Synthesis of diverse hydroxycinnamoyl phenylethanoid esters using

Subscriber access provided by TULANE UNIVERSITY

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

Synthesis of diverse hydroxycinnamoyl phenylethanoid esters using Escherichia coli

2

Min Kyung Song, A Ra Cho, Geun Young Sim, Joong-Hoon Ahn*

3

Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk

4

University, Seoul 05029, Republic of Korea

5 6

*Corresponding author

7

Phone: +82-2-45-3764; Fax: +82-2-3437-6106

8

E-mail: [email protected]

1

ACS Paragon Plus Environment


9

ABSTRACT

10

Caffeic acid phenethyl ester (CAPE) is an ester of a hydroxycinnamic acid

11

(phenylpropanoid) and a phenylethanoid (2-phenylethanol; 2-PE), which has long been used

12

in traditional medicine. Here, we synthesized 54 hydroxycinnamic acid-phenylethanoid esters

13

by feeding 64 combinations of hydroxycinnamic acids and phenylethanols to Escherichia coli

14

harboring the rice genes OsPMT and Os4CL. The same approach was applied for ester

15

synthesis with caffeic acid and eight different phenyl alcohols. Two hydroxycinnamoyl

16

phenethyl esters, p-coumaroyl tyrosol and CAPE, were also synthesized from glucose using

17

engineered E. coli by introducing genes for the synthesis of substrates. Consequently, we

18

synthesized approximately 393.4 mg/L p-coumaroyl tyrosol and 23.8 mg/L CAPE with this

19

approach. Overall, these findings demonstrate that the rice PMT and 4CL proteins can be used

20

for the synthesis of diverse hydroxycinnamoyl phenylethanoid esters owing to their

21

promiscuity and that further exploration of the biological activities of these compounds is

22

warranted.

23 24

KEYWORDS: Caffeic acid phenethyl ester, Hydroxycinnamic acids, Metabolic engineering,

25

Phenylethanoids

2


Page 2 of 30

Page 3 of 30

27


INTRODUCTION

28

Diverse compounds with beneficial biological activities are readily available in nature,

29

which can be exploited for medicinal and industrial purposes. In particular, the secondary

30

metabolites of plants, derived from certain core structures, show extensive structural diversity.

31

For example, polyphenols synthesized from either phenylalanine or tyrosine are among the

32

most abundant natural products found in plants,1 comprising at least four groups of compounds

33

based on the carbon skeleton2: hydroxybenzoic acids, hydroxycinnamic acids, stilbenes, and

34

flavonoids. Hydroxycinnamic acids with a C6-C3 carbon skeleton include p-coumaric acid,

35

caffeic acid, ferulic acid, and others, which exhibits diverse biological activities.

36

Phenylethanoids are a recently discovered group of polyphenols3 with a C6-C2 carbon skeleton;

37

typical members of this group include tyrosol and hydroxytyrosol.4, 5 Thus, the development

38

of engineering strategies for the enhanced synthesis of polyphenols and their derivatives could

39

enable the production of diverse bioactive products.

40

Ester formation between carboxylic acids and alcohols is a well-established chemical

41

reaction. Likewise, the formation of esters between hydroxycinnamic acids and

42

phenylethanoids extends the diversity of natural products. Caffeic acid phenethyl ester (CAPE)

43

is one such ester of caffeic acid and 2-phenylethanol (2-PE) and is a component of propolis,

44

derived from the honeybee.6 CAPE has also been used as a traditional medicine given its

45

diverse biological activities, including antioxidant, antimicrobial, anti-inflammatory,

46

anticancer, neuroprotective, and immunomodulatory activities.7-9 In addition, CAPE has

47

emerged as a candidate drug for the prevention of neurodegenerative diseases, such as

48

Alzheimer’s disease and Parkinson’s disease.10-12

49

Despite the application potential of CAPE, its biological synthetic pathway has not yet

50

been elucidated, although the chemical synthesis of its derivatives has been attempted.11, 13 3



Page 4 of 30

51

Determination of its biosynthetic pathway would allow for the rational design of a complete

52

CAPE pathway via assembly of relevant genes from various pathways or organisms. Based on

53

other known biological ester formation reactions, it can be speculated that acyl-coenzyme A

54

(CoA), which stores a large amount of energy in its thioester bond, may also serve as an acyl

55

donor for the formation of CAPE.14 In this scenario, caffeic acid would be activated by CoA

56

attachment, followed by conjugation with 2-PE. CoA is known to attach to hydroxycinnamic

57

acid via 4-cinnamate CoA ligase (4CL).15 Although the CAPE synthetic pathway is unknown,

58

those of its two substrates, caffeic acid and 2-PE, have been elucidated, and their biological

59

synthesis was successfully achieved using genes from various sources engineered in a

60

microbial system. Caffeic acid has been synthesized in Escherichia coli from tyrosine using

61

tyrosine ammonia lyase (TAL) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC),16 and

62

2-PE has been synthesized in both Kluyveromyces marxianus and E. coli from phenylpyruvate

63

using phenylpyruvate decarboxylase (encoded by aro10).17,

64

synthesized from phenylalanine by aromatic amino acid decarboxylases (AAADs).19 However,

65

the enzyme involved in the formation of esters from hydroxycinnamic acids and 2-PE remains

66

unknown, although a yeast gene was used to achieve the esterification of caffeic acid and 2-

67

PE.20 Therefore, to effectively synthesize CAPE and other phenylpropanoid hydroxycinnamate

68

esters (PHEs), it is necessary to comprehensively explore and identify the gene(s) responsible

69

for the esterification reaction. Accordingly, we focused on BAHD acyltransferase,21 also

70

known as HxxxD acyltransferase, as an enzyme that may potentially catalyze this reaction. We

71

cloned the BAHD transferase (PMT) gene and an E. coli strain harboring PMT and 4CL from

72

Oryza sativa was used to synthesize diverse PHEs. Furthermore, two PHEs (p-coumaroyl

73

tyrosol and CAPE) were synthesized from glucose using engineered E. coli.

74

4


18

In plants, 2-PE can be

Page 5 of 30


75

MATERIALS AND METHODS

76

Chemicals

77

Hydroxycinnamic acid derivatives (p-coumaric acid, caffeic acid, ferulic acid, 3-

78

methoxycinnamic acid, cinnamic acid, o-coumaric acid, m-coumaric acid, and 4-

79

methoxycinnamic acid) and benzyl alcohol derivatives (benzyl alcohol, 2-PE, 3-phenyl-1-

80

propanol, 4-phenyl-1-butanol, 5-phenyl-1-pentanol, 7-phenyl-1-heptanol, 8-phenyl-1-octanol,

81

and 9-phenyl-1-nonanol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tyrosol,

82

hydroxytyrosol,

83

methoxyphenethyl alcohol, 3-methoxyphenethyl alcohol, and 4-methoxyphenethyl alcohol

84

were also purchased from Sigma-Aldrich. CAPE was purchased from Tokyo Chemical

85

Industry (Tokyo, Japan).

2-hydroxyphenethyl

alcohol,

3-hydroxyphenethyl

alcohol,

2-

86 87

Plasmid construction

88

The p-coumarate monolignol transferase (PMT) genes from rice (O. sativa;

89

XM_015765814) and corn (Zea mays; ZM_BFc0019F21) were cloned using reverse

90

transcription-polymerase chain reaction (RT-PCR). cDNA was synthesized from the total RNA

91

of 3-week-old rice or corn using the Omniscript reverse transcriptase (Qiagen, Hilden,

92

Germany)

93

aagaattcaATGGGGTTCGCGGTGGT-3′

94

aagcggccggTTACTTGTCGAAGGCCTTGATCTC-3′;

95

aagaattcaATGGGCACCATCGTCGAC-3′ and 5′-aagcggccgcCTAGGCGGCCTGCTGGC-3′

96

(lowercase letters indicate EcoRI and NotI sites, respectively). The resulting PCR products

97

were sequenced and subcloned into the EcoRI/NotI sites of the pGEX 5X-3 or pET Duet1

98

plasmids (GE Healthcare, Oxford, UK) to obtain pG-OsPMT, pE-OsPMT, and pG-ZmPMT.

with

the

following

primers:

O.

sativa

PMT,

and

5


Z.

5′5′-

mays

PMT,

5′-


99

E. coli cells harboring both an empty pGEX vector and 4CL were used as a control. The 4CL

100

and AAS genes were cloned as previously described,15, 22 and the pC-AAS-4CL plasmid was

101

constructed by subcloning AAS into the EcoRI/NotI site of pCDF-Duet1 and subcloning 4CL

102

into the NdeI/XhoI site of pCDF-Duet 1. pA-SeTAL, pA-SeTAL-aroG-TyrA, and pA-SeTAL-

103

aroGfbr-TyrAfbr were constructed as previously described.23 HpaBC was cloned as previously

104

described16 and subcloned into the NdeI/XhoI site of pE-OsPMT.

105 106

Production of CAPE derivatives

107

To verify the production of hydroxycinnamic acid–phenylethanoid conjugates, E. coli

108

B-CP1 cells harboring both pG-OsPMT and pC-4CL were grown in Luria-Bertani (LB)

109

medium containing 50 μg/mL ampicillin and spectinomycin for 18 h at 37 C. A 1/50 volume

110

of the culture was then inoculated into fresh medium and incubated at 37 C until the optical

111

density at 600 nm (OD600) reached 0.8. Isopropyl--D-1-thiogalactopyranoside (IPTG) was

112

added at a final concentration of 1 mM, and the culture was further incubated at 18 C for 20

113

h. The cells were harvested and resuspended at OD600 = 3.0 in M9 medium containing 100 μM

114

of each substrate. The mixture was incubated at 30 C for 24 h, and the culture supernatant was

115

extracted with ethyl acetate. The reaction product was analyzed with high-performance liquid

116

chromatography (HPLC).

117

To synthesize p-coumaroyl tyrosol and CAPE from glucose, the E. coli strain was

118

grown in LB containing appropriate antibiotics at 37 C for 18 h, and then the culture was

119

inoculated into fresh medium and further incubated at 37 C for 4 h with shaking. The cells

120

were harvested and resuspended in M9 medium containing 1% yeast extract, antibiotics, and 1

121

mM IPTG. Dimethyl sulfoxide (DMSO) was added to the medium at a final concentration of

122

10%, and the culture was incubated at 30 C. 6


Page 6 of 30

Page 7 of 30

123


Analysis of reaction products

124

The culture, including cells, was extracted with ethyl acetate twice. The organic layer

125

was recovered, dried, and dissolved in methanol or DMSO, and then HPLC analysis was

126

carried out as described previously,16 except that ultraviolet detection was carried out at 270

127

nm or 320 nm. For matrix-assisted laser desorption/ionization-time-of-flight mass

128

spectrometry (MALDI-TOF MS), the sample was analyzed in methanol using gold

129

nanoparticles as a matrix. Equal volumes (1 μL) of the sample and matrix were pipetted into

130

the wells of a stainless steel 384-well target plate (Bruker Daltonics, Bremen, Germany), dried

131

in air at room temperature, and analyzed directly by mass spectrometry (MS) using an Autoflex

132

III MALDI-TOF mass spectrometer (Bruker Daltonics) equipped with a smart beam laser as

133

an ionization source. All spectra were acquired with a 19 kV accelerating voltage, 100 Hz

134

repetition rate, and an average of ~500 shots. High-resolution MS (Q Exactive mass

135

spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) was carried out as described

136

previously.24 In brief, the data-dependent top method was used to obtain mass spectra using an

137

electrospray ionization source in positive-ionization mode. The mass spectra were recorded in

138

the range of m/z 300–2,000 Da.

139

Nuclear magnetic resonance (NMR) spectroscopy was carried out as described by Yoon et

140

al.25 The NMR data were as follows: CAPE: 1H NMR (400 MHz, acetone) δ (ppm): 2.99 (2H,

141

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,

142

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

143

Hz).

144

p-coumaroyl tyrosol: 1H NMR (acetone-d6, 400 MHz) δ: 2.88 (2H, t, J = 6.9 Hz, H-

145

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),

146

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'), 7



147

7.58 (1H, d, J = 15.7 Hz, H-δ).

148

RESULTS

149

Screening of enzymes responsible for esterification of hydroxycinnamoyl-CoA and 2-PE

150

The esterification reaction that produces CAPE from caffeic acid and 2-PE requires a large

151

amount of energy,26 which was supplied by the attachment of CoA to caffeic acid. Accordingly,

152

we focused on BAHD acyltransferase,21 also known as HxxxD acyltransferase (PMT), as a

153

potential enzyme for catalyzing this reaction. PMT from rice has been shown to catalyze ester

154

formation between monolignols (p-coumaroyl alcohol, caffeoyl alcohol, coniferyl alcohol, and

155

sinapyl alcohol) and hydroxycinnamoyl-CoA.27 Given the structural similarity between

156

monolignols and phenylethanoids, we selected PMT as a candidate for the esterification

157

between caffeoyl-CoA and 2-PE. We tested the PMTs from rice (Oryza sativa) and corn (Zea

158

mays) for their ability to synthesize CAPE from 2-PE and caffeic acid. The genes encoding

159

these proteins were transformed into E. coli along with the 4CL gene (strains B-CP1 and B-

160

CP2). p-Coumaric acid, o-coumaric acid, m-coumaric acid, and caffeic acid were all tested as

161

hydroxycinnamic acids, and 2-PE was used as the phenylethanoid in all assessments. E. coli

162

B-CP1 produced a new product peak in the presence of p-coumaric acid, m-coumaric acid, and

163

caffeic acid (Fig. 1), while strain B-CP2 did not synthesize any product with any of the

164

substrates tested. In addition, as a control, E. coli harboring an empty pGEX vector and 4CL

165

did not produce any new compound. MS analysis demonstrated that the molecular masses of

166

the reaction products from B-CP1 corresponded with those of p-coumaric acid phenyl ester, m-

167

coumaric acid phenyl ester, and CAPE, respectively. Furthermore, the structure of the product

168

produced from caffeic acid and 2-PE using B-CP1 was confirmed to be CAPE using proton

169

NMR. These results indicated that OsPMT could be used to synthesize diverse PHEs. We used

170

OsPMT for subsequent analysis. 8


Page 8 of 30

Page 9 of 30


171 172

Synthesis of diverse CAPE derivatives using strain B-CP1

173

The engineered E. coli strain B-CP1 expressing OsPMT and 4CL was used as a

174

biocatalyst to synthesize hydroxycinnamoyl phenethyl esters. Initially, we tested eight

175

hydroxycinnamic acid derivatives (cinnamic acid, o-coumaric acid, m-coumaric acid, p-

176

coumaric acid, caffeic acid, ferulic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic

177

acid) with 2-PE. Among them, all of the combinations except for 3-methoxycinnamic acid and

178

2-PE resulted in a new peak, as observed by HPLC, which was not observed in the control E.

179

coli strain harboring only 4CL. The MS analysis of all combinations showed the expected

180

molecular mass (Table 2 and Supporting Information), demonstrating that the product from

181

each combination was the expected ester formed between the hydroxycinnamic acid and 2-PE.

182

Since Sim et al28 showed that 4CL can convert eight hydroxycinnamic acids into the

183

corresponding hydroxycinnamic acid-CoAs as the first step of the reaction, thereby facilitating

184

ester formation, we extended the substrates further to 2-PE derivatives, including tyrosol,

185

hydroxytyrosol,

186

methoxyphenethyl alcohol, 3-methoxyphenethyl alcohol, and 4-methoxyphenethyl alcohol.

187

Overall, 64 combinations of the eight phenylethanoids and eight hydroxycinnamic acids were

188

tested using the BCP-1 strain, resulting in the formation of 54 hydroxycinnamoyl phenethyl

189

esters (Table 2 and Supporting Information), as confirmed by MS analysis (Supporting

190

Information). p-Coumaric acid, m-coumaric acid, and caffeic acid formed esters with all of the

191

2-PE derivatives tested. 3-Methoxycinnamic acid, 4-methoxycinnamic acid, and cinnamic acid

192

formed esters with seven of the 2-PE derivatives, while ferulic acid formed esters with six of

193

the 2-PE derivatives. o-Coumaric acid was the poorest acyl group donor and formed a product

194

with only three of the 2-PE derivatives. Overall, these results suggested that OsPMT used

2-hydroxyphenethyl

alcohol,

2-(3-hydroxyphenyl)

9


alcohol,

2-


195

diverse substrates and can form esters. The observed differences in ester formation were related

196

to variations in the substrate specificity of OsPMT and not that of Os4CL, as all

197

hydroxycinnamic acid derivatives that were converted into hydroxycinnamic acid-CoA by

198

Os4CL formed an ester with at least one 2-PE derivative in all cases.

199

Moreover, we tested various phenyl alcohols (benzyl alcohol, 3-phenyl-1-propanol, 4-

200

phenyl-1-butanol, 5-phenyl-1-pentanol, 7-phenyl-1-heptanol, 8-phenyl-1-octanol, and 9-

201

phenyl-1-nonanol) as substrates along with caffeic acid in strain B-CP1. HPLC analysis of the

202

culture medium after ethyl acetate extraction from each combination of substrates showed a

203

new peak (Fig. 2), and the MS analysis of each product corresponded with the expected

204

molecular mass (Table 3 and Supporting Information). This indicated that OsPMT could also

205

use diverse phenyl alcohols as hydroxycinnamic acid acceptors, further demonstrating its broad

206

substrate range.

207 208 209

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-

210

Coumaroyl tyrosol is synthesized from p-coumaric acid and tyrosol, both of which were

211

derived from tyrosine: AAS synthesizes tyrosol from tyrosine,22 and TAL produces p-coumaric

212

acid from tyrosine. Thus, genes encoding these two proteins, along with 4CL and PMT, were

213

used to synthesize p-coumaroyl tyrosol from glucose. The resulting strain (B-CT1) was

214

examined for its ability to produce p-coumaroyl tyrosol. The strain B-CT1 produced a new

215

peak, and this product was determined to be p-coumaroyl tyrosol by NMR. We also observed

216

the synthesis of p-coumaroyl 2-PE, as AAS also converted phenylalanine into 2-PE.19

217

As both p-coumaric acid and tyrosol, the precursors for p-coumaroyl tyrosol, were

218

synthesized from tyrosine, we overexpressed genes (aroG and tyrA) that were known to 10


Page 10 of 30

Page 11 of 30


219

increase the tyrosine content in E. coli.29 Because AroG and TyrA are inhibited by tyrosine,14

220

feedback inhibition-free versions of aroG and tyrA (aroGfbr and tyrAfbr, respectively) were also

221

used. The combination of aroG, tyrA, aroGfbr, and tyrAfbr was tested (Table 1) for comparison

222

of the production of p-coumaroyl tyrosol in three strains (B-CT1–3). These three strains were

223

synthesized mainly p-coumaroyl tyrosol and p-coumaroyl 2-phenyl ethanol (Fig. 3A) but the

224

yields of these two compounds were different (Fig. 4). Strain B-CT3 synthesized

225

approximately 146.5 mg/L p-coumaroyl tyrosol, followed by B-CT2 (10.1 mg/L), and B-CT1

226

(1.6 mg/L). Thus, we further used strain B-CT3 to examine the production of p-coumaroyl

227

tyrosol for 84 h. p-Coumaroyl tyrosol was detected at 12 h, and its production rapidly increased

228

until 72 h, at which point approximately 392.4 mg/L was produced (Fig. 5A).

229

Next, CAPE was synthesized from glucose. Among the two precursors necessary for

230

the synthesis of CAPE, we previously reported the synthesis of caffeic acid using E. coli

231

harboring SeTAL and HpaBC16; SeTAL convertrd tyrosine into p-coumaric acid, which was

232

then hydroxylated by HpaBC to caffeic acid. 2-PE was synthesized using AAS (data not shown)

233

as, in addition to converting tyrosine into tyrosol, AAS was known to convert phenylalanine

234

into 2-PE.19 Therefore, the synthesis of CAPE required an additional gene, HpaBC, compared

235

to the synthesis of p-coumaroyl tyrosol. HpaBC was introduced into strain B-CT3, and the

236

resulting transformant (B-CP3) was used for the synthesis of CAPE from glucose. Strain B-

237

CP3 synthesized CAPE as a main product (P3 in Fig. 3B). Three additional byproducts (P1,

238

P2, and P4 in Fig. 3C) were also detected. By comparing the retention times of these products

239

with possible candidates synthesized from the combinations of eight hydroxycinnamic acids

240

and eight phenylethanoids, P1 and P4 were considered likely to be cafferoyl hydroxytyrosol

241

and p-coumaroyl 2-PE, respectively. These products could be expected because HpaBC can

242

hydroxylate tyrosol to produce hydroxytyrosol and can also convert p-coumaric acid into 11



243

caffeic acid. Three phenylethanoids (2-PE, tyrosol, and hydroxytyrosol) and two

244

hydroxycinnamates (p-coumaric acid and caffeic acid) may therefore be present in strain B-

245

CP3, and thus, theoretically, six conjugates could be synthesized in the cells. However, only

246

three compounds (CAPE, caffeoyl hydroxytyrosol, and p-coumaroyl 2-PE) were observed.

247

Substrate availability in the cell and the substrate preference of each enzyme were likely the

248

main factors determining the final products. Although P2 was always observed as a minor

249

product when caffeic acid was used as a substrate (data not shown), we could not confirm its

250

identity.

251

Using this strain, we examined the synthesis of the three compounds for 72 h. The

252

synthesis of CAPE dramatically increased until 36 h; its synthesis continued to increase until

253

72 h, at which point approximately 24.0 mg/L CAPE was synthesized (Fig. 5B). Caffeoyl

254

hydroxytyrosol was synthesized until 36 h, though its synthesis did not increase over time. The

255

synthesis of p-coumaroyl 2-PE decreased after 24 h, suggesting that some was converted into

256

CAPE. Previously, we showed that E. coli harboring HpaBC converted avenanthramide (avn)D

257

into avnF.30 In this reaction, p-coumaric acid, which formed part of avnD, was converted into

258

caffeic acid. 2-PE itself was not converted into tyrosol, as HpaBC only hydroxylated

259

compounds with a phenolic hydroxy group.31 Thus, p-coumaroyl 2-PE could be converted into

260

CAPE but not into p-coumaroyl tyrosol or caffeoyl tyrosol.

261 262

DISCUSSION

263

We successfully achieved the synthesis of 62 hydroxycinnamoyl phenylethanoid esters

264

owing to the substrate promiscuity of OsPMT and Os4CL. Os4CL attached CoA to diverse

265

phenylpropanoids, and OsPMT used diverse phenylpropanyl-CoAs and phenylalcohols to form

266

esters. Originally, OsPMT was reported as a catalyst for the synthesis of monolignol p12


Page 12 of 30

Page 13 of 30


267

coumaric acid conjugates27; however, we showed that it could use a broad array of other phenyl

268

alcohols, ranging from benzoyl alcohol to 9-phenyl-1-nonanol. This raises the possibility of

269

synthesizing more hydroxycinnamoyl phenylethanoid esters using other hydroxycinnamic

270

acids and phenyl alcohols. Based on the promiscuity of OsPMT and Os4CL, we estimated that

271

it would be possible to synthesize approximately 50 more esters. Other enzymes with similar

272

promiscuous properties were used to synthesize phenyl anthranilate32 in a manner similar to

273

combinatorial synthesis in chemistry. Such diversity would allow for further exploration of the

274

various biological activities of CAPE and its derivatives.

275

The solubility of CAPE in the medium had an influence on the final yield. Addition of

276

10% DMSO to the medium resulted in an approximately 3-fold greater production yield.

277

However, a DMSO concentration greater than 10% affected E. coli viability and therefore

278

reduced productivity. DMSO was known to inhibit the growth of bacteria33; however, 10%

279

DMSO did not have serious effects on bacterial growth.

280

During the synthesis of p-coumaroyl tyrosol from glucose, p-coumaroyl 2-PE was also

281

synthesized. Similarly, p-coumaroyl 2-PE and cafferoyl hydroxytyrosol were synthesized

282

during CAPE synthesis. Phenylalanine and tyrosine were both substrates provided by the host

283

and were used by AAS to synthesize 2-PE and tyrosol, respectively. TAL had a stronger

284

preference for tyrosine than for phenylalanine and therefore synthesized p-coumaric acid but

285

not cinnamic acid. In addition, HpaBC could hydroxylate compounds having phenolic hydroxy

286

group; it converted p-coumaric acid and tyrosol into caffeic acid and hydroxytyrosol,

287

respectively, and likely converted p-coumaroyl 2-PE into CAPE given that the concentration

288

of p-coumaroyl 2-PE decreased. Os4CL and OsPMT used all of the synthesized substrates,

289

including

290

hydroxycinnamates (p-coumaric acid and caffeic acid), to form hydroxycinnamoyl phenethyl

three

phenylethanoids

(2-PE,

tyrosol,

and

13


hydroxytyrosol)

and

two


291

esters. The final ratio of the products during the synthesis of either p-coumaroyl tyrosol or

292

CAPE seemed to reflect the substrate preference of each enzyme and/or the substrate

293

availability. The synthesis of not only CAPE but also caffeoyl tyrosol and caffeoyl

294

hydroxytyrosol was also observed during CAPE synthesis in another report.20

295

Esters are found in various biological systems.34 Plants are reservoirs of natural esters,

296

which include volatile compounds and cell wall components.35, 36 Therefore, plants contain

297

diverse genes for esterification enzymes. In vivo enzymes encoded by these genes generally act

298

on only a certain range of substrates, and such substrate specificity results in the synthesis of a

299

limited number of products. Therefore, the use of a simple heterologous system such as E. coli

300

offers an advantage in being able to test diverse substrates, most of which are not naturally

301

present in a plant and/or would not encounter these enzymes due to compartmental separation.

302

Therefore, the approach proposed herein may help extend the substrate range of certain

303

enzymes, allowing for the synthesis of diverse functional products.

304 305

Funding

306

This work was funded by a grant (NRF-2016R1A2B4014057) from the National Research

307

Foundation (NRF) funded by the Ministry of Education, Science and Technology and a grant

308

from the Next-Generation BioGreen 21 Program (PJ01326001), Rural Development

309

Administration, Republic of Korea.

310 311

Notes

312

The authors declare no competing financial interest.

313 314

ABBREVIATIONS USED 14


Page 14 of 30

Page 15 of 30


315

CAPE, caffeic acid phenethyl ester; 2-PE, 2-phenylethanol; CoA, coenzyme A; 4CL, 4-

316

cinnamate CoA ligase; TAL, tyrosine ammonia lyase; HpaBC, 4-hydroxyphenylacetate 3-

317

monooxygenase; AAAD, aromatic amino acid decarboxylase; PHE, phenylpropanoid

318

hydroxycinnamate esters; PMT, p-coumarate monolignol transferase; RT-PCR, reverse

319

transcription-polymerase chain reaction; IPTG, isopropyl-β-D-1-thiogalactopyranoside; LB

320

medium, Luria-Bertani medium; HPLC, high-performance liquid chromatography; MALDI-

321

TOF MS, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; NMR,

322

nuclear magnetic resonance

323 324

Supporting Information

325

The Supporting Information is available free of charge on the ACS Publications website.

326

Synthesized hydroxycinnamoyl phenylethanoids using Escherichia coli strain B-CP1(Table

327

S1); mass spectra of synthesized hydroxycinnamoyl phenylethanoids and caffeoyl phenyl

328

alcohols listed in Table 2, Table 3, Table S1. (PDF)

15



329

References

330

(1) Tsao, R. Chemistry and biochemistry of dietary polyphenols. Nutrients, 2010, 2, 1231-1246.

331

(2) Hardman, E. W. Diet components can suppress inflammation and reduce cancer risk. Nutr.

332

Res. Pract. 2014, 8, 233-240.

333

(3) Kim, S.-Y; Song, M. K; Jeon, J. H; Ahn, J.-H. Current status of microbial phenylethanoid

334

biosynthesis. J. Microbiol. Biotechnol. 2018, 28, 1225–1232.

335

(4) Xue, Z; Yang, B. Phenylethanoid glycosides: Research advances in their phytochemistry,

336

pharmacological activity and pharmacokinetics. Molecules 2016, 21, 991.

337

(5) Stark, A. H; Madar, Z. Olive oil as a functional food: epidemiology and nutritional

338

approaches. Nutr. Rev. 2002, 60,170–176.

339

(6) Viuda-Martos, M; Ruiz-Navajas, Y; Fernandez-Lopez, J; Perez-Alvares, J. A. Functional

340

properties of honey, propolis and royal jelly. J. Food Sci. 2008, 73, R117-124.

341

(7) Omene, C; Kalac, M; Wu, J; Marchi, E; Frenke, K; O’Connor, O. W. Propolis and its active

342

component, caffeic acid phenethyl ester (CAPE), modulate breast cancer therapeutic targets

343

via an epigenetically mediated mechanism of action. J. Cancer Sci. Ther. 2013, 5, 334-342.

344

(8) Murtaza, G; Karim, S; Akram, M. R; Khan, S. A; Azhar, S; Mumtaz, A; Asad, M. H. H. B.

345

Caffeic acid phenethyl ester and therapeutic potentials. Biomed. Res. Int. 2014, 2014: 145342.

346

(9) Armutcu, F; Akyol, S; Ustunsoy, S; Turan F. F. Therapeutic potential of caffeic acid

347

phenethyl ester and its anti-inflammatory and immunomodulatory effects (Review). Exp. Ther.

348

Med. 2015, 9, 1582–1588.

349

(10) Huang, Y; Jin, M; Pi, R; Zhang, J; Chen, M; Ouyang, Y; Liu, A; Chao, X; Liu, P; Liu, J;

350

Ramassamy, C; Qin, J. Protective effects of caffeic acid and caffeic acid phenethyl ester against

351

acrolein-induced neurotoxicity in HT22 mouse hippocampal cells. Neurosci. Lett. 2013,

352

535,146-151. 16


Page 16 of 30

Page 17 of 30


353

(11) Shi, H; Xie, D; Yang, R; Cheng, Y. Synthesis of caffeic acid phenethyl ester derivatives,

354

and their cytoprotective and neuritogenic activities in PC12 cells. J. Agric. Food Chem. 2014,

355

62, 5046–5053.

356

(12) Silva, R. B; Santos, N. A.G; Martins, N.M; Ferreira, D. A. S; Barbosa, F; Souza, V. C. O;

357

Kinoshita, A; Baffa, O; Del-Bel, E; Santos, A. C. Caffeic acid phenethyl ester protects against

358

the dopaminergic neuronal loss induced by 6-hydroxydopamine in rats. Neuroscience 2013,

359

233, 86– 94.

360

(13) Zhang, P; Tang, Y; Li, N. G; Zhu, Y; Duan, J. A. Bioactivity and chemical synthesis of

361

caffeic acid phenethyl ester and its derivatives. Molecules 2014, 19,16458-16476.

362

(14) Rodriguez, G. M; Tashiro, Y; Atsumi, S. Expanding ester biosynthesis in Escherichia coli.

363

Nature Chem. Biol. 2014, 10, 259-265.

364

(15) Lee, Y.‑J; Jeon, Y; Lee, J. S; Kim, B.‑G; Lee, C. H; Ahn, J.‑H. Enzymatic synthesis of

365

phenolic CoAs using 4‑coumarate:coenzyme A ligase (4CL) from rice. Bull. Korean Chem.

366

Soc. 2007, 28, 365–366.

367

(16) An, D. G.; Cha, M. N.; Nadarajan, S. P.; Kim, B. G.; Ahn, J.-H. Bacterial synthesis of four

368

hydroxycinnamic acids. Appl. Biol. Chem. 2016, 59, 173-179.

369

(17) Kim, Y.-Y; Lee, S.-W; Oh, M.-K. Biosynthesis of 2-phenylethanol from glucose with

370

genetically engineered Kluyveromyces marxianus. Enzyme Microb. Technol. 2014, 61-62, 44-

371

47.

372

(18) Kang, Z; Zhang, C; Du, G; Chen, J. Metabolic engineering of Escherichia coli for

373

production of 2-phenylethanol from renewable glucose. Appl. Biochem. Biotechnol. 2014, 172,

374

2012-2021.

375

(19) Kaminaga, Y; Schnepp, J; Peel, G; Kish, C. M; Ben-Nissan, G; Weiss, D; Orlova, I; Lavie,

376

O; Rhodes, D; Wood, K; Porterfield, D. M; Cooper, A. J; Schloss, J. V; Pichersky, E; Vainstein, 17



377

A; Dudareva, N. Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme

378

that catalyzes phenylalanine decarboxylation and oxidation. J. Biol. Chem. 2006, 281, 23357-

379

23366.

380

(20) Wang, J; Mahajani, M; Jackson, S. L; Yang, Y; Chen, M; Ferreira, E. M; Lin, Y; Yan, Y.

381

Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters

382

and amides. Met. Eng. 2017, 44, 89-99.

383

(21) D'Auria, J. Acyltransferases in plants: a good time to be BAHD. Cur. Opin. Plant Biol.

384

2006, 9, 331–340.

385

(22) Chung, D.; Kim, S. Y.; Ahn, J.-H. Production of three phenylethanoids, tyrosol,

386

hydroxytyrosol, and salidroside, using plant genes expressing in Escherichia coli. Sci. Rep.

387

2017, 7, 2578.

388

(23) Kim, M. J; Kim, B.-G; Ahn, J.-H. Biosynthesis of bioactive O-methylated flavonoids in

389

Escherichia coli. Appl. Microbiol. Biot. 2013, 97, 7195-7204.

390

(24) Kim, M; Kim, H; Lee, W; Lee, Y; Kwon, S.-W; Lee, J. Quantitative shotgun proteomics

391

analysis of rice anther proteins after exposure to high temperature. Int. J. Genomics, 2015, 2015,

392

238704.

393

(25) Yoon, J.-A; Kim, B.-G; Lee, W. J; Lim, Y; Chong, Y; Ahn, J.-H. Production of a novel

394

quercetin glycoside through metabolic engineering of Escherichia coli. Appl. Env. Microbiol.

395

2012, 78, 4256-4262.

396

(26) Dhake, K. P.; Thakare, D. D.; Bhanage, B. M. Lipase: a potential biocatalyst for the

397

synthesis of valuable flavour and fragrance ester compounds. Flavour Fragr. J. 2013, 28:71–

398

83.

399

(27) Withers, S; Lu, F; Kim, H; Zhu, Y; Ralph, J; Wilkerson, C. G. Identification of grass-

400

specific enzyme that acylates monolignols with p-coumarate. J. Biol. Chem. 2012, 287, 834718


Page 18 of 30

Page 19 of 30


401

8355.

402

(28) Sim, G. Y; Yang, S. M; Kim, B. G; Ahn, J.-H. Bacterial synthesis of N‑hydroxycinnamoyl

403

phenethylamines and tyramines. Microb. Cell Fact. 2015, 14:162.

404

(29) Juminaga, D; Baidoo, E. E, Redding-Johanson, A. M; Batth, T. S; Burd, H;

405

Mukhopadhyay, A; Petzold, C. J; Keasling, J. D. Modular engineering of L-tyrosine production

406

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

avenanthramides using engineered Escherichia coli. Microb. Cell Fact. 2018, 17, 46.

409

(31) Koma, D; Yamanaka, H; Moriyoshi, K; Ohmoto, T; Sakai, K. Production of aromatic

410

compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway.

411

Appl. Environ. Microbiol. 2012, 78, 6203-6216.

412

(32) Eudes, A; Teixeira Benites, V; Wang, G; Baidoo, E. E; Lee, T. S; Keasling, J. D; Loqué,

413

D. Precursor-directed combinatorial biosynthesis of cinnamoyl, dihydrocinnamoyl, and

414

benzoyl anthranilates in Saccharomyces cerevisiae. PLoS One 2015, 10, e0138972.

415

(33) Ansel, H. C; Norred, W. P; Roth, I. L. Antimicrobial activity of dimethyl sulfoxide against

416

Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. J. Pharm. Sci. 1969, 58,

417

836-839.

418

(34) Barney, B. M. The sweet smell of biosynthesis. Nature Chem. Biol. 2014, 10, 346-347.

419

(35) Baldwin, I. T. Plant volatiles. Cur. Biol. 2010, 20, R392-R397.

420

(36) Domergue, F; Kosma, D. K. (2017) Occurrence and biosynthesis of alkyl

421

hydroxycinnamates in plant lipid barriers. Plants (Basel), 2017, 6, 25.

422

19



423

Figure Legends

424 425

Figure 1. Synthesis of hydroxycinnamic acid–2-PE derivatives using strain B-CP1. A, Four

426

standards (caffeic acid, p-coumaric acid, m-coumaric acid, and 2-PE) used; B, Reaction product

427

from 2-PE and m-coumaric acid; C, Reaction product from 2-PE and p-coumaric acid; D,

428

Reaction product from 2-PE and caffeic acid. Substrates dissolved in dimethyl sulfoxide

429

(DMSO) were fed to strain B-CP1 at the final concentration of 100 μM. The resulting mixture

430

was incubated at 30 C for 24 h and analyzed by HPLC (S1, S2, and S3 are m-coumaric acid,

431

p-coumaric acid, and caffeic acid, respectively; P1, P2, and P3 are reaction products).

432 433

Figure 2. Synthesis of caffeoyl phenyl alcohols using strain B-CP1. A, Caffeic acid standard;

434

B, Standard 4-phenyl-1-butanol and 5-phenyl-1-pentanol; C, Reaction product (P3) from 4-

435

phenyl-1-butanol and caffeic acid; D, Reaction product (P2) from 5-phenyl-1-pentanol and

436

caffeic acid. Strain B-CP1 was used for synthesis. Caffeic acid was detected at 310 nm. The

437

phenylalcohols and the reaction products were detected at 310 nm. The molecular masses of

438

the reaction products were determined using MALDI-TOF MS.

439 440

Figure 3. A, Synthesis of p-coumaroyl tyrosol from glucose using strain B-CT3. In addition to

441

p-coumaroyl tyrosol (P1), p-coumaroyl 2-phenylethanol (P2) was synthesized due to the

442

substrate specificity of AAS. B, CAPE (caffeic acid phenethyl ester) standard; C, Synthesis of

443

CAPE from glucose using strain B-CP3. CAPE and p-coumaroyl 2-phenylethanol were

444

synthesized. P1, caffeoyl hydroxytyrosol; P2, unidentified metabolite; P3, CAPE; P4, p-

445

coumaroyl 2-PE.

446 20


Page 20 of 30

Page 21 of 30


447

Figure 4. Synthesis of p-coumaroyl tyrosol from glucose using strains harboring different

448

constructs. The synthesis of p-coumaroyl tyrosol was highest in strain B-CT3, followed by B-

449

CT2 and B-CT1.

450 451

Figure 5. A, Production of p-coumaroyl tyrosol using strain B-CT3; B, Production of CAPE

452

using strain B-CP3.

21



Page 22 of 30

Table 1. Plasmids and strains used in the present study Plasmids or E. coli strain Plasmids

Relevant properties or genetic marker

Source reference

f1 ori, Ampr

Novagen

pG-OsPMT pE-OsPMT pG-ZmPMT pC-4CL pC-AAS

pGEX5X-3 + PMT from Oryza sativa pET Duet1 + PMT from O. sativa pGEX5X-3 + PMT from Zea mays pCDFduet + 4CL gene from O. sativa pCDFduet + AAS gene from Petroselinum crispum

This study This study This study This study This study

pA-SeTAL

pACYCDuet harboring TAL from Saccharothrix Kim et al (2013) espanaensis pACYCDuet harboring TAL from S. Kim et al (2013) espanaensis, aroG and TyrA from E. coli pACYCDuet harboring TAL from S. Kim et al (2013) espanaensis, aroGfbr and TyrAfbr from E. coli

pGEX5X-3 pETDuet pCDFDuet

pA-SeTAL-aroGTyrA pA-SeTAL-aroGfbrTyrAfbr pE-PMT-HpaBC

or

pETDuet + PMT from O. sativa and HpaBC from E. coli pCDFduet + AAS gene from P. crispum and 4CL from O. sativa

This study

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

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

pC-AAS-4CL

B-CT2 B-CT3

22


Chung (2017)

et

al

Page 23 of 30


Table 2. Synthesized caffeoyl phenylethanoids using Escherichia coli strain B-CP1 OH HO

R1 O

R2

O Donor

Acceptor

R3 Structure of synthesized compound

R1

R2

R3

Theoretical mass [M+Na]+

Measured mass [M+Na]+

Mass spectrum # in supporting information

caffeic acid

2-phenylethanol

H

H

H

307.1049

306.283

1

caffeic acid

tyrosol

H

H

OH

323.0998

322.257

2

caffeic acid

hydroxytyrosol

H

OH

OH

339.0947

338.641

3

caffeic acid

2-hydroxyphenethyl alcohol

OH

H

H

323.0998

322.650

4

caffeic acid

2-(3-hydroxyphenyl) alcohol

H

OH

H

323.0998

322.262

5

caffeic acid

2-methoxyphenethyl alcohol

OCH3

H

H

337.1154

336.265

6

caffeic acid


H

OCH3

H

337.1154

336.277

7

caffeic acid


H

H

OCH3

337.1154

336.278

8

1)[M+H]+

ND, not synthesized

23



Page 24 of 30

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

Benzyl alcohol

293.0892

292.526

9

Caffeic acid

2-Phenylethanol

307.1049

306.656

10

Caffeic acid

3-phenyl-1-propanol

321.1205

320.622

11

Caffeic acid

4-phenyl-1-butanol

335.1362

334.620

12

Caffeic acid

5-phenyl-1-pentanol

349.1518

348.608

13

Caffeic acid

7-phenyl-1-heptanol

377.1831

376.712

14

Caffeic acid

8-phenyl-1-octanol

391.1988

390.692

15

Caffeic acid

9-phenyl-1-nonanol

405.2144

404.675

16

24


Page 25 of 30


A

150 100

m-Coumaric acid

p-Coumaric acid

2-PE

Caffeic acid

50

UV: 270nm

Absorbance(mAU)

0

B

60

P1

40 S1

20 0

UV: 270nm

C

750 500

P2 S2

250

UV: 320nm

0

D

600

P2

400 S3

200 0

UV: 320nm

0

2

4

6

8

10

12

Time (min)

Figure 1

25


14

16

18

20


300

A

200

Page 26 of 30

Caffeic acid

100 UV: 310nm

Absorbance (mAU)

0

B

2000

5-phenyl-1-pentanol

4-phenyl-1-butanol

1000 UV: 213nm

0 2500 2000

C P1

1000

UV: 213nm

0

D

2500 2000 1500 1000 500 0

P2 UV: 213nm

0

2

4

6

8

10

12

Time (min) Figure 2

26


14

16

18

20


Absorbance (mAU)

Page 27 of 30

2000 1750 1500 1250 1000 750 500 250 0 600

A

P1

P2

B

500

Caffeic acid phenethyl ester

375 250 125 0

C

300

P3 200

E. coli metabolite

Caffeic acid

P2

100

P1

P4

0

UV: 320nm

0

2

4

6

8

10

12

Time (min) Figure 3

27


14

16

18

20


160

p-Coumaroyl tyrosol (mg/L)

140 120 100 80 60 40 20 0

B-CT1

B-CT2

B-CT3

Figure 4

28


Page 28 of 30


A

B p-Coumaroyl tyrosol p-Coumaroyl 2-phenylethanol Cell growth

7

350

6

300

5

250

4

200

3

150

2

100

1

50 0

Caffeic acid phenethyl ester p-Coumaroyl 2-phenylethanol Cafferoyl hydroxytyrosol Cell growth

7

5

15

4 3

10

2 5

1 0

0

10 20 30 40 50 60 70 80 90 Time (h)

8

6 20

0

0

0

30 25

Concentration(mg/L)

400

Concentration(mg/L)

8

Growth (O.D600)

450

10 20 30 40 50 60 70 80 Time (h)

Figure 5

29


Growth (OD600)

Page 29 of 30


Page 30 of 30

TOC GRAPHIC R6 R5

R7

HO

Phenylethanoids R5, R6, R6= H, OH, or OCH3 R6

O OH

R2

R4 R3

R1

Os4CL

R2

R4

R5

O

O

R1

R1

S CoA

OsPMT

R3

Hydroxycinnamic acids

Hydroxycinnamoyl-CoA

R1, R2, R3, R4 = H, OH, or OCH3

R1, R2, R3, R4 = H, OH, or OCH3

R7

O

R2

R4 R3

hydroxycinnamoyl phenylethanoids

30


Synthesis of diverse hydroxycinnamoyl phenylethanoid esters using

Recommend Documents