–3). These three strains were
synthesized mainly p-coumaroyl tyrosol and p-coumaroyl 2-phenyl ethanol (Fig. 3A) but the
yields of these two compounds were different (Fig. 4). Strain B-CT3 synthesized
approximately 146.5 mg/L p-coumaroyl tyrosol, followed by B-CT2 (10.1 mg/L), and B-CT1
(1.6 mg/L). Thus, we further used strain B-CT3 to examine the production of p-coumaroyl
tyrosol for 84 h. p-Coumaroyl tyrosol was detected at 12 h, and its production rapidly increased
until 72 h, at which point approximately 392.4 mg/L was produced (Fig. 5A).
Next, CAPE was synthesized from glucose. Among the two precursors necessary for
the synthesis of CAPE, we previously reported the synthesis of caffeic acid using E. coli
harboring SeTAL and HpaBC16; SeTAL convertrd tyrosine into p-coumaric acid, which was
then hydroxylated by HpaBC to caffeic acid. 2-PE was synthesized using AAS (data not shown)
as, in addition to converting tyrosine into tyrosol, AAS was known to convert phenylalanine
into 2-PE.19 Therefore, the synthesis of CAPE required an additional gene, HpaBC, compared
to the synthesis of p-coumaroyl tyrosol. HpaBC was introduced into strain B-CT3, and the
resulting transformant (B-CP3) was used for the synthesis of CAPE from glucose. Strain B-
CP3 synthesized CAPE as a main product (P3 in Fig. 3B). Three additional byproducts (P1,
P2, and P4 in Fig. 3C) were also detected. By comparing the retention times of these products
with possible candidates synthesized from the combinations of eight hydroxycinnamic acids
and eight phenylethanoids, P1 and P4 were considered likely to be cafferoyl hydroxytyrosol
and p-coumaroyl 2-PE, respectively. These products could be expected because HpaBC can
hydroxylate tyrosol to produce hydroxytyrosol and can also convert p-coumaric acid into
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caffeic acid. Three phenylethanoids (2-PE, tyrosol, and hydroxytyrosol) and two
hydroxycinnamates (p-coumaric acid and caffeic acid) may therefore be present in strain B-
CP3, and thus, theoretically, six conjugates could be synthesized in the cells. However, only
three compounds (CAPE, caffeoyl hydroxytyrosol, and p-coumaroyl 2-PE) were observed.
Substrate availability in the cell and the substrate preference of each enzyme were likely the
main factors determining the final products. Although P2 was always observed as a minor
product when caffeic acid was used as a substrate (data not shown), we could not confirm its
identity.
Using this strain, we examined the synthesis of the three compounds for 72 h. The
synthesis of CAPE dramatically increased until 36 h; its synthesis continued to increase until
72 h, at which point approximately 24.0 mg/L CAPE was synthesized (Fig. 5B). Caffeoyl
hydroxytyrosol was synthesized until 36 h, though its synthesis did not increase over time. The
synthesis of p-coumaroyl 2-PE decreased after 24 h, suggesting that some was converted into
CAPE. Previously, we showed that E. coli harboring HpaBC converted avenanthramide (avn)D
into avnF.30 In this reaction, p-coumaric acid, which formed part of avnD, was converted into
caffeic acid. 2-PE itself was not converted into tyrosol, as HpaBC only hydroxylated
compounds with a phenolic hydroxy group.31 Thus, p-coumaroyl 2-PE could be converted into
CAPE but not into p-coumaroyl tyrosol or caffeoyl tyrosol.
DISCUSSION
We successfully achieved the synthesis of 62 hydroxycinnamoyl phenylethanoid esters
owing to the substrate promiscuity of OsPMT and Os4CL. Os4CL attached CoA to diverse
phenylpropanoids, and OsPMT used diverse phenylpropanyl-CoAs and phenylalcohols to form
esters. Originally, OsPMT was reported as a catalyst for the synthesis of monolignol p-
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coumaric acid conjugates27; however, we showed that it could use a broad array of other phenyl
alcohols, ranging from benzoyl alcohol to 9-phenyl-1-nonanol. This raises the possibility of
synthesizing more hydroxycinnamoyl phenylethanoid esters using other hydroxycinnamic
acids and phenyl alcohols. Based on the promiscuity of OsPMT and Os4CL, we estimated that
it would be possible to synthesize approximately 50 more esters. Other enzymes with similar
promiscuous properties were used to synthesize phenyl anthranilate32 in a manner similar to
combinatorial synthesis in chemistry. Such diversity would allow for further exploration of the
various biological activities of CAPE and its derivatives.
The solubility of CAPE in the medium had an influence on the final yield. Addition of
10% DMSO to the medium resulted in an approximately 3-fold greater production yield.
However, a DMSO concentration greater than 10% affected E. coli viability and therefore
reduced productivity. DMSO was known to inhibit the growth of bacteria33; however, 10%
DMSO did not have serious effects on bacterial growth.
During the synthesis of p-coumaroyl tyrosol from glucose, p-coumaroyl 2-PE was also
synthesized. Similarly, p-coumaroyl 2-PE and cafferoyl hydroxytyrosol were synthesized
during CAPE synthesis. Phenylalanine and tyrosine were both substrates provided by the host
and were used by AAS to synthesize 2-PE and tyrosol, respectively. TAL had a stronger
preference for tyrosine than for phenylalanine and therefore synthesized p-coumaric acid but
not cinnamic acid. In addition, HpaBC could hydroxylate compounds having phenolic hydroxy
group; it converted p-coumaric acid and tyrosol into caffeic acid and hydroxytyrosol,
respectively, and likely converted p-coumaroyl 2-PE into CAPE given that the concentration
of p-coumaroyl 2-PE decreased. Os4CL and OsPMT used all of the synthesized substrates,
including three phenylethanoids (2-PE, tyrosol, and hydroxytyrosol) and two
hydroxycinnamates (p-coumaric acid and caffeic acid), to form hydroxycinnamoyl phenethyl
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295
296
297
298
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301
302
303
304
305
306
307
308
309
310
311
312
313
314
esters. The final ratio of the products during the synthesis of either p-coumaroyl tyrosol or
CAPE seemed to reflect the substrate preference of each enzyme and/or the substrate
availability. The synthesis of not only CAPE but also caffeoyl tyrosol and caffeoyl
hydroxytyrosol was also observed during CAPE synthesis in another report.20
Esters are found in various biological systems.34 Plants are reservoirs of natural esters,
which include volatile compounds and cell wall components.35, 36 Therefore, plants contain
diverse genes for esterification enzymes. In vivo enzymes encoded by these genes generally act
on only a certain range of substrates, and such substrate specificity results in the synthesis of a
limited number of products. Therefore, the use of a simple heterologous system such as E. coli
offers an advantage in being able to test diverse substrates, most of which are not naturally
present in a plant and/or would not encounter these enzymes due to compartmental separation.
Therefore, the approach proposed herein may help extend the substrate range of certain
enzymes, allowing for the synthesis of diverse functional products.
Funding
This work was funded by a grant (NRF-2016R1A2B4014057) from the National Research
Foundation (NRF) funded by the Ministry of Education, Science and Technology and a grant
from the Next-Generation BioGreen 21 Program (PJ01326001), Rural Development
Administration, Republic of Korea.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
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316
317
318
319
320
321
322
323
324
325
326
327
328
CAPE, caffeic acid phenethyl ester; 2-PE, 2-phenylethanol; CoA, coenzyme A; 4CL, 4-
cinnamate CoA ligase; TAL, tyrosine ammonia lyase; HpaBC, 4-hydroxyphenylacetate 3-
monooxygenase; AAAD, aromatic amino acid decarboxylase; PHE, phenylpropanoid
hydroxycinnamate esters; PMT, p-coumarate monolignol transferase; RT-PCR, reverse
transcription-polymerase chain reaction; IPTG, isopropyl-β-D-1-thiogalactopyranoside; LB
medium, Luria-Bertani medium; HPLC, high-performance liquid chromatography; MALDI-
TOF MS, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; NMR,
nuclear magnetic resonance
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Synthesized hydroxycinnamoyl phenylethanoids using Escherichia coli strain B-CP1(Table
S1); mass spectra of synthesized hydroxycinnamoyl phenylethanoids and caffeoyl phenyl
alcohols listed in Table 2, Table 3, Table S1. (PDF)
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330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
References
(1) Tsao, R. Chemistry and biochemistry of dietary polyphenols. Nutrients, 2010, 2, 1231-1246.
(2) Hardman, E. W. Diet components can suppress inflammation and reduce cancer risk. Nutr.
Res. Pract. 2014, 8, 233-240.
(3) Kim, S.-Y; Song, M. K; Jeon, J. H; Ahn, J.-H. Current status of microbial phenylethanoid
biosynthesis. J. Microbiol. Biotechnol. 2018, 28, 1225–1232.
(4) Xue, Z; Yang, B. Phenylethanoid glycosides: Research advances in their phytochemistry,
pharmacological activity and pharmacokinetics. Molecules 2016, 21, 991.
(5) Stark, A. H; Madar, Z. Olive oil as a functional food: epidemiology and nutritional
approaches. Nutr. Rev. 2002, 60,170–176.
(6) Viuda-Martos, M; Ruiz-Navajas, Y; Fernandez-Lopez, J; Perez-Alvares, J. A. Functional
properties of honey, propolis and royal jelly. J. Food Sci. 2008, 73, R117-124.
(7) Omene, C; Kalac, M; Wu, J; Marchi, E; Frenke, K; O’Connor, O. W. Propolis and its active
component, caffeic acid phenethyl ester (CAPE), modulate breast cancer therapeutic targets
via an epigenetically mediated mechanism of action. J. Cancer Sci. Ther. 2013, 5, 334-342.
(8) Murtaza, G; Karim, S; Akram, M. R; Khan, S. A; Azhar, S; Mumtaz, A; Asad, M. H. H. B.
Caffeic acid phenethyl ester and therapeutic potentials. Biomed. Res. Int. 2014, 2014: 145342.
(9) Armutcu, F; Akyol, S; Ustunsoy, S; Turan F. F. Therapeutic potential of caffeic acid
phenethyl ester and its anti-inflammatory and immunomodulatory effects (Review). Exp. Ther.
Med. 2015, 9, 1582–1588.
(10) Huang, Y; Jin, M; Pi, R; Zhang, J; Chen, M; Ouyang, Y; Liu, A; Chao, X; Liu, P; Liu, J;
Ramassamy, C; Qin, J. Protective effects of caffeic acid and caffeic acid phenethyl ester against
acrolein-induced neurotoxicity in HT22 mouse hippocampal cells. Neurosci. Lett. 2013,
535,146-151.
16
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Page 17 of 30
Journal of Agricultural and Food Chemistry
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
(11) Shi, H; Xie, D; Yang, R; Cheng, Y. Synthesis of caffeic acid phenethyl ester derivatives,
and their cytoprotective and neuritogenic activities in PC12 cells. J. Agric. Food Chem. 2014,
62, 5046–5053.
(12) Silva, R. B; Santos, N. A.G; Martins, N.M; Ferreira, D. A. S; Barbosa, F; Souza, V. C. O;
Kinoshita, A; Baffa, O; Del-Bel, E; Santos, A. C. Caffeic acid phenethyl ester protects against
the dopaminergic neuronal loss induced by 6-hydroxydopamine in rats. Neuroscience 2013,
233, 86– 94.
(13) Zhang, P; Tang, Y; Li, N. G; Zhu, Y; Duan, J. A. Bioactivity and chemical synthesis of
caffeic acid phenethyl ester and its derivatives. Molecules 2014, 19,16458-16476.
(14) Rodriguez, G. M; Tashiro, Y; Atsumi, S. Expanding ester biosynthesis in Escherichia coli.
Nature Chem. Biol. 2014, 10, 259-265.
(15) Lee, Y.‑J; Jeon, Y; Lee, J. S; Kim, B.‑G; Lee, C. H; Ahn, J.‑H. Enzymatic synthesis of
phenolic CoAs using 4‑coumarate:coenzyme A ligase (4CL) from rice. Bull. Korean Chem.
Soc. 2007, 28, 365–366.
(16) An, D. G.; Cha, M. N.; Nadarajan, S. P.; Kim, B. G.; Ahn, J.-H. Bacterial synthesis of four
hydroxycinnamic acids. Appl. Biol. Chem. 2016, 59, 173-179.
(17) Kim, Y.-Y; Lee, S.-W; Oh, M.-K. Biosynthesis of 2-phenylethanol from glucose with
genetically engineered Kluyveromyces marxianus. Enzyme Microb. Technol. 2014, 61-62, 44-
47.
(18) Kang, Z; Zhang, C; Du, G; Chen, J. Metabolic engineering of Escherichia coli for
production of 2-phenylethanol from renewable glucose. Appl. Biochem. Biotechnol. 2014, 172,
2012-2021.
(19) Kaminaga, Y; Schnepp, J; Peel, G; Kish, C. M; Ben-Nissan, G; Weiss, D; Orlova, I; Lavie,
O; Rhodes, D; Wood, K; Porterfield, D. M; Cooper, A. J; Schloss, J. V; Pichersky, E; Vainstein,
17
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Page 18 of 30
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
A; Dudareva, N. Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme
that catalyzes phenylalanine decarboxylation and oxidation. J. Biol. Chem. 2006, 281, 23357-
23366.
(20) Wang, J; Mahajani, M; Jackson, S. L; Yang, Y; Chen, M; Ferreira, E. M; Lin, Y; Yan, Y.
Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters
and amides. Met. Eng. 2017, 44, 89-99.
(21) D'Auria, J. Acyltransferases in plants: a good time to be BAHD. Cur. Opin. Plant Biol.
2006, 9, 331–340.
(22) Chung, D.; Kim, S. Y.; Ahn, J.-H. Production of three phenylethanoids, tyrosol,
hydroxytyrosol, and salidroside, using plant genes expressing in Escherichia coli. Sci. Rep.
2017, 7, 2578.
(23) Kim, M. J; Kim, B.-G; Ahn, J.-H. Biosynthesis of bioactive O-methylated flavonoids in
Escherichia coli. Appl. Microbiol. Biot. 2013, 97, 7195-7204.
(24) Kim, M; Kim, H; Lee, W; Lee, Y; Kwon, S.-W; Lee, J. Quantitative shotgun proteomics
analysis of rice anther proteins after exposure to high temperature. Int. J. Genomics, 2015, 2015,
238704.
(25) Yoon, J.-A; Kim, B.-G; Lee, W. J; Lim, Y; Chong, Y; Ahn, J.-H. Production of a novel
quercetin glycoside through metabolic engineering of Escherichia coli. Appl. Env. Microbiol.
2012, 78, 4256-4262.
(26) Dhake, K. P.; Thakare, D. D.; Bhanage, B. M. Lipase: a potential biocatalyst for the
synthesis of valuable flavour and fragrance ester compounds. Flavour Fragr. J. 2013, 28:71–
83.
(27) Withers, S; Lu, F; Kim, H; Zhu, Y; Ralph, J; Wilkerson, C. G. Identification of grass-
specific enzyme that acylates monolignols with p-coumarate. J. Biol. Chem. 2012, 287, 8347-
18
ACS Paragon Plus Environment

Page 19 of 30
Journal of Agricultural and Food Chemistry
401
402
8355.
(28) Sim, G. Y; Yang, S. M; Kim, B. G; Ahn, J.-H. Bacterial synthesis of N‑hydroxycinnamoyl
403
404
405
406
phenethylamines and tyramines. Microb. Cell Fact. 2015, 14:162.
(29) Juminaga, D; Baidoo, E. E, Redding-Johanson, A. M; Batth, T. S; Burd, H;
Mukhopadhyay, A; Petzold, C. J; Keasling, J. D. Modular engineering of L-tyrosine production
in Escherichia coli. Appl. Environ. Microbiol. 2012, 78, 89-98.
407
(30) Lee, S. J; Sim, G. Y; Kang, H; Yeo, W. S; Kim, B.‑G; Ahn, J.‑H. Synthesis of
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
avenanthramides using engineered Escherichia coli. Microb. Cell Fact. 2018, 17, 46.
(31) Koma, D; Yamanaka, H; Moriyoshi, K; Ohmoto, T; Sakai, K. Production of aromatic
compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway.
Appl. Environ. Microbiol. 2012, 78, 6203-6216.
(32) Eudes, A; Teixeira Benites, V; Wang, G; Baidoo, E. E; Lee, T. S; Keasling, J. D; Loqué,
D. Precursor-directed combinatorial biosynthesis of cinnamoyl, dihydrocinnamoyl, and
benzoyl anthranilates in Saccharomyces cerevisiae. PLoS One 2015, 10, e0138972.
(33) Ansel, H. C; Norred, W. P; Roth, I. L. Antimicrobial activity of dimethyl sulfoxide against
Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. J. Pharm. Sci. 1969, 58,
836-839.
(34) Barney, B. M. The sweet smell of biosynthesis. Nature Chem. Biol. 2014, 10, 346-347.
(35) Baldwin, I. T. Plant volatiles. Cur. Biol. 2010, 20, R392-R397.
(36) Domergue, F; Kosma, D. K. (2017) Occurrence and biosynthesis of alkyl
hydroxycinnamates in plant lipid barriers. Plants (Basel), 2017, 6, 25.
19
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Figure Legends
Figure 1. Synthesis of hydroxycinnamic acid–2-PE derivatives using strain B-CP1. A, Four
standards (caffeic acid, p-coumaric acid, m-coumaric acid, and 2-PE) used; B, Reaction product
from 2-PE and m-coumaric acid; C, Reaction product from 2-PE and p-coumaric acid; D,
Reaction product from 2-PE and caffeic acid. Substrates dissolved in dimethyl sulfoxide
(DMSO) were fed to strain B-CP1 at the final concentration of 100 μM. The resulting mixture
was incubated at 30 C for 24 h and analyzed by HPLC (S1, S2, and S3 are m-coumaric acid,
p-coumaric acid, and caffeic acid, respectively; P1, P2, and P3 are reaction products).
Figure 2. Synthesis of caffeoyl phenyl alcohols using strain B-CP1. A, Caffeic acid standard;
B, Standard 4-phenyl-1-butanol and 5-phenyl-1-pentanol; C, Reaction product (P3) from 4-
phenyl-1-butanol and caffeic acid; D, Reaction product (P2) from 5-phenyl-1-pentanol and
caffeic acid. Strain B-CP1 was used for synthesis. Caffeic acid was detected at 310 nm. The
phenylalcohols and the reaction products were detected at 310 nm. The molecular masses of
the reaction products were determined using MALDI-TOF MS.
439
440
441
442
443
444
445
446
Figure 3. A, Synthesis of p-coumaroyl tyrosol from glucose using strain B-CT3. In addition to
p-coumaroyl tyrosol (P1), p-coumaroyl 2-phenylethanol (P2) was synthesized due to the
substrate specificity of AAS. B, CAPE (caffeic acid phenethyl ester) standard; C, Synthesis of
CAPE from glucose using strain B-CP3. CAPE and p-coumaroyl 2-phenylethanol were
synthesized. P1, caffeoyl hydroxytyrosol; P2, unidentified metabolite; P3, CAPE; P4, p-
coumaroyl 2-PE.
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452
Figure 4. Synthesis of p-coumaroyl tyrosol from glucose using strains harboring different
constructs. The synthesis of p-coumaroyl tyrosol was highest in strain B-CT3, followed by B-
CT2 and B-CT1.
Figure 5. A, Production of p-coumaroyl tyrosol using strain B-CT3; B, Production of CAPE
using strain B-CP3.
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Table 1. Plasmids and strains used in the present study
Plasmids or E. coli Relevant properties or genetic marker
Source
or
strain
reference
Plasmids
pGEX5X-3
pETDuet
f1 ori, Ampr
Novagen
pCDFDuet
pG-OsPMT
pE-OsPMT
pG-ZmPMT
pC-4CL
pGEX5X-3 + PMT from Oryza sativa
pET Duet1 + PMT from O. sativa
pGEX5X-3 + PMT from Zea mays
pCDFduet + 4CL gene from O. sativa
This study
This study
This study
This study
pC-AAS
pCDFduet + AAS gene from Petroselinum This study
crispum
pA-SeTAL
pACYCDuet harboring TAL from Saccharothrix Kim et al (2013)
espanaensis
pA-SeTAL-aroG-
TyrA
pACYCDuet harboring TAL from S. Kim et al (2013)
espanaensis, aroG and TyrA from E. coli
pA-SeTAL-aroGfbr- pACYCDuet harboring TAL from S. Kim et al (2013)
TyrAfbr
espanaensis, aroGfbr and TyrAfbr from E. coli
pE-PMT-HpaBC
pC-AAS-4CL
pETDuet + PMT from O. sativa and HpaBC from This study
E. coli
pCDFduet + AAS gene from P. crispum and 4CL Chung et al
from O. sativa
(2017)
Strains
BL21 (DE3)
-
-
F- ompT hsdSB(rB mB ) gal dcm lon (DE3)
Novagen
B-CP1
B-CP2
BL21 (DE3) harboring OsPMT and 4CL
BL21 (DE3) harboring ZmPMT and 4CL
This study
This study
B-CP3
BL21 (DE3) harboring pA-SeTAL-aroGfbr- This study
TyrAfbr, pC-AAS-4CL, and pE-PMT-HpaBC
B-CT1
B-CT2
B-CT3
BL21 (DE3) harboring pA-SeTAL, pC-AAS- This study
4CL, and pE-PMT
BL21 (DE3) harboring pA-SeTAL-aroG-TyrA, This study
pC-AAS-4CL, and pE-PMT
BL21 (DE3) harboring pA-SeTAL-aroGfbr- This study
TyrAfbr, pC-AAS-4CL, and pE-PMT
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Table 2. Synthesized caffeoyl phenylethanoids using Escherichia coli strain B-CP1
OH
HO
R1
O
R2
O
R3
Donor
Acceptor
Structure of synthesized compound
Theoretical mass [M+Na]+
Measured mass [M+Na]+
Mass spectrum # in
supporting information
R1
R2
R3
caffeic acid
caffeic acid
caffeic acid
caffeic acid
2-phenylethanol
tyrosol
H
H
H
H
H
OH
OH
H
307.1049
323.0998
339.0947
323.0998
306.283
322.257
338.641
322.650
1
2
3
4
hydroxytyrosol
H
OH
H
2-hydroxyphenethyl alcohol
2-(3-hydroxyphenyl) alcohol
2-methoxyphenethyl alcohol
OH
caffeic acid
caffeic acid
caffeic acid
H
OCH3
H
OH
H
H
H
H
323.0998
337.1154
337.1154
322.262
336.265
336.277
5
6
7
3-methoxyphenethyl
alcohol
OCH3
4-methoxyphenethyl alcohol
caffeic acid
1)[M+H]+
H
H
OCH3
337.1154
336.278
8
ND, not synthesized
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Table 3. Synthesized caffeoyl phenyl alcohols using Escherichia coli strain B-CP1
Donor
Acceptor
Theoretica1 mass
[M+Na]+
Measured mass
[M+Na]+
Mass spectrum # in
supporting information
Caffeic acid
Caffeic acid
Benzyl alcohol
2-Phenylethanol
293.0892
307.1049
292.526
306.656
9
10
Caffeic acid
Caffeic acid
Caffeic acid
Caffeic acid
Caffeic acid
Caffeic acid
3-phenyl-1-propanol
4-phenyl-1-butanol
5-phenyl-1-pentanol
7-phenyl-1-heptanol
8-phenyl-1-octanol
9-phenyl-1-nonanol
321.1205
335.1362
349.1518
377.1831
391.1988
405.2144
320.622
334.620
348.608
376.712
390.692
404.675
11
12
13
14
15
16
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m-Coumaric acid
150
100
50
p-Coumaric acid
A
B
2-PE
Caffeic acid
UV: 270nm
0
60
40
20
0
P1
S1
UV: 270nm
C
D
750
500
250
0
P2
S2
UV: 320nm
600
400
200
0
P2
S3
UV: 320nm
0
2
4
6
8
10
12
14
16
18
20
Time (min)
Figure 1
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300
200
100
A
B
C
D
Caffeic acid
UV: 310nm
UV: 213nm
0
2000
5-phenyl-1-pentanol
4-phenyl-1-butanol
1000
0
2500
2000
P1
1000
0
UV: 213nm
2500
2000
1500
1000
500
P2
UV: 213nm
0
0
2
4
6
8
10
12
14
16
18
20
Time (min)
Figure 2
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2000
1750
1500
1250
1000
750
500
250
0
A
B
C
P1
P2
600
500
375
250
125
0
Caffeic acid phenethyl ester
300
200
100
0
P3
E. coli metabolite
Caffeic acid
P2
P1
P4
UV: 320nm
0
2
4
6
8
10
12
14
16
18
20
Time (min)
Figure 3
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160
140
120
100
80
60
40
20
0
B-CT1
B-CT2
B-CT3
Figure 4
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A
B
30
8
7
6
5
4
3
2
1
0
450
400
350
300
250
200
150
100
50
8
Caffeic acid phenethyl ester
p-Coumaroyl tyrosol
p-Coumaroyl 2-phenylethanol
Cell growth
p-Coumaroyl 2-phenylethanol
Cafferoyl hydroxytyrosol
Cell growth
7
6
5
4
3
2
1
0
25
20
15
10
5
0
0
0
10 20 30 40 50 60 70 80
Time (h)
0
10 20 30 40 50 60 70 80 90
Time (h)
Figure 5
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