Metabolic engineering of Escherichia coli for production of 2

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Metabolic engineering of Escherichia coli for production of 2-phenylethanol and 2-phenylethylacetate from glucose Daoyi Guo, Lihua Zhang, Sijia Kong, Zhijie Liu, Xun Li, and Hong Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01594 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Journal of Agricultural and Food Chemistry

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Metabolic engineering of Escherichia coli for production of 2-phenylethanol and

2

2-phenylethylacetate from glucose

3

Daoyi Guo*§, Lihua Zhang§, Sijia Kong§, Zhijie Liu #, Xun Li§, Hong Pan*§

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Corresponding author: Tel: +86-797-8393536; E-mail address: [email protected]

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and [email protected]

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§

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Normal University, Ganzhou 341000, China

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#

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Laboratory of Industrial Microbiology, Hubei Collaborative Innovation Center for

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Key Laboratory of Organo-Pharmaceutical Chemistry, Jiangxi Province, Gannan

Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key

Industrial Fermentation, Hubei University of Technology, Wuhan 430068, China

11 12 13 14 15 16 17 18 19 20 21 22

ACS Paragon Plus Environment


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ABSTRACT

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The rose-like odor 2-phenylethanol (2-PE) and its more fruit-like ester

26

2-phenylethylacetate (2-PEAc) are two important aromatic compounds and have wide

27

applications. In the past, 2-PE and 2-PEAc were mainly produced from

28

L-phenylalanine. In this study, the Escherichia coli was engineered to de novo

29

biosynthesis of 2-PE and 2-PEAc from glucose. Firstly, overexpression of deregulated

30

3-deoxy-D-arabinoheptulosonate-7-phosphate

31

mutase/prephenate dehydratase pheAfbr for increasing phenylpyruvate production in E.

32

coli. Subsequently, heterologous expression of decarboxylase kdc and overexpression

33

of reductase yjgB for the conversion of phenylpyruvate to 2-PE. The engineered strain

34

DG01 produced 578 mg/L of 2-PE. Finally, heterologous expression of an

35

aminotransferase aro8 to redirect the metabolic flux to phenylpyruvate. 1016 mg/L of

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2-PE was accumulated in the engineered strain DG02. Alcohol acetyltransferase ATF1

37

from S. cerevisiae can esterify a wide variety of alcohols including 2-PE. We have

38

further demonstrated the biosynthesis of 2-PEAc from glucose by overexpressing atf1

39

for the subsequent conversion of 2-PE to 2-PEAc. The engineered strain DG03

40

produced 687 mg/L 2-PEAc.

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Keywords:

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2-phenylethylacetate.

metabolic

engineering;

synthase

Escherichia

43 44


aroGfbr

coli;

and

chorismate

2-phenylethanol;

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45 46

INTRODUCTION

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The rose-like odor 2-phenylethanol (2-PE) and its more fruit-like ester

48

2-phenylethylacetate (2-PEAc) are high-value flavor and fragrance compounds, and

49

have a wide range of applications in the cosmetics, perfumery, and food industries.

50

Although the production of 2-PE and 2-PEAc by chemical synthesis has advantages in

51

cost, consumers prefer more natural or bio products in the area of flavor 1. However,

52

such natural extracts of aroma compounds obtained from plant sources by physical

53

processes are costly on account of low extraction yields 2, 3. In the past decades, great

54

efforts have been made in the biosynthesis of 2-PE and 2-PEAc from L-phenylalanine

55

4-6

56

maximum titer of 2-PE and/or 2-PEAc. However, in order to achieve economic

57

production of 2-PE and/or 2-PEAc, cheaper carbon substrate other than

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L-phenylalanine should be used. In recent years, construction of microbial cell

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factories that are able to produce value-added products directly from renewable

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carbon sources has made significant progress 7-10. Thus, the engineering of the diverse

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microbial pathways to biogenerate large quantities of aroma compounds presents an

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attractive alternative to extractions from plant sources.

. In those studies, L-phenylalanine is used as a starting substrate to reach the

63

In this study, two de novo biosynthetic pathways for the production of 2-PE and

64

2-PEAc were constructed in Escherichia coli (Figure 1). This study described here

65

shows the way for the development of an economical process for biosynthesis of 2-PE

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and 2-PEAc directly from glucose.



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MATERIALS AND METHODS

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Enzymes, chemicals, and strains

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Benzyl alcohol and phenethyl propionate were purchased from Sigma-Aldrich

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(Steinheim, Germany). Restriction endonucleases and DNA polymerases were

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purchased from NEB (New England Biolabs). T4 DNA ligase, plasmid extraction kits,

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PCR purification kits and DNA extraction kits were purchased from Fermentas

74

(Burlington, Canada). E. coli DH5α and E. coli BL21 (DE3) were purchased from

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TaKaRa (Dalian, China). The E. coli DH-5α was used for plasmid construction and

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screening. E. coli BL21 (DE3) was used as a host for fermentation to synthesize 2-PE

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and 2-PEAc. All other chemical reagents were analytically pure and purchased from

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Sinopharm Chemical Reagent Co., Ltd ( Shanghai, China).

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Plasmid construction

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All the primers used in this study are listed in Table 1. The strains and plasmids

81

used in this study are listed in Table 2. AroGfbr (D146N, GenBank WP_001109196.1)

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was synthesized by Genewiz Biotech Co. Ltd and amplified by PCR using primers

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aroGfbr-XbaI and aroGfbr-SpeI-BamHI, and ligated into pET28a(+) via XbaI and

84

BamHI to yield plasmid pDG11. PheAfbr (a truncated pheA, lacking the R-domain

85

sequence for feedback inhibition by L-phenylalanine, GenBank NP_311489.1) was

86

amplified by PCR from E. coli MG1655 genomic DNA using primers pheAfbr-XbaI

87

and pheAfbr-SpeI-SalI, and ligated into pET28a(+) via XbaI and SalI to give pDG12.

88

The XbaI-SalI fragments of pheAfbr from pDG12 was inserted into SpeI and SalI sites


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of pDG11 to give pDG13. The XbaI-SalI fragments of aroGfbr and pheAfbr from

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pDG13 was inserted into XbaI- and XhoI sites of pBBRMCS1 to give pDG14.

91

Plasmid pPG34, which co-expression of decarboxylase kdc from Saccharomyces

92

cerevisiae YPH499 and reductase yjgB from E. coli, was constructed in our previous

93

study11. Aro8 gene from S. cerevisiae was amplified by PCR using the primer pair

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aro8-XbaI and aro8-NheI-BamHI, and ligated into pPG34 via NheI and XhoI to yield

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plasmid pDG15. Plasmid pPG37, which co-expression of kdc, yjgB, aro8 and atf1 was

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constructed in our previous study11.

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Shake flask cultures

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Overnight cultures (1 mL) of recombinant E. coli strains were transferred to 100

99

mL modified M9 medium with 20 g/L glucose as previously described by Guo12 and

100

shaken at 30°C. 36 mg/L chloromycetin and 50 mg/L kanamycin were added to the

101

medium, when needed. When the OD600 reached about 0.6-0.8, IPTG was added to

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the medium for induction of gene expression at a final concentration of 0.1 mM.

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Samples were taken at regular intervals after induction and analyzed by GC/MS.

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Analytical methods

105

Extraction and GC/MS analysis of 2-PE and 2-PEAc was carried out as previously

106

described by Guo12. Glass beads were added to 10 mL culture broth for breaking the

107

cells by vigorous shaking. 2-PE and 2-PEAc were extracted using ethyl acetate (15

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mL). After the centrifuge stratification, the upper ethyl acetate phase was withdrawn,

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evaporated to near dryness, and redissolved in 0.6 mL ethyl acetate. A 1 µL portion of

110

the ethyl acetate phase was analysed after a split 10:1 injection on an Agilent 7890A



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GC equipped with an Agilent 5975 MS detector and an Agilent HP–5MS capillary

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column. The following temperature program was applied: 80°C for 1 min, an increase

113

of 20°C/min to 250°C, and 250°C for 2 min. Benzyl alcohol and phenethyl propionate

114

were used as internal standards for quantitative 2-PE and 2-PEAc, respectively.

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Results

116

Developing a fermentation protocol for 2-PE production from glucose

117

Phenylpyruvate is the precursor substrate for the biosynthesis of 2-PE. Recently,

118

microbial 2-PE biosynthesis from L-phenylalanine was developed based on

119

transamination of L-phenylalanine to phenylpyruvate by aminotransferase

120

However, L-phenylalanine is an expensive precursor compared to glucose. To obtain

121

2-PE from glucose, strains needs to have a strong capacity for phenylpyruvate

122

synthesis. In this study, a biosynthetic pathway for 2-PE was established from glucose

123

in E. coli. The overall pathway includes the upstream pathway from glucose to

124

phenylpyruvate and the downstream pathway from phenylpyruvate to 2-PE.

13

.

125

The phenylpyruvate can be de novo biosynthesized from glucose via

126

L-phenylalanine pathway. However the efficiency is quite low because of feedback

127

inhibition in L-phenylalanine pathway 14. Overexpression of feedback resistant mutant

128

of

129

bifunctional enzyme chorismate mutase/prephenate dehydratase (pheAfbr) is an

130

essential strategy to overproduce phenylpyruvate. In this study, an artificial pathway

131

was constructed for 2-PE biosynthesis in E. coli. This pathway comprised four gene

132

products: feedback-resistant mutants of aroGfbr and pheAfbr for the efficient

3-deoxy-D-arabinoheptulosonate-7-phosphate

synthase


(aroGfbr)

and

the

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overproduction of phenylpyruvate, 2-keto acid decarboxylase kdc for the conversion

134

of phenylpyruvate to phenylacetaldehyde and aldehyde reductase yjgB for the

135

conversion of phenylacetaldehyde to 2-PE. The resulting E. coli strain DG01 was

136

cultured in a modified M9 medium in shake flasks and produced up to 578 ± 15.1

137

mg/L 2-PE within 30 h (Table 3). 2-PE yield and productivity were calculated to be

138

28.9 mg/g glycerol and 19.3 mg/L/h, respectively (Table 4). This proved that the

139

synthetic pathway was functionally expressed in E. coli. Therefore, this strain could

140

be used as a starting strain for further improvement of the production of 2-PE.

141

Overexpression of aro8 gene to increase 2-PE production

142

To further improve the production of 2-PE, inactivation of competing pathways

143

or redirection of the byproducts to the desired intermediates is an effective strategy.

144

Phenylpyruvate is precursor substrate for the biosynthesis of 2-PE. Therefore, we

145

assumed that increasing phenylpyruvate availability would improve 2-PE production.

146

Because phenylpyruvate can be converted to L-phenylalanine by aminotransferase

147

tyrB catalysis, the knockout of tyrB gene may be able to promote 2-PE biosynthesis.

148

However, a latest report showed that the knockout of tyrB gene caused the poor cell

149

growth and eventually leaded to a dramatically decrease in 2-PE production. In this

150

study, the heterologous overexpression of an aminotransferase aro8 that redirect

151

phenylalanine to phenylpyruvate was used to improve the production of 2-PE. The

152

resulting E. coli strain DG02 produced up to 1016 ± 52.9 mg/L 2-PE within 30 h

153

(Table 3). 2-PE yield and productivity were calculated to be 50.8 mg/g glucose and

154

33.9 mg/L/h, respectively (Table 4). This proves that overexpression of aro8 can



155

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effectively improve 2-PE production.

156

Besides 2-PE, the significant formation of 2-PEAc, 4-hydroxyphenethyl alcohol,

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4-hydroxyphenethylacetate, 3-indolylethanol and 3-indolylacetate were also observed

158

with this recombinant E. coli, as revealed by GC/MS analysis (Figure 2). We

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hypothesize that the precursor substrates for 4-hydroxyphenethyl alcohol and

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3-indolylethanol

161

4-Hydroxyphenylpyruvic acid and tryptophan pathway intermediate indole-3-pyrucic

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acid, respectively. 4-Hydroxyphenylpyruvic acid and indole-3-pyrucic acid can be

163

converted to 4-hydroxyphenylacetaldehyde and indole-3-acetaldehyde by 2-keto acid

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decarboxylase kdc. Then 4-hydroxyphenylacetaldehyde and indole-3-acetaldehyde

165

were converted to 4-hydroxyphenethyl alcohol and 3-indolylethanol alcohol by

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aldehyde reductase yjgB. Finally, 4-hydroxyphenethyl alcohol and 3-indolylethanol

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alcohol were converted to 4-hydroxyphenethylacetate and 3-indolylacetate by an

168

intrinsic alcohol acetyltransferase-like enzyme of E. coli15.

169

Developing a fermentation protocol for 2-PEAc production from glucose

alcohol

are

from

tyrosine

pathway

intermediate

170

Compared with 2-PE, 2-PEAc is a more fruit-like odor which is also widely used

171

in fields of foods and cosmetics. In theory, 2-PEAc can be obtained from

172

esterification of 2-PE in a 2-PE -producing microbe under catalysis of alcohol

173

acyltransferase. Although our study shows that an alcohol acyltransferase-like enzyme

174

exists in E. coli, the enzyme gene sequence has not been reported yet. Several studies

175

showed that alcohol acetyltransferase ATF1 from S. cerevisiae can esterify a wide

176

variety of alcohols including 2-PE

16, 17

. In this study, a fermentative route for


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177

biosynthesis of 2-PEAc was created by further heterologous expression of atf1 for the

178

conversion of 2-PE to 2-PEAc in the 2-PE-producing strain. The resulting E. coli

179

strain DG03 produced up to 687 ± 62.7 mg/L 2-PEAc within 30 h (Table 3). 2-PEAc

180

yield and productivity were calculated to be 34.36 mg/g glucose and 22.9 mg/L/h,

181

respectively (Table 4). This proves that ATF1 has a high catalytic activity.

182

DISCUSSION

183

The rose-like odor 2-PE and its more fruit-like ester 2-PEAc are two important

184

perfumery compounds. Great efforts have been made on engineered E. coli or yeast

185

for the biosynthesis of 2-PE and 2-PEAc from L-phenylalanine

186

engineered S. cerevisiae for the production of 2-PE from L-phenylalanine and 4.8 g/L

187

2-PE was produced in a medium containing 10 g/L L-phenylalanine after 195 h 4.

188

Hwang et al. engineered E. coli for the whole-cell bioconversion of L-phenylalanine

189

and 4.7 g/L 2-PE was produced in a medium containing 100 mM L-phenylalanine 22.

190

A cofactor self-sufficient system was developed for enhanced production of 2-PE in E.

191

coli 23. 2.2 g/L 2-PE and 1.3 g/L 2-PEAc was produced from 9 g/L L-phenylalanine in

192

Kluyveromyces marxianus CBS 600 by the use of in situ product removal (ISPR)

193

technique

194

used to reduce the cost of production of 2-PE and 2-PEAc. A pathway was

195

constructed for the biosynthesis of 2-PE from glucose in E. coli. This pathway

196

comprised four gene products: a tyrosine-sensitive DAHP synthase aroF and pheAfbr

197

from E. coli, phenylpyruvate decarboxylase kdc from Pichia pastoris GS115 and

198

alcohol dehydrogenase adh1 from S. cerevisiae S288c25. However, the titer and yield

18-21

. Kim et al.

24

. However, compared with L-phenylalanine, cheaper substrate should be



199

of 2-PE (0.285 g/L and 14.3 mg/g) produced from 20 g/L glucose by this engineered

200

E. coli is low. Thus, screening of more active candidate enzymes (such as high

201

activity phenylpyruvate decarboxylase) and replacement of wild-type aroF to a

202

feedback resistant mutant of DAHP synthase in the future is a promising strategy for

203

enhancing 2-PE production. A engineered Kluyveromyces marxianus strain produced

204

up to 1.3 g/L of 2-PE from 20 g/L glucose after 72 h cultivation by overexpressing a

205

feedback resistant DAHP synthase aroGfbr from Klebsiella pneumonia 26. Because this

206

strain requires a long fermentation process for production of 2-PE, the productivity

207

(18.1 mg/L/h) is relatively low.

208

Compared with yeast, E. coli has a faster growth rate. Here, we attempted to

209

engineer E. coli for biosynthesis of 2-PE from glucose. The overall pathway includes

210

the upstream pathway from glucose to phenylpyruvate and the downstream pathway

211

from phenylpyruvate to 2-PE. The precursor substrate phenylpyruvate can be de novo

212

synthesized from glucose via L-phenylalanine pathway. Among the L-phenylalanine

213

pathway, the enzyme DAHP synthase and the bifunctional enzyme chorismate

214

mutase/prephenate dehydratase PheA are the two key rate-limiting steps toward the

215

synthesis of phenylpyruvate27-29. E. coli has three DAHP synthase isoenzymes which

216

are encoded by aroF, aroG and aroH. Because AroG contributes most of the overall

217

DAHPS activity (about 80%), it is often used as the major candidate enzyme for

218

biosynthesis of aromatic amino acids

219

variants aroGfbr and pheAfbr have been identified, most of them are thermally unstable

220

30, 32-34

30, 31

. Although many feedback resistant (fbr)

. The fermentation performances of six aroGfbr mutants were compared and it is


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221

found that aroGfbr (D146N) with a good thermostability is the best mutant for

222

L-phenylalanine accumulation35. A truncated PheA (lacking the R-domain sequence

223

for feedback inhibition by L-phenylalanine) could retain the native catalytic activities

224

and thermostability, but release the L-phenylalanine feedback inhibition

225

study, the aroGfbr (D146N) and the truncated pheA mentioned above were chosen and

226

overexpressed to increase the formation of phenylpyruvate from glucose. Our group

227

has reported that the 2-keto acid decarboxylase KDC from S. cerevisiae and aldehyde

228

reductase YjgB from E. coli can efficiently convert phenylpyruvate to 2-PE (>82%)11.

229

So, the two enzymes mentioned above were chosen to make up the downstream

230

pathway from phenylpyruvate to 2-PE. The resulting E. coli strain DG01 produced up

231

to 578 ± 15.1 mg/L 2-PE.

35, 36

. In this

232

The phenylpyruvate availability is limited due to the endogenous conversion of

233

phenylpyruvate to L-phenylalanine by aminotransferase TyrB in E. coli. A previous

234

study showed that knockout of the tyrB gene will cause cell growth to be inhibited

235

and eventually leads to a dramatically decrease in 2-PE production

236

we

237

aminotransferase aro8. The resulting E. coli strain DG02 produced up to 1016 ± 52.9

238

mg/L 2-PE which is the highest titer reported for the de novo biosynthesis of 2-PE by

239

metabolically engineered E. coli so far. Recently, Koma et al reported the biosynthesis

240

of 2-PE from glucose through co-expression of aroGfbr, pheAfbr, ipdC and aldehyde

241

reductase gen (adhC, yqhD, yjgB or yahK) 37. The titer of 2-PE is about 5.7-6.5 mM

242

(695-793 mg/L). Finally, we have further demonstrated the biosynthesis of 2-PEAc

redirect

L-phenylalanine

to

phenylpyruvate

by


25

. In this study,

overexpression

of

an


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243

from glucose by overexpressing atf1 for the subsequent conversion 2-PE to 2-PEAc.

244

To our knowledge, this is the first report on the biosynthesis of 2-PEAc directly from

245

a renewable carbon source. The resulting E. coli strain DG03 produced up to 687 ±

246

62.7 mg/L 2-PEAc. Our group has reported that the biosynthesis of 2-PEAc from

247

L-phenylalanine by engineered E. coli. We found that a large amount of 2-PE failed to

248

be effectively converted to 2-PEAc. We hypothesize that the reason for the low

249

conversion is the imbalance of the two precursor substrates, 2-PE and acetyl-CoA.

250

2-PE is rapidly synthesized and secreted to the outside of the cell in a synthetic

251

system using L-phenylalanine as a precursor substrate and thus could not be esterified

252

in time to 2-PEAc.

253

The aim of this research was to create a functional synthetic pathway in E. coli

254

and develop the completely fermentative route for producing 2-PE and 2-PEAc from

255

glucose. It has been demonstrated that 2-PE and 2-PEAc has inhibitory effects on cell

256

growth of E. coli when their concentrations are higher than 1.0 g/L

257

improvement of the yield of 2-PE and 2-PEAc by synthetic biology technology may

258

be hindered by product toxicity. Several studies showed that in situ product removal

259

(ISPR) technique (such as two-phase culture system) can effectively overcome the

260

limitation by product toxicity, which resulted in significant improvement of their yield

261

38-40

262

optimization (For example, removal of the product, optimization of culture medium,

263

fermentation temperature and pH) will be needed to improve the production of 2-PE

264

and 2-PEAc.

25

. Further

. Thus, future efforts to further strain improvement and fermentative process


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265

Funding information

266

This work was supported by National Natural Science Foundation of China

267

(81460312).

268

Compliance with ethical standards

269

Conflict of interest The authors declare that they have no competing interests.

270

Ethics approval

271

or animals performed by any of the authors.

272

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13. Etschmann, M. M. W.; Sell, D.; Schrader, J., Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium. Biotechnology Letters 2003, 25, 531-536. 14. Gallardo, E.; De, D. S.; Zamora, R.; Derdelinckx, G.; Delvaux, F. R.; Hidalgo, F. J., Influence of lipids in the generation of phenylacetaldehyde in wort-related model systems. Journal of Agricultural & Food Chemistry 2008, 56, 3155-9. 15. Liu, W.; Xu, X.; Zhang, R.; Cheng, T.; Cao, Y.; Li, X.; Guo, J.; Liu, H.; Xian, M., EngineeringEscherichia colifor high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture. Biotechnology for Biofuels 2016, 9, 58. 16. Guo, D. Y.; Pan, H.; Li, X., Metabolic engineering of Escherichia coli for production of biodiesel from fatty alcohols and acetyl-CoA. Applied Microbiology & Biotechnology 2015, 99, 7805-7812. 17. Rodriguez, G. M.; Tashiro, Y.; Atsumi, S., Expanding ester biosynthesis in Escherichia coli. Nature Chemical Biology 2014, 10, 259. 18. Hazelwood, L. A.; Daran, J. M.; van Maris, A. J.; Pronk, J. T.; Dickinson, J. R., The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Applied & Environmental Microbiology 2008, 74, 2259-2266. 19. Eshkol, N.; Sendovski, M.; Bahalul, M.; Katzezov, T.; Kashi, Y.; Fishman, A., Production of 2-phenylethanol from L-phenylalanine by a stress tolerant Saccharomyces cerevisiae strain. Journal of Applied Microbiology 2009, 106, 534-42. 20. Morrissey, J. P.; Etschmann, M. M. W.; Schrader, J.; Billerbeck, G. M., Cell factory applications of the yeast Kluyveromyces marxianus for the biotechnological production of natural flavour and fragrance molecules. Yeast 2015, 32, 3-16. 21. Stark, D.; Münch, T.; Sonnleitner, B.; Marison, I. W.; Stockar, U. V., Extractive Bioconversion of 2-Phenylethanol from l -Phenylalanine by Saccharomyces cerevisiae. Biotechnol Prog 2002, 18, 514-523. 22. Hwang, J. Y.; Park, J.; Seo, J. H.; Cha, M.; Cho, B. K.; Kim, J.; Kim, B. G., Simultaneous synthesis of 2-phenylethanol and L-homophenylalanine using aromatic transaminase with yeast Ehrlich pathway. Biotechnology & Bioengineering 2009, 102, 1323-9. 23. Wang, P.; Yang, X.; Lin, B.; Huang, J.; Tao, Y., Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol. Metabolic Engineering 2017, 44, 143. 24. Etschmann, M. M.; Sell, D.; Schrader, J., Production of 2-phenylethanol and 2-phenylethylacetate from L-phenylalanine by coupling whole-cell biocatalysis with organophilic pervaporation. Biotechnology & Bioengineering 2005, 92, 624-34. 25. Kang, Z.; Zhang, C.; Du, G.; Chen, J., Metabolic Engineering of Escherichia coli for Production of 2-phenylethanol from Renewable Glucose. Applied Biochemistry & Biotechnology 2014, 172, 2012-21. 26. Kim, T. Y.; Lee, S. W.; Oh, M. K., Biosynthesis of 2-phenylethanol from glucose with genetically engineered Kluyveromyces marxianus. Enzyme & Microbial Technology 2014, 61-62, 44. 27. Báezviveros, J. L.; Osuna, J.; Hernándezchávez, G.; Soberón, X.; Bolívar, F.; Gosset, G., Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli. Biotechnology & Bioengineering 2004, 87, 516-524. 28. Doroshenko, V. G.; Tsyrenzhapova, I. S.; Krylov, A. A.; Kiseleva, E. M.; Ermishev, V. Y.; Kazakova, S. M.; Biryukova, I. V.; Mashko, S. V., Pho regulon promoter-mediated transcription of the key pathway gene aroGFbr improves the performance of an L-phenylalanine-producing Escherichia coli strain. Applied Microbiology & Biotechnology 2010, 88, 1287. 29. Kikuchi, Y.; Tsujimoto, K.; Kurahashi, O., Mutational analysis of the feedback sites of


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phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli. Appl Environ Microbiol 1997, 63, 761-762. 30. Sprenger, G. A., From scratch to value: engineering Escherichia coli wild type cells to the production of l -phenylalanine and other fine chemicals derived from chorismate. Applied Microbiology & Biotechnology 2007, 75, 739-49. 31. Rodriguez, A.; Martínez, J. A.; Flores, N.; Escalante, A.; Gosset, G.; Bolivar, F., Engineering Escherichia coli to overproduce aromatic amino acids and derived compounds. Microbial Cell Factories 2014, 13, 126. 32. Chandran, S. S.; Yi, J.; Draths, K. M.; Von, D. R.; Weber, W.; Frost, J. W., Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnology Progress 2003, 19, 808. 33. Flores, N.; Xiao, J.; Berry, A.; Bolivar, F.; Valle, F., Pathway engineering for the production of aromatic compounds in Escherichia coli. Nature Biotechnology 1996, 14, 620. 34. Gerigk, M.; Bujnicki, R.; Ganpo‐Nkwenkwa, E.; Bongaerts, J.; Sprenger, G.; Takors, R., Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains. Biotechnology & Bioengineering 2002, 80, 746. 35. Liu, S. P.; Xiao, M. R.; Zhang, L.; Xu, J.; Ding, Z. Y.; Gu, Z. H.; Shi, G. Y., Production of l -phenylalanine from glucose by metabolic engineering of wild type Escherichia coli W3110. Process Biochemistry 2013, 48, 413-419. 36. Zhang, S.; Pohnert, G.; Kongsaeree, P.; Wilson, D. B.; Clardy, J.; Ganem, B., Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins. Febs Journal 1998, 273, 6248. 37. Koma, D.; Yamanaka, H.; Moriyoshi, K.; Ohmoto, T.; Sakai, K., Production of aromatic compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway. Applied & Environmental Microbiology 2012, 78, 6203-16. 38. Etschmann, M. M. W.; Schrader, J., An aqueous–organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast. Applied Microbiology & Biotechnology 2006, 71, 440. 39. Gao, F.; Daugulis, A. J., Bioproduction of the aroma compound 2-phenylethanol in a solid-liquid two-phase partitioning bioreactor system by Kluyveromyces marxianus. Biotechnology & Bioengineering 2009, 104, 332-9. 40. Mei, J.; Min, H.; Lü, Z., Enhanced biotransformation of l -phenylalanine to 2-phenylethanol using an in situ product adsorption technique. Process Biochemistry 2009, 44, 886-890.

377 378 379 380 381 382



383 384 385

Table 1. Primers used in this study

386

Primer name aroGfbr-XbaI

Sequence (5’-3’) GTATCTAGAAAGAGGAGATATAATGAATTATCAGAA CGACGATTTACGC

aroGfbr-SpeI-BamHI

TATGGATCCACTAGTTTACCCGCGACGCGCTTTTA

pheAfbr-XbaI

GTATCTAGAAAGAGGAGATATAATGACATCGGAAAA CCCGTTACTG

pheAfbr-SpeI-SalI

ATATGTCGACACTAGTTCACAACGTGGTTTTCGCCGG A

aro8-XbaI

AACTCTAGATTTAAGAAGGAGATATAATGATGACTT TACCTGAATCAAAAGACTTTTC

aro8-NheI-BamHI

ACAGGATCCGCTAGCCTATTTGGAAATACCAAATTCT TCGTATAA

387 388 389 390 391 392 393


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394 395 396 397

Table 2. Plasmids and strains used in this study

398

Plasmids/strains

Properties

Source/reference

pDG11

pET28a; pT7: aroGfbr

This study

pDG12

pET28a; pT7: pheAfbr

This study

pDG13

pET28a; pT7: aroGfbr and pheAfbr

This study

pDG14

pBBRMCS1; pT7: aroGfbr and pheAfbr

This study

pPG34

pET28a; pT7: kdc and yjgB

11

pDG15

pET28a; pT7: kdc, yjgB and aro8

This study

pPG37

pET28a; pT7: kdc, yjgB, aro8 and atf1

11

DG01

BL21/ pDG14/ pPG34

This study

DG02

BL21/ pDG14/ pDG15

This study

DG03

BL21/ pDG14/ pPG37

This study

Plasmids

Strains

399 400 401 402



403 404 405 406

Table 3. 2-PE and 2-PEAc production in engineered E. coli strains. Data are the

407

mean values of three independent experiments ± SD. Strains

2-PE (mg/L)

2-PEAc (mg/L)

DG01

578 ± 15.1

188 ± 12.2

DG02

1016 ± 52.9

246 ± 47.8

DG03

46 ± 10.7

687 ± 62.7

408 409 410 411 412 413 414 415 416 417 418 419 420 421


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422 423 424 425 426

Table 4. The yield and productivity calculations in engineered E. coli strains in shake

427

flasks for 30 h. Yield calculated by milligrams 2-PE or 2-PEAc produced divided by

428

grams glucose consumed. Productivity calculated by concentration of 2-PE or 2-PEAc

429

produced per hour. Data are the mean values of three independent experiments ± SD. Strains

Yield (mg/g)

Productivity (mg/L/h)

DG01

28.9 ± 0.75

19.3 ± 0.50

DG02

50.8 ± 2.64

33.9 ± 1.76

DG03

34.36 ± 3.14

22.9 ± 2.09

430 431 432 433 434 435 436 437 438 439



440 441 442 443

Figure legends:

444

Figure.1. Engineered pathways for production of 2-PE and 2-PEAc from glucose.

445

Figure.2. GC/MS analyses of 2-PE and 2-PEAc from engineered E. coli strains.

446

Identified substances: 1, benzyl alcohol (internal standard); 2, 2-PE; 3, 2-PEAc; 4,

447

phenethyl propionate (internal standard); 5, 4-hydroxyphenethyl alcohol; 6,

448

4-hydroxyphenethylacetate; 7, 3-indolylethanol; 8, 3-indolylacetate.

449 450 451 452 453 454 455 456 457 458 459 460 461


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462 463 464 465

466 467

Figure.1. Engineered pathways for production of 2-PE and 2-PEAc from glucose.

468 469 470 471 472 473



474 475 476 477

478 479

Figure.2. GC/MS analyses of 2-PE and 2-PEAc from engineered E. coli strains.

480

Identified substances: 1, benzyl alcohol (internal standard); 2, 2-PE; 3, 2-PEAc; 4,

481

phenethyl propionate (internal standard); 5, 4-hydroxyphenethyl alcohol; 6,

482

4-hydroxyphenethylacetate; 7, 3-indolylethanol; 8, 3-indolylacetate.

483 484 485


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TOC graphic 224x186mm (300 x 300 DPI)


Metabolic engineering of Escherichia coli for production of 2

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