Efficient synthesis of hydroxytyrosol from L-DOPA using engineered

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Biotechnology and Biological Transformations

Efficient synthesis of hydroxytyrosol from LDOPA using engineered Escherichia coli whole cells Chaozhi Li, Pu Jia, Yajun Bai, Tai-Ping Fan, Xiaohui Zheng, and yujie cai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01856 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 31, 2019

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Journal of Agricultural and Food Chemistry

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Efficient synthesis of hydroxytyrosol from L-DOPA using

2

engineered Escherichia coli whole cells

3

Chaozhi Lia, Pu Jiab, Yajun Baib, Tai-ping Fanc, Xiaohui Zhengb*, Yujie Caia*

4

a

5

Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China

6

b

College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, China

7

c

Department of Pharmacology, University of Cambridge, Cambridge CB2 1T, UK

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

8 9

First author: Chaozhi Li and Pu Jia contributed equally to this work

10

a*

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The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

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Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China

13

Tel.: +86-18961727911

14

Fax: +86-0551-85327725

15

E-mail: [email protected]

16

Address: Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China

17

b*

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E-mail: [email protected]

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Address: College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069,

20

China

Corresponding authors: Yujie Cai

Xiaohui Zheng

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Abstract

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Hydroxytyrosol is a high value-added compound with a variety of biological and

24

pharmacological activities. In this study, a whole-cell catalytic method for the synthesis

25

of hydroxytyrosol was developed: aromatic amino acid aminotransferase (TyrB), L-

26

glutamate dehydrogenase (GDH), α-keto acid decarboxylase (PmKDC), and aldehyde

27

reductase (YahK) were co-expressed in Escherichia coli to catalyze the synthesis of

28

hydroxytyrosol from L-DOPA. The plasmids with different copy numbers were used

29

to balance the expression of the four enzymes, and the most appropriate strains (pRSF-

30

yahK-tyrB, pCDF-gdh-Pmkdc) were identified. After determining the optimum

31

temperature (35 ℃) and pH (7.5) for whole-cell catalysis, the yield of hydroxytyrosol

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reached 36.33 mM (5.59 g/L) and the space-time yield reached 0.70 g L−1 h−1.

33 34

Key words:

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L-DOPA, hydroxytyrosol, Escherichia coli, whole-cell catalysis, co-expression

36 37 38 39 40 41 42


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Introduction

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Hydroxytyrosol (3, 4-dihydroxyphenylethanol) is a natural polyphenolic

45

compound, which has high fat- and water-solubility. It is an excellent antioxidant with

46

many biological and pharmacological activities, such as anticancer, antibacterial, and

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anti-inflammatory properties 1-4, so it is a promising compound for the pharmaceutical

48

industry 5. Hydroxytyrosol mainly exists in olives in the form of oleuropein, hence

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hydroxytyrosol can be extracted from olives or the wastewater from olive processing

50

6-8,

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but the source of raw materials is limited and the yield is low. Several methods of chemical synthesis have been developed. The starting

52

materials,

53

dihydroxybenzaldehyde

54

converted to hydroxytyrosol by one or more steps in the presence of a chemical catalyst.

55

However, there are problems with many of these methods, such as an expensive

56

substrate, harsh conditions, complicated steps, or low yield, so that they are not suitable

57

for large-scale industrial production.

58

such

as

3,4-dihydroxyphenylacetic 11,

tyrosol

12-14,

acid

9,

catechol10,

and 3,4-dimethoxyphenylacetic acid

3,415,

are

Some biosynthesis methods have also been reported. For example, Espin et al.

59

used mushroom tyrosinase to convert tyrosol into hydroxytyrosol

60

Serratia marcescens

61

can

62

Pseudomonas mendocina KR1 can convert 2-phenylethanol into hydroxytyrosol

63

Orenes-Piñero et al. transferred the phenol hydroxylase gene from Geobacillus

convert

17,

tyrosol

Pseudomonas aeruginosa into

hydroxytyrosol.

18,

16.

In addition,

and Pseudomonas putida F6

Toluene-4-monooxygenase


19

from 20.


64

thermoglucosidasius into Escherichia coli, to transform tyrosol into hydroxytyrosol 21.

65

Satoh et al. cloned tyrosine hydroxylase from mouse into E. coli to convert tyrosine to

66

hydroxytyrosol 22. Li et al. designed an artificial pathway to obtain hydroxytyrosol from

67

simple carbon sources, such as glucose and glycerol, through a series of steps 23. Wei

68

et al. designed multiple-pathway networks to synthesize hydroxytyrosol from tyrosine

69

based on HpaBC mutants 24. However, the efficiency of these methods is not high or

70

the substrate is expensive. The task to find methods for the production of

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hydroxytyrosol that are economical, simple, and efficient is still challenging.

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In this study, a pathway for the production of hydroxytyrosol from L-DOPA, using

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engineered E. coli whole cells is proposed. Aromatic amino acid aminotransferase

74

(TyrB), L-glutamate dehydrogenase (GDH), α-keto acid decarboxylase (PmKDC), and

75

aldehyde reductase (YahK) were co-expressed in E. coli. First, L-DOPA is converted

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to 3,4-dihydroxyphenylpyruvic acid through the transamination of TyrB, which is

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further converted to 3,4-dihydroxyphenylacetaldehyde by decarboxylation of PmKDC,

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and finally, 3, 4-dihydroxyphenylacetaldehyde is reduced by YahK to hydroxytyrosol.

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The transamination and the reduction were coupled by GDH to achieve cofactor

80

regeneration (Fig. 1).

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Materials and methods

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Strains, plasmids, and reagents

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The bacterial strains and plasmids used in this study were listed in Table 1. E. coli

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JM109 and E. coli BL21 (DE3), both from TaKaRa (Dalian, China), were used as 4 ACS Paragon Plus Environment

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cloning host and expression host, respectively. The pRSFDuet-1, pETDuet-1, and

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pCDFDuet-1 expression vectors were all from Novagen (Darmstadt, Germany).

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Molecular biological reagents such as T4 DNA ligase, PrimeSTAR HS DNA

88

polymerase, restriction endonuclease, Taq DNA polymerase, plasmid miniprep kit, and

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DNA gel extraction kit were from TaKaRa. Isopropyl β-D-1-thiogalactopyranoside

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(IPTG), ampicillin, streptomycin, and kanamycin were from Sangon Biotech (Shanghai,

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China). L-DOPA and hydroxytyrosol were from Sigma-Aldrich (St. Louis, MO, USA).

92

Other chemical reagents are analytically pure and of an analytical grade.

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Pathway and plasmid construction

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The synthesis pathway of hydroxytyrosol constructed in this study is shown in Fig.

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1. The gdh gene from Clostridium difficile ATCC 9689 (GenBank ID: M65250)25 was

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synthesized by Sangon Biotech. The tyrB, Pmkdc, yahK were cloned from E. coli BL21

97

(DE3) (GenBank ID: ACT45717)26, Proteus mirabilis JN458 (GenBank ID:

98

KY441412)27 and E. coli BL21 (DE3) (GenBank ID: AAC73428.1)28, respectively. The

99

genes were amplified by PCR and three plasmids (pRSFDuet-1, pETDuet-1,

100

pCDFDuet-1) used in this study were extracted by the plasmid miniprep kit (TaKaRa).

101

The plasmids and genes were digested, and then the genes were ligated to the plasmids

102

with T4 DNA ligase. The recombinant plasmids were transformed into E. coli JM109

103

and verified by colony PCR and sequencing. All successfully constructed plasmids

104

were transformed into E. coli BL21 (DE3) to express the genes. The plasmids and

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strains constructed in this study are shown in Table 1. 5 ACS Paragon Plus Environment


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Growth media, culture conditions, and preparation of whole-cell catalysts

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The constructed strains were inoculated into a test tube containing 3 mL Luria-

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Bertani (LB) medium and cultured for 12 h, and then a 250-mL Erlenmeyer flask

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containing 50 mL LB medium was inoculated with 1 mL of culture and incubated at 37

110

℃, 200 rpm. When the optical density (OD) at 600 nm reached 0.6, IPTG was added to

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a final concentration of 0.4 mM, and cultured at 15 ℃, 200 rpm for 24 h. Corresponding

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antibiotics were added at the appropriate working concentration throughout the culture

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process. The bacteria were collected by centrifugation (8000×g, 10 min), washed with

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pH 6.0 phosphate buffer (PB) and resuspended to obtain the whole-cell catalyst, which

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was stored at 4 ℃.

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Whole-cell catalysis and optimization of reaction conditions

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The reaction system was 6 mL of PB containing whole-cell catalysts 20 g/L (wet

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cells), L-DOPA 40.61 mM, L-glutamate 30 mM, and NAD+ 30 mM. The reaction was

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carried out in a 50-mL Erlenmeyer flask at 30 ℃, 100 rpm for 6 h and terminated by

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boiling in a water bath for 2 min. In order to optimize the reaction conditions, different

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temperatures and pHs were set to explore the optimum temperature and pH of the

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whole-cell catalyst.

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Enzyme activity assays

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The activity of TyrB was assayed by detecting the increase of hydroxytyrosol

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when excess GDH, PmKDC, YahK, and L-DOPA were added. Excess L-glutamate and

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NAD+ were added when assaying the activity of GDH and then the increase of NADH 6 ACS Paragon Plus Environment

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was measured at 340 nm (Shimadzu UV-1800, Suzhou, China). The enzyme activity of

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PmKDC was determined by adding excess 3,4-dihydroxyphenylpyruvic acid, NADH,

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and YahK, and detecting the increase in hydroxytyrosol concentration. In determining

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the activity of yahK, excess 3,4-dihydroxyphenylpyruvic acid, NADH, and PmKDC

131

were added to detect the increase in hydroxytyrosol concentration. One unit of enzyme

132

activity (U) was defined as the amount of enzyme required for the increase of 1μmol

133

hydroxytyrosol (NADH) per min. The results were converted to units of enzyme

134

activity contained in 1 mL of the whole-cell catalyst. All assays were repeated three

135

times.

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Analytical methods

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Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS, Waters

138

Acquity UPLC and Waters MALDI SYNAPT Q-TOF MS, Milford, USA) was used to

139

confirm the formation of hydroxytyrosol. The amount of hydroxytyrosol was

140

determined by high-performance liquid chromatography (HPLC, PerkinElmer Flexar,

141

Waltham, USA). The analytical conditions of HPLC were as follows: PerkinElmer

142

Series 200 UV/VIS detector, Waters SunFire C18 column (5 μm, 4.6 × 250 mm);

143

column temperature 35 ℃, detection wavelength 280 nm, injection quantity 10 μL, flow

144

rate 1 mL/min. The mobile phase A was 100% acetonitrile and the mobile phase B was

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water containing 0.1% formic acid. The mobile phase was changed from A/B=10/90 to

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A/B=100/0 for 15 min, and a linear gradient was performed.

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Results 7 ACS Paragon Plus Environment


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Confirmation of hydroxytyrosol

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The whole-cell catalytic reaction was carried out with strain 1 and the reaction

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solution was assayed by UPLC-MS with Electron Spray Ionization (ESI) under

151

negative ion mode. [M-H]- appeared at m/z=153, in accordance with the molecular

152

weight of hydroxytyrosol and the results of the standard, which indicated that the

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synergistic effect of the four enzymes produced hydroxytyrosol (Fig. 2). From the

154

results of the UPLC-MS assay, we also know that hydroxytyrosol has a strong

155

absorption under UV light at 280 nm, which influenced the detection conditions for the

156

detection of hydroxytyrosol by HPLC in subsequent experiments.

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Comparison of the ability of different strains to produce hydroxytyrosol

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Plasmids with different copy numbers have previously been used to balance the

159

expression of enzymes in a multi-enzyme system 29-30. To achieve the best performance

160

for the present pathway, we used three plasmids with different copy numbers:

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pRSFDuet-1, pETDuet-1, and pCDFDuet-1. A series of strains were constructed (Table

162

1), and their production capacity and enzyme activity were compared (Fig. 3, Fig. 4).

163

Firstly, strains 1–4 were constructed. Under the premise that the overexpression of

164

yahK was strong, gdh, tyrB, and Pmkdc were strongly overexpressed (strains 2, 3, and

165

4). In strains 1–4, the enzyme activity of TyrB and the amount of hydroxytyrosol

166

increased significantly when tyrB was strongly overexpressed but the amount of

167

hydroxytyrosol did not change significantly after the activities of GDH and PmKDC

168

increased. Therefore, it was determined that TyrB was the bottleneck for this pathway, 8 ACS Paragon Plus Environment

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and the enzyme activities of GDH and PmKDC are relatively sufficient. In addition,

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strain 5 (tyrB, Pmkdc, gdh were strongly overexpressed but yahK was modestly

171

overexpressed in comparison), strain 6 (yahK and tyrB were strongly overexpressed,

172

gdh and Pmkdc were modestly overexpressed in comparison) and strain 7 (all genes

173

were strongly overexpressed) were constructed. Compared with strain 7, the amount of

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hydroxytyrosol in strain 5 decreased significantly after the overexpression of yahK was

175

reduced, which indicated that strong overexpression of yahK was also essential. Finally,

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strains 3, 6, and 7, which exhibited high yields, were compared and strain 3 was

177

determined to be the best strain for the production of hydroxytyrosol, with a yield that

178

reached 24.31 mM (3.74 g/L). Subsequently, we used sodium dodecyl sulfate-

179

polyacrylamide gel electrophoresis (SDS-PAGE) to verify the expression of the four

180

enzymes in strain 3 (Fig. 5). Because the molecular weights of TyrB, GDH, and YahK

181

are similar, they are difficult to distinguish well in SDS-PAGE, hence, their expression

182

is mainly verified by enzyme activity assays.

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Optimization of pH and temperature of whole-cell catalysis

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In order to improve the production of hydroxytyrosol, the optimum pH and

185

temperature of whole-cell catalysis were determined in strain 3. Hydroxytyrosol was

186

synthesized at different pHs (5–9), and the yield reached the highest when pH was 7.5

187

(Fig. 6a). Moreover, hydroxytyrosol was synthesized at temperatures of 20 ℃, 25 ℃,

188

30 ℃, 35 ℃, 40 ℃, and 45 ℃. The optimum temperature was found to be 35 ℃ (Fig.

189

6b). To sum up, the optimum conditions for the synthesis of hydroxytyrosol in strain 3 9 ACS Paragon Plus Environment


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are pH 7.5 and 35 ℃.

191

Production of hydroxytyrosol under optimal conditions

192

Based on the above studies, strain 3 was used to catalyze the synthesis of

193

hydroxytyrosol under the optimum conditions, and the time course of the synthesis of

194

hydroxytyrosol was studied (Fig. 7). Considering that a high substrate concentration is

195

beneficial to the improvement of volumetric productivity and process economy, but that

196

the solubility of L-DOPA in water is only about 3 g/L (15.23 mM), we used

197

supersaturated L-DOPA (8 g/L). Therefore, before the supply of undissolved L-DOPA

198

was exhausted, its concentration was almost unchanged. When all the L-DOPA was

199

dissolved, its concentration decreased gradually, and the concentration of

200

hydroxytyrosol increased gradually with the progress of the reaction. After 8 h, L-

201

DOPA was almost exhausted and the concentration of hydroxytyrosol reached the

202

maximum value. Ultimately, up to 36.33 mM hydroxytyrosol (5.59 g/L) was obtained

203

from 40.61 mM L-DOPA (8 g/L), and the space-time yield was up to 0.70 g L−1 h−1.

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Discussion

205

Whole-cell biocatalysis has been widely used in the synthesis of many compounds

206

31-36. Firstly, the whole-cell catalyst has better operability and economy than the isolated

207

enzyme 31; secondly, the whole-cell catalyst has better stability and can be reused in

208

several bioconversions with little loss in activity 34; finally, the tolerance of the whole-

209

cell catalyst to substrate and product was higher than that of the free-enzyme system 35.

210

These characteristics suggest that the whole-cell catalyst is more suitable for industrial 10 ACS Paragon Plus Environment

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processes than the free-enzyme system. In addition, the growth stage of the bacterial

212

strain is not suitable for synthesis of hydroxytyrosol because of its extensive

213

antibacterial activity 37. The whole-cell catalysis can separate the growth stage of the

214

strain from the synthesis stage of hydroxytyrosol, avoiding the inhibition of

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hydroxytyrosol on the growth of the strain.

216

Here, we designed a pathway to synthesize hydroxytyrosol from L-DOPA through

217

three steps: transamination, decarboxylation, and reduction (Fig. 1). On the one hand,

218

α-ketoglutaric acid is consumed as an amino receptor in the transamination, and L-

219

glutamate is produced at the same time. On the other hand, NADH is consumed as the

220

reduction equivalent in the reduction, and NAD+ is produced at the same time.

221

Therefore, we used L-glutamate dehydrogenase to couple the transamination and

222

reduction, achieving the regeneration of both α-ketoglutaric acid and NADH. Usually,

223

the regeneration of NADH uses glucose dehydrogenase or formate dehydrogenase

224

39.

225

impurity (gluconic acid) into the reaction system, which leads to an increase in the

226

complexity of product purification. Formate dehydrogenase does not produce new

227

impurities, but its activity and stability are not high. In this pathway, the use of

228

transaminase instead of deaminase or amino acid oxidase makes the synthesis of

229

hydroxytyrosol do not need oxygen, which is conducive to prevent hydroxytyrosol from

230

being oxidized.

231

38-

Glucose dehydrogenase has high activity and stability. However, it introduces a new

In the multi-enzyme cascade reaction, if the activity of each enzyme cannot be 11 ACS Paragon Plus Environment


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well coordinated, bottleneck or excess enzyme activity will appear, which will usually

233

lead to the accumulation of metabolic intermediates and affect the production of the

234

final product. Therefore, it is necessary to take measures to balance the expression of

235

the enzymes in this study. One way to balance the expression of enzymes is to use

236

plasmids with different copy numbers 29-30. In this study, tyrB and yahK were strongly

237

overexpressed in pRSFDuet-1 (high copy number), while gdh and Pmkdc with

238

sufficient activity were weakly overexpressed in pCDFDuet-1 (low copy number),

239

which made the whole synthesis pathway achieve the highest performance (strain 3).

240

Interestingly, the production of strain 6 (pRSF-yahK-tyrB, pET-gdh-Pmkdc) and strain

241

7 (pRSF-tyrB-gdh-Pmkdc-yahK) was slightly lower than that of strain 3 (Fig. 3). This

242

phenomenon may be as a result of strong overexpression of too many enzymes, leading

243

to a heavy metabolic burden on the strain 40, which in turn affected the expression of

244

rate-limiting enzymes. In strain 3, the enzyme with excess activity was weakly

245

overexpressed, with only the rate-limiting enzyme being strongly overexpressed, so that

246

more intracellular resources and energy would be given to the rate-limiting enzyme.

247

After the optimization of pH and temperature of the whole-cell catalysis, strain 3

248

synthesized 36.33 mM hydroxytyrosol (5.59 g/L) from 40.61 mM L-DOPA within 8 h,

249

and the space-time yield was up to 0.70 g L−1 h−1, characteristics which were

250

significantly better than those reported in previous studies(Table 2). However, there are

251

some endogenous enzymes in E. coli that degrade phenolic compounds, for example,

252

HpaD (3, 4-dihydroxyphenylacetic acid 2, 3-dioxygenase) and MhpB (2,312 ACS Paragon Plus Environment

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dihydroxyphenlypropionic acid 1, 2-dioxygenase) can destroy the catechol structure of

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phenolic compounds and cause their ring opening41, FeaB (phenylacetaldehyde

255

dehydrogenase)

256

dihydroxyphenylacetic acid23, so that L-DOPA, 3, 4-dihydroxyphenylpyruvic acid, 3,

257

4-dihydroxyacetaldehyde and hydroxytyrosol are likely to be degraded. Therefore, if

258

these endogenous enzymes are knocked out, a better result may be obtained.

may

oxidize

3,

4-dihydroxyphenylacetaldehyde

to

3,

4-

259

In conclusion, hydroxytyrosol was synthesized efficiently from L-DOPA by the

260

catalysis of engineered E. coli co-expressing aromatic amino acid aminotransferase, L-

261

glutamate dehydrogenase, α-keto acid decarboxylase, and aldehyde reductase. At the

262

same time, cofactor regeneration was achieved. At present, there are many methods to

263

produce L-DOPA 50, which is easy to obtain commercially and is cheap relative to the

264

high value of hydroxytyrosol. As far as we know, this is the first whole-cell catalytic

265

synthesis of hydroxytyrosol from L-DOPA. This method has the advantages of simple

266

operation, high efficiency, and good economy, and has great potential for industrial

267

application.

268

Abbreviations GDH

L-glutamate dehydrogenase

IPTG

isopropyl β-D-1-thiogalactopyranoside

PB

phosphate buffer

UPLC-MS

ultra-performance liquid chromatography-mass spectrometry



269

HPLC

high-performance liquid chromatography

ESI

Electron Spray Ionization

SDS-PAGE

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Funding

270

We thank the National Key Scientific Instrument and Equipment Development

271

Project of China (2013YQ17052504), the Program for Changjiang Scholars and

272

Innovative Research Team in the University of Ministry of Education of China

273

(IRT_15R55), the seventh group of Hundred-Talent Program of Shanxi Province

274

(2015), and The Key Project of Research and Development Plan of Shaanxi

275

(2017ZDCXL-SF-01-02-01) for financial support.


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429

Figure Captions

430

Fig. 1. The synthesis pathway of hydroxytyrosol. TyrB, aromatic amino acid

431

aminotransferase;

432

decarboxylase; YahK, aldehyde reductase.

GDH,

L-glutamate

dehydrogenase;

PmKDC,

α-keto

acid

433 434

Fig. 2. UPLC-MS assay of hydroxytyrosol. (a) Mass spectrometry of hydroxytyrosol.

435

(b) UV absorption spectra of hydroxytyrosol.

436 437

Fig. 3. Comparison of the hydroxytyrosol production in each strain.

438 439

Fig. 4. Comparison of the activities of four enzymes in each strain.

440 441

Fig. 5. SDS-PAGE assay of crude enzymes in strain 3 and four enzymes. (M) the

442

molecular-weight marker. E. coli BL21 (DE3) harboring (Lane 1) pRSF-tyrB,

443

(Lane 2) pRSF-gdh, (Lane 3) pRSF-Pmkdc, (Lane 4) pRSF-yahK, and (Lane 5)

444

pRSF-yahK-tyrB and pCDF-gdh-Pmkdc (strain 3).

445 446

Fig. 6. Effects of different conditions on the yield of hydroxytyrosol from strain 3. The

447

effect of (a) different pHs and (b) temperatures on the production of hydroxytyrosol.

448 449

Fig. 7. Time course of hydroxytyrosol synthesis from L-DOPA in strain 3. 21 ACS Paragon Plus Environment


450

Tables

451

Table 1. Strain and plasmids used in this study

Page 22 of 28

Strain and plasmids

Description

Source

pRSFDuet-1

Double T7 promoters, RSF ori, KanR

Novagen

pETDuet-1

Double T7 promoters, pBR322 ori, AmpR

Novagen

pCDFDuet-1

Double T7 promoters, CDF13 ori, SmR

Novagen

pRSF-yahK

pRSFDuet-1 carrying yahK

This study

pCDF-tyrB-gdh-Pmkdc

pCDFDuet-1 carrying tyrB, gdh and Pmkdc

This study

pRSF-yahK-tyrB

pRSFDuet-1 carrying yahK and tyrB

This study

pCDF-gdh-Pmkdc

pCDFDuet-1 carrying gdh and Pmkdc

This study

pRSF-yahK-Pmkdc

pRSFDuet-1 carrying yahK and Pmkdc

This study

pCDF-tyrB-gdh

pCDFDuet-1 carrying tyrB and gdh

This study

pRSF-yahK-gdh

pRSFDuet-1 carrying yahK and gdh

This study

pCDF-tyrB-Pmkdc

pCDFDuet-1 carrying tyrB and Pmkdc

This study

pRSF-tyrB-gdh-Pmkdc

pRSFDuet-1 carrying tyrB, gdh and Pmkdc

This study

pET-yahK

pETDuet-1 carrying yahK

This study

pET-gdh-Pmkdc

pETDuet-1 carrying gdh and Pmkdc

This study

pRSF-tyrB-gdh-Pmkdc-yahK

pRSFDuet-1 carrying tyrB, gdh, Pmkdc and yahK

This study

Strain 1

E.coli BL21(DE3)/(pRSF-yahK, pCDF-tyrB-gdh-Pmkdc)

This study

Strain 2

E.coli BL21(DE3)/(pRSF-yahK-gdh, pCDF-tyrB-Pmkdc)

This study

Strain 3

E.coli BL21(DE3)/(pRSF-yahK-tyrB, pCDF-gdh-Pmkdc)

This study

Strain 4

E.coli BL21(DE3)/(pRSF-yahK-Pmkdc, pCDF-tyrB-gdh)

This study

Strain 5

E.coli BL21(DE3)/(pRSF-tyrB-gdh-Pmkdc, pET-yahK)

This study

Strain 6

E.coli BL21(DE3)/(pRSF-yahK-tyrB, pET-gdh-Pmkdc)

This study

Strain 7

E.coli BL21(DE3)/(pRSF-tyrB-gdh-Pmkdc-yahK)

This study

452 453 454 455 456 457 458


Page 23 of 28

459


Table 2. Comparison of different hydroxytyrosol biosynthesis method Year

Substrate

Concentration

Space-time yield

Reference

2001

tyrosol

16 mM

591.3 mg L-1 h-1

16

2004

tyrosol

3.84 g/L

548.6 mg L-1 h-1

18

2005

tyrosol

1.6 g/L

228.6 mg L-1 h-1

17

2006

tyrosol

4 g/L

571.4 mg L-1 h-1

42

2006

tyrosol

0.8 mM

49.3 mg L-1 h-1

19

2007

tyrosol

2.30 mM

44.3 mg L-1 h-1

43

2009

tyrosol

3.22 mM

49.6 mg L-1 h-1

44

2012

2-phenylethanol

133 mg/L

54 mg L-1 h-1

20

2012

tyrosine

0.19 mM

1.5 mg L-1 h-1

22

2013

tyrosol

≦5 mM

≦77 mg L-1 h-1

2014

3-nitrophenethyl alcohol

0.121 g/L

121 mg L-1 h-1

45

2014

3,4-dihydroxyphenylacetic acid

27.9 mM

204.6 mg L-1 h-1

46

2017

tyrosine

208 mg/L

6.9 mg L-1 h-1

47

2018

tyrosine

268.3 mg/L

8.9 mg L-1 h-1

48

2018

tyrosol

37.39 mM

213.3-319.9 mg L-1 h-1

49

2018

tyrosine

1243 mg/L

25.9 mg L-1 h-1

23

2019

tyrosine

1890 mg/L

52.5 mg L-1 h-1

24

2019

L-DOPA

5.59 g/L

698.8 mg L-1 h-1

This work

460


21


462

Figure graphics

463

Fig. 1.

464 465

Fig. 2.

466 467 468


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469


Fig. 3.

470 471

Fig. 4.

472 473 474

Fig. 5.



475 476

Fig. 6.

477 478


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480


Fig. 7.

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496



497

Graphic for table of contents

498


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Efficient synthesis of hydroxytyrosol from L-DOPA using engineered

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