Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl

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Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl Alcohol in Escherichia coli Zhenya Chen, Xiaolin Shen, Jian Wang, Jia Wang, Ruihua Zhang, Justin Forrest Rey, Qipeng Yuan, and Yajun Yan ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 7, 2017

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ACS Synthetic Biology

Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl Alcohol in

1

Escherichia coli

2 3 ‡

†‡

† Zhenya Chen, , Xiaolin Shen, , Jian Wang,

4

Justin Forrest Rey,

5

§

§

†‡

Jia Wang, , Ruihua Zhang,

§

Qipeng Yuan,*,†,‡ and Yajun Yan*,§

6 7 8

†

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, Beijing 100029, China

9 10 11

‡

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing

University of Chemical Technology, Beijing 100029, China

12 13

§

College of Engineering, The University of Georgia, Athens, GA 30602, USA

14 15

*

16

Qipeng Yuan

17

15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China

18

E-mail: [email protected]; telephone: +86-10-64437610

Corresponding authors:

19 20

Yajun Yan

21

146 Riverbend Research Lab South, The University of Georgia, Athens, GA 30602, USA

22

E-mail: [email protected]; telephone: +1-706-542-8293

1

ACS Paragon Plus Environment


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23

ABSTRACT

24

Vanillyl alcohol is a phenolic alcohol and is used as a flavoring agent in foods and beverages.

25

In this paper, we propose a novel artificial pathway for microbial production of vanillyl

26

alcohol from simple carbon sources. The pathway extends from 4-hydroxybenzoic acid

27

(4-HBA), and needs only three heterologous enzymes, p-hydroxybenzoate hydroxylase

28

(PobA), carboxylic acid reductase (CAR) and caffeate O-methyltransferase (COMT). First,

29

we examined the promiscuous activity of COMT towards 3,4-dihydroxybenzyl alcohol and

30

found a kcat value of 0.097 s-1. Meanwhile, 499.36 mg/L vanillyl alcohol was produced by

31

COMT in vivo catalysis when fed with 1000 mg/L 3,4-dihydroxybenzyl alcohol. In the

32

following experiment, de novo biosynthesis of vanillyl alcohol was carried out and 240.69

33

mg/L vanillyl alcohol was produced via modular optimization of pathway genes. This work

34

was to date the first achievement for microbial production of vanillyl alcohol. Additionally,

35

the present study demonstrates the application of enzyme promiscuity of COMT in the design

36

of an artificial pathway for the production of high-value methylated aromatic compounds.

37 38

KEYWORDS: vanillyl alcohol, aromatic compounds, shikimate pathway, caffeate

39

O-methyltransferase, enzyme promiscuity, microbial synthesis

2


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40

INTRODUCTION

41

Vanillyl alcohol (4-Hydroxy-3-methoxybenzyl alcohol), a widely used flavoring agent, is a

42

natural phenolic compound, existing in several diverse plants, such as Gastrodia elata

43

Blume1-3 and Vanilla planifolia.4 Vanillyl alcohol displays a variety of biological activities.

44

For instance, the alcohol exhibits 65% antioxidant activity by β-carotene-linoleate assay and

45

90% by DPPH assay,4 significant anti-angiogenic activity in the chick chorioallantoic

46

membrane (CAM), anti-inflammatory activity and anti-nociceptive activity in mice,5

47

inhibition of cell growth in food spoilage yeasts,6 and anti-asthmatic activity in guinea pig.7

48

In addition, vanillyl alcohol possesses anticonvulsive and free radical scavenging activities in

49

ferric chloride-induced epileptic seizures in Sprague-Dawley rats.8 So far, the main approach

50

for vanillyl alcohol production is via direct extraction from various plants. However, these

51

approaches are limited by the supply of raw materials, harsh reaction conditions, and low

52

yields. By addressing these limitations, microbial-based biosynthesis can be an appealing

53

alternative approach for vanillyl alcohol production.

54 55

Microbial-based metabolic engineering is a powerful biotechnological platform, and has been

56

considered as an eco-friendly approach for production of many high-value compounds from

57

simple carbon sources,9 such as amino acids,10,11 flavonoids,12-14 fatty acids,15

58

phenylpropanoic acids,16 terpenoids,17,18 coumarins,19,20 monolignols,21 isoprenes,22 alkanes,23

59

and other biofuels.24,25 So far, only the biosynthesis of vanillin, which is a precursor of

60

vanillyl alcohol and also known as an active component of Gastrodia elata Blume, has been

61

achieved in different microbes. Hansen et al. designed an artificial pathway for vanillin 3



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production from glucose in Schizosaccharomyces pombe, which was extended from

63

3-dehydroshikimate (3-DHS), an intermediate in the shikimate pathway. To convert 3-DHS

64

into vanillin, three heterologous enzymes were introduced into the host, 3-dehydroshikimate

65

dehydratase from Podospora pauciseta, carboxylic acid reductase (CAR) from Nocardia

66

genus, and catechol O-methyltransferase from Homo sapiens. Unfortunately, the vanillin titer

67

was only 65 mg/L.26 Subsequently, in order to improve the vanillin production and decrease

68

its toxicity, Brochado et al. employed an additional glycosyltransferase into the

69

vanillin-producing S. cerevisiae strain, resulting in 500 mg/L vanillin β-D-glucoside

70

production in batch cultivation via in silico metabolic engineering strategy.27 Notably, using

71

this biosynthetic pathway only accumulated trace amounts of vanillin. Recently, an

72

alternative route was constructed by mimicking a natural pathway of plants to achieve

73

vanillin production from different carbon sources in E. coli. In this route, five enzymes,

74

tyrosine

75

O-methyltransferase (COMT), trans-feruloyl-CoA synthetase (FCS), and enoyl-CoA

76

hydratase/aldolase (ECH), were arranged accordingly to convert L-tyrosine to vanillin.

77

Grafting this artificial pathway into a tyrosine-overproducing strain only enabled the host to

78

produce 97.2 mg/L vanillin from L-tyrosine, 19.3 mg/L from glucose, 13.3 mg/L from xylose

79

and 24.7 mg/L from glycerol.28 Although the biosynthesis of vanillin was achieved, the titer

80

of vanillin was low due to the long pathway, low catalytic activities of pathway enzymes, and

81

the instability of vanillin.

ammonia-lyase

(TAL),

4-coumarate

3-hydroxylase

(C3H),

caffeate

82 83

To overcome these issues and achieve vanillyl alcohol production from simple carbon sources, 4


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84

a novel biosynthetic pathway was assembled in the present study (Figure 1). Notably, this

85

artificial pathway extends from 4-hydroxybenzoic acid (4-HBA), an endogenous compound

86

in E. coli generated from the shikimate pathway. Four heterologous proteins,

87

p-hydroxybenzoate

88

phosphopantetheinyl transferase (Sfp) and COMT, were introduced into E. coli. Combined

89

with two endogenous enzymes, chorismate lyase (UbiC) and alcohol dehydrogenase (ADH),

90

E. coli achieves the vanillyl alcohol de novo biosynthesis. COMT, with caffeic acid and

91

caffeyl alcohol as native substrates, was tested due to substrate similarity to catalyze

92

3,4-dihydroxybenzyl alcohol for vanillyl alcohol production. Remarkably, the results of the in

93

vitro enzyme assay and in vivo whole-cell bioconversion experiment of COMT indicated this

94

attempt achieved the desired effect. COMT (Km = 0.52 ± 0.04 mM, kcat = 0.097 ± 0.002 s-1,

95

with 3,4-dihydroxybenzyl alcohol as substrate) could produce 499.36 ± 43.75 mg/L vanillyl

96

alcohol in vivo when fed with 1000 mg/L 3,4-dihydroxybenzyl alcohol. Hence, COMT was

97

used to substitute the catechol O-methyltransferase, which has low activity.26,29 Based on that,

98

expression of the above-mentioned non-natural pathway (Figure 1) in E. coli enabled

99

generation of 66.94 ± 9.14 mg/L vanillyl alcohol from simple carbon sources. Further,

100

modular optimization of pathway genes enhanced vanillyl alcohol production to 240.69 ±

101

22.20 mg/L, which is the highest titer of microbial-based vanillyl alcohol achieved so far.

hydroxylase

(PobA),

CAR,

the

5


CAR

maturation

factor


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102

RESULTS AND DISCUSSION

103 104

Design of a novel biosynthetic pathway for vanillyl alcohol production

105

Vanillyl alcohol is a widely occurring aromatic metabolite, and the main approach for its

106

production is extraction from a diverse set of plants. In consideration of the limitation of the

107

plant resources and harsh reaction conditions of the extraction processes, biosynthetic vanillyl

108

alcohol is essential to substitute for the naturally occurring vanillyl alcohol. Therefore, we

109

proposed an artificial pathway for de novo production of vanillyl alcohol (Figure 1). In this

110

pathway, 4-HBA is catalyzed by PobA from Pseudomonas aeruginosa, CAR from

111

Mycobacterium marinum, endogenous ADHs and COMT from Arabidopsis thaliana

112

sequentially in order to produce the end-product, vanillyl alcohol. 4-HBA is an endogenous

113

compound in E. coli generated from the shikimate pathway. PobA, p-hydroxybenzoate

114

hydroxylase, catalyzes the first step of hydroxylating 4-HBA into 3,4-dihydroxybenzoic acid.

115

CAR (carboxylic acid reductase), when coupled with its activator Sfp (CAR maturation

116

factor phosphopantetheinyl transferase), demonstrates catalytic versatility towards benzoic

117

acids and fatty acids for generation of corresponding aldehydes.30-32 Thus, we inferred CAR

118

would permit conversion of 3,4-dihydroxybenzoic acid into 3,4-dihydroxybenzaldehyde,

119

which was not stable and would be further converted into 3,4-dihydroxybenzyl alcohol by

120

endogenous ADHs. The final step is methylation of 3,4-dihydroxybenzyl alcohol into vanillyl

121

alcohol, which might be uncertain for lack of efficient methyltransferase. Given the low

122

activity of the characterized catechol O-methyltransferase from Homo sapiens,26,29 COMT

123

from A. thaliana was tested here to convert 3,4-dihydroxybenzyl alcohol into vanillyl alcohol

124

because of its strong activity in methylating caffeyl alcohol into coniferyl alcohol21 and

125

substrate similarity between caffeyl alcohol and 3,4-dihydroxybenzyl alcohol (Figure 2A).

126

Assembly of the above heterologous genes in E. coli would enable the establishment of a 6


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127

novel artificial pathway for the biosynthesis of vanillyl alcohol from renewable carbon

128

sources.

129 130

Enzymatic activity assay of COMT

131

To examine our assumption that COMT is able to catalyze 3,4-dihydroxybenzyl alcohol into

132

vanillyl alcohol, the specific activity and kinetic parameters of purified COMT towards

133

3,4-dihydroxybenzyl alcohol were determined. The plasmid pET-COMT was transferred into

134

E. coli BL21 Star (DE3), resulting in strain CZY10, used for expressing COMT with an

135

N-terminal multi-histidine tag. After expression, COMT was purified to homogeneity, as

136

verified by SDS-PAGE analysis (Figure 2B). Two phenolic compounds, 3,4-dihydroxybenzyl

137

alcohol and 3,4-dihydroxybenzoic acid, similar to its native substrate caffeyl alcohol and

138

caffeic acid, respectively, were used as substrates to test the capability of COMT at adding

139

the methyl group to the 3-hydroxyl group. As the results shown in Table 1, when using

140

3,4-dihydroxybenzyl alcohol as the substrate, COMT has a specific activity of 0.140 ± 0.003

141

µmol/min/mg protein, which is 4-fold higher than that of 3,4-dihydroxybenzoic acid (0.031 ±

142

0.001 µmol/min/mg protein). HPLC analysis of the reaction product confirmed the generation

143

of vanillyl alcohol, suggesting that COMT could indeed add a methyl group to the 3-hydroxyl

144

group of 3,4-dihydroxybenzyl alcohol. Unexpectedly, HPLC analysis of the reaction product

145

from 3,4-dihydroxybenzoic acid confirmed the production of only isovanillic acid, indicating

146

COMT methylated at the 4-hydroxyl group of 3,4-dihydroxybenzoic acid (Figure 2A). The

147

capability of methylation was consistent with COMT towards native substrate, caffeic

148

acid.33,34 Differently, the isoferulic acid, formed by methylation at the 4-hydroxyl group of

149

caffeic acid, only occupied less than 5% of the methylated products.33,34 These results

150

suggested that COMT preferred 3,4-dihydroxybenzyl alcohol over 3,4-dihydroxybenzoic acid

151

as the substrate, and the catalytic mechanisms towards the two substrates might be different. 7



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152 153

To further investigate the kinetic properties of COMT, kinetic parameters towards

154

3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzyl alcohol were measured. As shown in

155

Table 2 and Figure S1, COMT has a Km of 1.73 ± 0.09 mM and a kcat of 0.021 ± 0.0007 s-1

156

towards 3,4-dihydroxybenzoic acid, with isovanillic acid as the only product. Notably,

157

COMT has a 3-fold lower Km value towards 3,4-dihydroxybenzyl alcohol (0.52 ± 0.04 mM)

158

(Figure S2), when compared with the Km value towards 3,4-dihydroxybenzoic acid, with

159

vanillyl alcohol as the product, indicating that COMT possesses about 3-fold higher substrate

160

affinity

161

3,4-dihydroxybenzyl alcohol was measured as 0.097 ± 0.002 s-1, a 5-fold higher value

162

compared with that of COMT towards 3,4-dihydroxybenzoic acid. This comparison

163

illustrated that the activity of COMT towards 3,4-dihydroxybenzyl alcohol was 5-fold higher

164

than COMT towards 3,4-dihydroxybenzoic acid. The kcat/Km of COMT towards

165

3,4-dihydroxybenzyl

166

3,4-dihydroxybenzoic acid (0.012 mM-1·s-1). Overall, COMT demonstrated higher specificity

167

and

168

3,4-dihydroxybenzoic acid.

towards

catalytic

3,4-dihydroxybenzyl

alcohol

activity

(0.19

towards

alcohol.

-1

-1

mM ·s )

The

was

kcat

16-fold

3,4-dihydroxybenzyl

of

COMT

higher

alcohol

than

than

towards

towards

towards

169 170

Bioconversion of 3,4-dihydroxybenzoic acid or 3,4-dihydroxybenzyl alcohol into

171

corresponding products

172

To investigate the applicability of COMT for microbial production of vanillyl alcohol,

173

whole-cell bioconversion experiments were carried out to test its in vivo catalytic efficiency.

174

Firstly, we incubated BW25113 (F‫ )׳‬with various concentrations of vanillyl alcohol (0, 1, 3 and

175

5 g/L) to test its toxicity. As the results shown in Figure S3, when cultivating BW25113 (F‫ )׳‬in

8


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176

the 1 g/L vanillyl alcohol medium, the OD600 value had no significant difference compared

177

with the value of the strain cultivated in 0 g/L vanillyl alcohol medium (as control), indicating

178

less than 1 g/L vanillyl alcohol had a negligible effect on the cell growth. Meanwhile, when

179

treating the strains with 3 g/L or 5 g/L of vanillyl alcohol, the OD600 values slightly decreased

180

to 5.84 ± 0.19 and 4.97 ± 0.04, respectively, compared with the value of control (6.19 ± 0.15)

181

after 24 hours cultivation. Interestingly, the OD600 values increased to comparable values to the

182

control from 24 to 48 h. These results suggested that more than 1 g/L vanillyl alcohol inhibited

183

the cell growth at the beginning of incubation, and as cultivation continued, this inhibition

184

effect can be relieved. Afterward, strain CZY11, containing plasmid pZE-COMT, was used for

185

conducting these feeding experiments. When fed with 1000 mg/L 3,4-dihydroxybenzoic acid,

186

the isovanillic acid titer raised with the increase of cell density in the first 24 hours (Figure 3A).

187

During this period, the isovanillic acid titer increased rapidly between 12 h and 24 h, and

188

reached 157.56 ± 0.54 mg/L at 24 h with an OD600 value of 8.21 ± 0.10. Meanwhile, only 2.31

189

± 0.04 mg/L vanillic acid was accumulated. During the next 12 h, we observed the decrease of

190

isovanillic acid and vanillic acid production and the OD600 decreased slightly to 7.81 ± 0.08.

191

The results suggest that COMT is able to convert 3,4-dihydroxybenzoic acid into isovanillic

192

acid in vivo, consistent with in vitro assay results. In addition, even though vanillic acid was not

193

detected with in vitro enzyme assay, COMT can convert a trace amount of

194

3,4-dihydroxybenzoic acid into vanillic acid.

195 196

As a comparison, to achieve the vanillyl alcohol biosynthesis in strain CZY11, 1000 mg/L

197

3,4-dihydroxybenzyl alcohol was fed into the medium at 5.5 h. As Figure 3B shows, the titer

198

of vanillyl alcohol increased stably between 5.5 h and 12 h with the trend of cell growth. 9



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199

Although the cells stopped growing, the vanillyl alcohol titer raised to 499.36 ± 43.75 mg/L

200

in the subsequent 12 hours. A trace amount of isovanillyl alcohol (13.67 ± 2.04 mg/L) was

201

also detected. Compared with 157.56 ± 0.54 mg/L isovanillic acid production by CZY11

202

when fed with 1000 mg/L 3,4-dihydroxybenzoic acid, a 3-fold higher vanillyl alcohol titer

203

was achieved when feeding the similar amount of 3,4-dihydroxybenzyl alcohol into cultures.

204

The titer of vanillyl alcohol and isovanillyl alcohol decreased when the cultivation extended

205

to 36 h. Similar trends were also observed in production of isovanillic acid and vanillic acid

206

(Fig. 3A), probably due to the automatic or enzymatic degradation of products. Additionally,

207

the

208

3,4-dihydroxybenzyl alcohol into vanillyl alcohol in vivo as it does in vitro. Specifically, even

209

we were not able to detect the isovanillyl alcohol with in vitro enzyme assay; but, COMT can

210

convert a small amount of 3,4-dihydroxybenzyl alcohol to isovanillyl alcohol in vivo.

211

Furthermore, we explored the initial in vivo activities of COMT towards these two substrates.

212

As shown in Figure 3C, when feeding 3,4-dihydroxybenzoic acid, the initial in vivo activity

213

of COMT was 3.30 ± 0.80 µM/h/OD. This value was calculated based on the isovanillic acid

214

titer at 9 h in the conversion experiment, since the product was not observed at 6.5 h. As a

215

comparison, the initial in vivo activity of COMT, when feeding 3,4-dihydroxybenzyl alcohol,

216

was 11.56 ± 2.18 µM/h/OD, approximately 3.5 fold higher.

feeding

experiments

indicated

COMT

has

the

capability

of

methylating

217 218

In order to test the efficiency of CAR, we carried out the conversion experiment with strain

219

CZY12, containing plasmid pCS-CS. As the results shown in Figure 3D, strain CZY12

220

completely consumed 1000 mg/L 3,4-dihydroxybenzoic acid generating 821.43 ± 6.79 mg/L

221

3,4-dihydroxybenzyl alcohol in 24 h and the cell density gradually increased throughout the

222

cultivation, reaching 6.19 ± 0.2 at 36 h. These results suggest that CAR and its activator Sfp,

223

coupled with endogenous ADHs, can effectively convert 3,4-dihydroxybenzoic acid into 10


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224

3,4-dihydroxybenzyl alcohol in vivo. To further enhance the conversion efficiency of CZY12,

225

we employed the use of another strain, CZY13, with the expression of an alcohol

226

dehydrogenase (ADH6), to conduct the feeding experiments. As a result, 812.08 ± 51.48

227

mg/L 3,4-dihydroxybenzyl alcohol was produced from 1000 mg/L 3,4-dihydroxybenzoic acid

228

in 24 h (Figure 3E), which was similar to strain CZY12. Cells grew continuously and the

229

OD600 was 5.52 ± 0.16 at 36 h, lower than that of strain CZY12. These results indicate that

230

over-expression of alcohol dehydrogenase did not improve conversion efficiency and instead

231

caused growth stress to the host. Most notably, endogenous ADHs were sufficient to reduce

232

generated aldehydes to alcohols, as our previous study reported.35

233

234

Based upon the above conclusions that CAR and endogenous ADHs could efficiently convert

235

3,4-dihydroxybenzoic acid into 3,4-dihydroxybenzyl alcohol and COMT could efficiently

236

convert 3,4-dihydroxybenzyl alcohol into vanillyl alcohol in vivo, we tested the efficiency of

237

the downstream pathway of the vanillyl alcohol de novo biosynthesis. To achieve this goal, the

238

whole pathway (Figure 1) was split at 3,4-dihydroxybenzoic acid into both upstream and

239

downstream pathways. The downstream pathway plasmids pZE-COMT and pCS-CS were

240

co-transferred into E. coli BW25113 (F‫)׳‬, generating strain CZY14. Conversion experiments

241

were carried out to examine the capability of the downstream pathway. When 1000 mg/L

242

3,4-dihydroxybenzoic acid was fed to the cultures at 5.5 h, both cell growth and vanillyl

243

alcohol titer had an increased trend during the first 24 h (Figure 3F). In the next 12 hours, the

244

trend of cell growth was opposite to that of the vanillyl alcohol titer. The titer of vanillyl

245

alcohol increased to 210.17 ± 19.44 mg/L at 36 h, while the OD600 decreased to 6.45 ± 0.15 and

246

the 3,4-dihydroxybenzyl alcohol titer also decreased mainly because of the conversion of 11



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247

3,4-dihydroxybenzyl alcohol to vanillyl alcohol by COMT. Compared with CZY11, CZY14

248

with COMT, CAR and Sfp co-expression produced 2.4-fold lower vanillyl alcohol

249

concentrations. Moreover, we observed 820.05 ± 18.04 mg/L 3,4-dihydroxybenzyl alcohol

250

production at 12 h and 4.38 ± 0.99 mg/L isovanillyl alcohol accumulation at 36 h, indicating

251

that CAR and endogenous ADHs can efficiently convert 3,4-dihydroxybenzoic acid into

252

3,4-dihydroxybenzyl alcohol, resulting in only trace amounts of by-product isovanillyl alcohol

253

production. Overall, the results of the downstream pathway bioconversion experiment

254

demonstrated that COMT, CAR and Sfp, accompanied with endogenous ADHs, can

255

effectively catalyze 3,4-dihydroxybenzoic acid into vanillyl alcohol, proving that they have

256

potential to be used for vanillyl alcohol de novo biosynthesis.

257 258

De novo production of vanillyl alcohol

259

We combined the upstream and downstream pathways to achieve vanillyl alcohol de novo

260

production (Figure 1). To enhance chorismate conversion into 4-HBA, chorismate lyase

261

(UbiC) with high catalytic activity32,36 was over-expressed. For the purpose of achieving

262

vanillyl alcohol biosynthesis from simple carbon sources, plasmid pZE-CUP was created

263

(Figure 4) and co-transferred with plasmid pCS-CS into E. coli BW25113 (F‫)׳‬, generating

264

strain CZY15. Shake flask fermentation with CZY15 allowed production of vanillyl alcohol.

265

As shown in Figure 5A, vanillyl alcohol was produced with a stable increase during the

266

whole cultivation process. The cell density raised in the first 36 hours, synchronizing with the

267

increase of vanillyl alcohol titer. At the end of the fermentation, the vanillyl alcohol titer

268

reached 66.94 ± 9.14 mg/L with an OD600 of 7.83 ± 0.16, with a trace amount of isovanillyl

269

alcohol produced. Likewise, 149.65 ± 14.42 mg/L 3,4-dihydroxybenzyl alcohol was

270

accumulated. To increase the precursor supply, we over-expressed aroL, ppsA, tktA and 12


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271

aroGfbr in order to boost the availability of 3,4-dihydroxybenzyl alcohol. Strain CZY16

272

containing plasmids pZE-CUP-APTA and pCS-CS (Figure 4) was used for shake flask

273

fermentation to demonstrate this purpose. As shown in Figure 5B, within 36 h, the

274

3,4-dihydroxybenzyl alcohol titer was enhanced to 314.97 ± 4.45 mg/L and the vanillyl

275

alcohol titer increased to 118.08 ± 19.60 mg/L as well, which was 1.8-fold higher than that of

276

CZY15. Overall, introducing this artificial pathway into E. coli enabled the production of

277

vanillyl alcohol from simple carbon sources, and the titer was enhanced by redirecting more

278

carbon flux into the vanillyl alcohol pathway.

279 280

Enhancement of vanillyl alcohol production via modular optimization

281

Strain CZY15 containing plasmids pZE-CUP and pCS-CS produced limited vanillyl alcohol,

282

possibly because the COMT, ubiC and pobA were not optimally expressed in E. coli. To

283

modulate expression levels of both enzymes, we put the COMT and ubiC on the high-copy

284

number plasmid under two separated operons. Additionally, in light of the high activity of

285

PobA, pobA was fixed on the low-copy number plasmid to balance the whole metabolic

286

pathway. Thus, the plasmids pZE-C-U and pSA-PobA were constructed and introduced into E.

287

coli BW25113 (F‫ )׳‬with pCS-CS, resulting in strain CZY17. Shake flask fermentation with

288

strain CZY17 allowed production of vanillyl alcohol with a maximum titer of 240.69 ± 22.20

289

mg/L at 36 h (Figure 5C), which represents a 3.6-fold increase when compared to strain

290

CZY15. Additionally, after modular optimization, cells grew faster and the titer of the

291

intermediate, 3,4-dihydroxybenzyl alcohol, reached 282.53 ± 5.06 mg/L at the same time

292

point. However, with further over-expression of aroL, ppsA, tktA and aroGfbr, CZY18 could

293

only produce 83.88 ± 5.77 mg/L vanillyl alcohol, while the titer of 3,4-dihydroxybenzyl

294

alcohol did not have an obvious improvement (Figure 5D). This was most likely due to the

295

stress of imbalanced gene expression in the host. These results suggest that modular 13



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296

optimization of COMT, UbiC and PobA was an efficient approach and contributed to the titer

297

enhancement of vanillyl alcohol.

14


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298

Conclusion

299

In this study, we constructed a novel pathway for de novo biosynthesis of vanillyl alcohol.

300

This artificial pathway only needs three heterologous enzymes: PobA, CAR and COMT.

301

Compared with the native pathway for production of the precursor (vanillin) in plants,28 our

302

designed pathway for vanillyl alcohol production was more efficient due to less reaction steps

303

and high activity of pathway enzymes. First, we adopted the concept of enzyme promiscuity

304

to validate the activity of COMT towards 3,4-dihydroxybenzyl alcohol via in vitro enzyme

305

assay. As a result, COMT has a kcat value of 0.097 ± 0.002 s-1 towards 3,4-dihydroxybenzyl

306

alcohol. Meanwhile, 499.36 mg/L vanillyl alcohol was produced by COMT in vivo catalysis

307

when fed with 1000 mg/L 3,4-dihydroxybenzyl alcohol. Our following feeding experiment

308

was conducted to examine the conversion efficiency of CAR towards 3,4-dihydroxybenzoic

309

acid and observed 821.43 ± 6.79 mg/L 3,4-dihydroxybenzyl alcohol was produced from 1000

310

mg/L 3,4-dihydroxybenzoic acid.

311 312

Afterward, we grafted the artificial pathway into E. coli and observed 66.94 ± 9.14 mg/L

313

vanillyl alcohol was produced from simple carbon sources. In addition, we employed a

314

simplified method of modular optimization to balance the whole metabolic pathway enzyme

315

expression. After using this approach, the vanillyl alcohol titer was enhanced to 240.69 ±

316

22.20 mg/L. In conclusion, we established a novel biosynthetic pathway to achieve vanillyl

317

alcohol production and validated the promiscuity of COMT. However, the activity of COMT

318

limited the production of vanillyl alcohol. Recently, protein engineering for target enzyme

319

modification has been known as an efficient and economical approach for improving the

320

activity of rate-limiting enzymes in the metabolic engineering field.37-41 To address the issue

321

of COMT activity, future work may focus on improving the activity via a protein engineering

322

approach. 15



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323

METHODS

324 325

Media, strains and plasmids

326

Luria-Bertani (LB) medium containing 10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract,

327

was used for cell inoculation, propagation and protein expression. Modified M9 (M9Y)

328

medium containing 10 g glycerol, 2.5 g glucose, 6 g Na2HPO4, 0.5 g NaCl, 3 g KH2PO4, 1 g

329

NH4Cl, 2 mmol MgSO4, 0.1 mmol CaCl2 and 5 g yeast extract per liter was used for feeding

330

experiments and de novo production of vanillyl alcohol. When needed, ampicillin, kanamycin

331

and chloramphenicol were added to the medium to the final concentration of 100, 50 and 34

332

µg/mL, respectively. E. coli XL1-Blue was used for plasmid construction and propagation,

333

while E. coli BL21 Star (DE3) was used for COMT expression and purification. E. coli

334

BW25113 (F‫ )׳‬was used for feeding experiments and de novo biosynthesis of vanillyl alcohol.

335

Plasmids pZE12-luc, pCS27 and pSA74 which are high-, medium-, and low-copy number

336

plasmids, respectively, were used for pathway construction. Plasmid pETDuet-1 was used for

337

COMT expression and purification. The details of the strains and plasmids, used in this study,

338

were included in Table 3.

339 340

DNA manipulation

341

Plasmids pCS-APTA and pZE-COMT were constructed in our previous studies19,21. In order

342

to measure the in vitro activity of COMT, pET-COMT was constructed by inserting gene

343

COMT, amplified by PCR from pZE-COMT, to pETDuet-1 using BamHI and HindIII.

344

Plasmids pZE-COMT, pCS-CS and pZE-ADH6 were used for feeding experiments. To create 16


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345

plasmid pCS-CS, car encoding carboxylic acid reductase and sfp encoding CAR maturation

346

factor phosphopantetheinyl transferase were amplified from Mycobacterium marinum and

347

Bacillus subtilis, respectively and cloned into pCS27 using KpnI, NdeI and BamHI. Gene

348

encoding ADH6 was amplified from Saccharomyces cerevisiae genome DNA and cloned

349

into pZE-luc using KpnI and XbaI, generating plasmids pZE-ADH6. To achieve de novo

350

production of vanillyl alcohol, other plasmids pSA-PobA, pZE-CUP, pZE-CU, pZE-C-U,

351

pZE-CUP-APTA and pZE-CU-APTA were created. To construct pSA-PobA, gene pobA，

352

encoding p-hydroxybenzoate hydroxylase, was amplified from Pseudomonas aeruginosa

353

genome and cloned into pSA74 using KpnI and HindIII. Genes COMT amplified from

354

pZE-COMT, ubiC amplified from E. coli BL21 Star (DE3) genome, and pobA amplified from

355

Pseudomonas aeruginosa genome were cloned into pZE-luc using KpnI, PstI, SphI and XbaI,

356

resulting in pZE-CUP. Genes COMT and ubiC were cloned into pZE-luc using KpnI, PstI and

357

XbaI, generating pZE-CU. The expressing cassette PLlacO1-APTA was amplified from

358

pCS-APTA and inserted into pZE-CUP and pZE-CU between SpeI and SacI, yielding

359

plasmids pZE-CUP-APTA and pZE-CU-APTA. Plasmid pZE-C-U was constructed by

360

inserting the expressing cassette PLlacO1-UbiC to pZE-COMT using SpeI and SacI.

361 362

In vitro COMT enzyme assay

363

E. coli BL21 Star (DE3) carrying pET-COMT (CZY10) was pre-inoculated in 3 mL LB

364

medium containing ampicillin and grown aerobically at 37 °C, respectively. After 12 h, 500

365

µL of preinoculum was transferred into 50 mL of fresh LB containing ampicillin and cultured

366

until OD600 reached around 0.6 at 37 °C, and then induced overnight with 0.5 mM IPTG at 17



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367

30 °C. Cells were then harvested and lysed by a beads beater. The recombinant proteins with

368

an N-terminal multi-histidine tag were purified using His-Spin protein miniprep kit (ZYMO

369

RESEARCH).42 Pierce BCA Protein Assay Kit (Thermo Scientific) was used for measuring

370

the protein concentrations. The COMT assays were carried out by mimicking catechol

371

O-methyltransferase activity assay, described by Kunjapur et al.29 A 1 mL reaction system

372

contained 100 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 2 mM S-adenosyl-L-methionine tosylate,

373

0.5 µM purified COMT and 0-2000 µM substrate (3,4-dihydroxybenzoic acid or

374

3,4-dihydroxybenzyl alcohol). The reactions were conducted at 30 °C for 30 min (for

375

3,4-dihydroxybenzoic acid) and 15 min (for 3,4-dihydroxybenzyl alcohol) and terminated by

376

adding 10 µL 100% HCl. The reaction rates of COMT were calculated according to the

377

product formation, which were measured by HPLC. The kinetic parameters were estimated

378

with OriginPro8.5 through non-linear regression of the Michaelis-Menten equation.

379 380

Toxicity test

381

Single colonies of E. coli BW25113 (F‫ )׳‬were pre-inoculated into 3 mL of LB medium and

382

cultured overnight at 37 °C. 200 µL overnight cultures were inoculated into 20 mL M9Y

383

medium containing 0 g/L, 1 g/L, 3 g/L and 5g/L vanillyl alcohol, respectively. The cultures

384

were cultivated at 37 °C for 48 h. Samples were collected at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h,

385

24 h, 36 h and 48 h and the cell growth was confirmed by measuring OD600.

386 387

Feeding experiments

388

In order to conduct in vivo conversion experiments, E. coli BW25113 (F‫)׳‬43 was transformed 18


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389

with plasmid pZE-COMT, generating strain CZY11, E. coli BW25113 (F‫ )׳‬was transformed

390

with plasmid pCS-CS, generating strain CZY12, E. coli BW25113 (F‫ )׳‬was co-transformed

391

with plasmids pCS-CS and pZE-ADH6, generating strain CZY13, and E. coli BW25113 (F‫)׳‬

392

was co-transformed with plasmids pZE-COMT and pCS-CS, generating strain CZY14.

393

Single colonies were pre-inoculated into 3 mL of LB medium containing ampicillin and

394

cultured overnight at 37 °C. 200 µL overnight cultures were inoculated into 20 mL M9Y

395

medium containing ampicillin. The cultures were cultivated at 37 °C until OD600 reached 0.6

396

and then induced with IPTG (a final concentration of 0.5 mM) at 30 °C. After 3 h induction,

397

for strain CZY11, 1000 mg/L 3,4-dihydroxybenzoic acid or 3,4-dihydroxybenzyl alcohol was

398

fed into the cultures. For strains CZY12, CZY13 and CZY14, 3,4-dihydroxybenzoic acid was

399

fed into the cultures at a final concentration of 1000 mg/L. Samples were collected at the time

400

when substrates were added (5.5 h), 9 h, 12 h, 24 h, and 36 h; cell growth was confirmed by

401

measuring OD600 and the products and intermediates were analyzed by HPLC. Additional

402

samples were taken at 6.5 h to calculate the initial in vivo activity of COMT.

403 404

De novo production of vanillyl alcohol

405

E. coli BW25113 (F‫ )׳‬containing plasmids pZE-CUP and pCS-CS (CZY15), E. coli

406

BW25113 (F‫ )׳‬containing plasmids pZE-CUP-APTA and pCS-CS (CZY16), E. coli

407

BW25113 (F‫ )׳‬containing plasmids pZE-C-U, pCS-CS and pSA-PobA (CZY17), and E. coli

408

BW25113 (F‫ )׳‬containing plasmids pZE-CU-APTA, pCS-CS and pSA-PobA (CZY18) were

409

used for de novo biosynthesis of vanillyl alcohol. Transformants were pre-inoculated in 3 mL

410

LB overnight and then 200 µL samples were inoculated into 20 mL M9Y medium containing 19



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Page 20 of 38

411

suitable antibiotics and 0.5 mM IPTG. The cultures were cultivated at 30 °C and samples

412

were collected every 12 h until 48 h. OD600 values were measured and the concentrations of

413

the products were analyzed by HPLC.

414 415

HPLC analysis

416

3,4-Dihydroxybenzoic acid and vanillic acid were purchased from Alfa Aesar. Isovanillic acid

417

and isovanillyl alcohol were purchased from Sigma-Aldrich. 3,4-Dihydroxybenzyl alcohol

418

and vanillyl alcohol were purchased from VWR and TCI AMERICA, respectively. These six

419

compounds all have over 95% purities and were used as standards. HPLC (Dionex Ultimate

420

3000), equipped with a reverse phase ZORBAX SB-C18 column and an Ultimate 3000

421

Photodiode Array Detector, was used for analysis and quantification of standards and samples.

422

The column temperature was set to 28 °C. Flowing phase contains solvent A (water with 0.1%

423

formic acid) and solvent B (100% methanol) with a flow rate of 1 mL/min. The following

424

gradients were used: 5% to 50% solvent B for 20 min, 100% solvent B for 2 min, 100% to 5%

425

solvent B for 2 min and 5% solvent B for an additional 5 min. 3,4-Dihydroxybenzoic acid,

426

3,4-dihydroxybenzyl alcohol, vanillic acid, vanillyl alcohol, isovanillic acid and isovanillyl

427

alcohol were quantified based on their peak areas at specific wavelengths (260 nm for

428

3,4-dihydroxybenzoic

429

3,4-dihydroxybenzyl alcohol, vanillyl alcohol and isovanillyl alcohol).

acid,

vanillic

acid

and

isovanillic

20


acid,

280

nm

for

Page 21 of 38

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430

AUTHOR INFORMATION

431

Corresponding Authors

432

*

433

*

(Q.Y.) Tel: +86-10-64437610. E-mail: [email protected]. (Y.Y.) Tel: 001-706-542-8293. E-mail: [email protected].

434 435

Author Contributions

436

Z.C. conceived this study, designed and conducted the experiments. X.S., J(ian). W., J(ia). W.

437

and R.Z. participated in the research. Z.C. analyzed the data and wrote the manuscript. Q.Y.

438

and Y.Y. directed the research. J. R. and Y.Y. revised the manuscript.

439 440

ACKNOWLEDGEMENT

441

This work was supported by the National Natural Science Foundation of China (21406010,

442

21606012 and 21636001), the Programme of Introducing Talents of Discipline to Universities

443

(“111” project, B13005), the Program for Changjiang Scholars and Innovative Research

444

Team in Universities in China (No. IRT13045)，the Academic Leader of Beijing Polytechnic

445

(DTR201601), and the Key Project of Beijing Polytechnic (YZK028). We also acknowledge

446

the College of Engineering, The University of Georgia, Athens and the International Joint

447

Graduate Training Program of Beijing University of Chemical Technology.

21



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generation pathway for improving lysine production of Corynebacterium glutamicum

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by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate

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dehydrogenase. Metab. Eng. 25, 30-37.

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(38) Chen, W., Zhang, S., Jiang, P., Yao, J., He, Y., Chen, L., Gui, X., Dong, Z., and Tang, S.

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(2015) Design of an ectoine-responsive AraC mutant and its application in metabolic

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engineering of ectoine biosynthesis. Metab. Eng. 30, 149-155.

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(39) Chen, Z., and Zeng, A. (2016) Protein engineering approaches to chemical biotechnology. Curr. Opin. Biotech. 42, 198-205.

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(40) Wang, X., Wang, G., Li, X., Fu, J., Chen, T., Wang, Z., and Zhao, X. (2016) Directed

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evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in

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metabolic engineering. J. Biotechnol. 231, 115-121.

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(41) Zhang, L., Liang, Y., Wu, W., Tan, X., and Lu, X. (2016) Microbial synthesis of propane

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by engineering valine pathway and aldehyde-deformylating oxygenase. Biotechnol.

563

Biofuels 9, 80.

564

(42) Lin, Y., Sun, X., Yuan, Q., and Yan, Y. (2014) Extending shikimate pathway for the

565

production of muconic acid and its precursor salicylic acid in Escherichia coli. Metab.

566

Eng. 23, 62-69.

567 568

(43) Atsumi, S., Hanai, T., and Liao, J. C. (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86-89.

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Page 27 of 38

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570


Figure legends

571 572

Figure 1. The novel biosynthetic pathway of vanillyl alcohol production. Black-colored

573

arrows indicate the native pathways in E. coli; blue-colored arrow indicates the heterologous

574

steps;

575

3-deoxy-D-arabinoheptulosonate 7-phosphate; 4-HBA, 4-hydroxybenzoic acid; PpsA,

576

phosphoenolpyruvate

577

2-dehydro-3-deoxyphosphoheptonate aldolase; AroL, shikimate kinase II; UbiC, chorismate

578

lyase; PobA, p-hydroxybenzoate hydroxylase; CAR, carboxylic acid reductase; Sfp, the CAR

579

maturation factor phosphopantetheinyl transferase; ADHs, alcohol dehydrogenases; COMT,

580

caffeate O-methyltransferase.

PEP,

phosphoenolpyruvate;

E4P,

synthetase;

D-erythrose

TktA,

4-phosphate;

transketolase;

DAHP,

AroG,

581 582

Figure 2. The catalytic reactions and SDS-PAGE of COMT. (A): The catalytic reactions of

583

COMT towards different substrates. (B): SDS-PAGE of COMT. The black-colored arrow

584

directs the band of COMT and lane M demonstrates the protein molecular weight marker.

585

586

Figure 3. Production of vanillyl alcohol from different substrates and initial in vivo activities

587

of COMT towards different substrates. The substrates (a final concentration of 1000 mg/L)

588

were supplemented to the cell cultures at 5.5 h. For (A) and (B), strain CZY11 was used, and

589

3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzyl alcohol were fed into medium,

590

respectively. (C): The initial in vivo activities of COMT towards 3,4-dihydroxybenzoic acid

591

and 3,4-dihydroxybenzyl alcohol. For (D), strain CZY12 was used and 3,4-dihydroxybenzoic

592

acid was fed into medium. For (E), strain CZY13 was used and 3,4-dihydroxybenzoic acid

593

was fed into medium. For (F), strain CZY14 was used and 3,4-dihydroxybenzoic acid was 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

594

fed into medium. Three independent experiments were conducted to generate the data.

595 596

Figure 4. Plasmid constructs for de novo production of vanillyl alcohol, including plasmids

597

pZE-CUP, pCS-CS, pZE-CUP-APTA, pZE-C-U, pSA-PobA and pZE-CU-APTA.

598 599

Figure 5. Microbial production of vanillyl alcohol. For (A), strain CZY15 was used. For (B),

600

strain CZY16 was used. For (C), strain CZY17 was used. For (D), strain CZY18 was used.

601

Three independent experiments were conducted to generate the data.

602

28


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603

Table 1. Specific activities of COMT towards 3,4-dihydroxybenzoic acid and

604

3,4-dihydroxybenzyl alcohol. Two independent experiments were conducted to generate the

605

data. Substrate

Specific activity (µmol/min/mg protein)

3,4-Dihydroxybenzoic acid

0.031 ± 0.001

3,4-Dihydroxybenzyl alcohol

0.140 ± 0.003

606

29



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Page 30 of 38

607

Table 2. Kinetic parameters of COMT towards 3,4-dihydroxybenzoic acid and

608

3,4-dihydroxybenzyl alcohol. Two independent experiments were conducted to generate the

609

data. 610

Substrate

Km (mM)

kcat (s-1)

kcat/Km -1 (mM-1·s 611)

3,4-Dihydroxybenzoic acid

1.73 ± 0.09

0.021 ± 0.0007

0.012

3,4-Dihydroxybenzyl alcohol

0.52 ± 0.04

0.097 ± 0.002

0.19

612 613 614

30


Page 31 of 38

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615


Table 3. Plasmids and strains used in this study Plasmids and strains

Description

Source

pETDuet-1

pT7, PBR322 ori, Ampr

Novagen

pZE12-luc

PLlacO1, colE ori, luc, Ampr

19

pCS27

PLlacO1, P15A ori, Kanr

19

pSA74

PLlacO1, pSC101 ori, Cm r

19

pET-COMT

pETDuet-1 containing COMT from Arabidopsis thaliana

This study

pZE-COMT

pZE12-luc containing COMT

21

pZE-ADH6

pZE12-luc containing ADH6 from Saccharomyces cerevisiae

This study

pZE-CUP

pZE12-luc containing ubiC from E. coli, COMT from A. thaliana,

This study

Plasmids

and pobA from Pseudomonas aeruginosa pZE-CU

pZE12-luc containing ubiC and COMT

This study

pZE-C-U

pZE12-luc containing ubiC and COMT, two operons

This study

pCS-APTA

pCS27 containing aroL, ppsA, tktA and aroG from E. coli

19

pZE-CUP-APTA

pZE12-luc containing PLlacO1-CUP and PLlacO1-APTA

This study

pZE-CU-APTA

pZE12-luc containing PLlacO1-CU and PLlacO1-APTA

This study

pCS-CS

pCS27 containing car from Mycobacterium marinum and sfp from

This study

fbr

Bacillus subtilis pSA-PobA

pSA74 containing pobA

This study

recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac

Stratagene

Strains XL1-Blue

[F' proAB lacIqZ△M15 Tn10 (Tetr)] BL21Star (DE3) ‫׳‬

BW25113 (F )

F- ompT hsdSB (rB-mB-) gal dcm (DE3)

Invitrogen

rrnBT14 ∆lacZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78 F‫׳‬

43

[traD36 proAB lacIqZ∆M15 Tn10(Tetr)] CZY10

BL21Star (DE3) with pET-COMT ‫׳‬

This study

CZY11

BW25113 (F ) with pZE-COMT

This study

CZY12

BW25113 (F‫ )׳‬with pCS-CS

This study

CZY13

BW25113 (F‫ )׳‬with pCS-CS and pZE-ADH6

This study

‫׳‬

CZY14

BW25113 (F ) with pZE-COMT and pCS-CS

This study

CZY15

BW25113 (F‫ )׳‬with pZE-CUP and pCS-CS

This study

CZY16

BW25113 (F‫ )׳‬with pZE-CUP-APTA and pCS-CS

This study

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CZY17 CZY18

BW25113 (F‫ )׳‬with pZE-C-U, pCS-CS and pSA-PobA ‫׳‬

BW25113 (F ) with pZE-CU-APTA, pCS-CS and pSA-PobA

616

32


Page 32 of 38

This study This study

Page 33 of 38

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Table of Contents Graphic 27x9mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. The novel biosynthetic pathway of vanillyl alcohol production. Black-colored arrows indicate the native pathways in E. coli; blue-colored arrow indicates the heterologous steps; PEP, phosphoenolpyruvate; E4P, D-erythrose 4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; 4-HBA, 4hydroxybenzoic acid; PpsA, phosphoenolpyruvate synthetase; TktA, transketolase; AroG, 2-dehydro-3deoxyphosphoheptonate aldolase; AroL, shikimate kinase II; UbiC, chorismate lyase; PobA, phydroxybenzoate hydroxylase; CAR, carboxylic acid reductase; Sfp, the CAR maturation factor phosphopantetheinyl transferase; ADHs, alcohol dehydrogenases; COMT, caffeate O-methyltransferase. 273x104mm (300 x 300 DPI)


Page 34 of 38

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Figure 2. The catalytic reactions and SDS-PAGE of COMT. (A): The catalytic reactions of COMT towards different substrates. (B): SDS-PAGE of COMT. The black-colored arrow directs the band of COMT and lane M demonstrates the protein molecular weight marker. 366x135mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Production of vanillyl alcohol from different substrates and initial in vivo activities of COMT towards different substrates. The substrates (a final concentration of 1000 mg/L) were supplemented to the cell cultures at 5.5 h. For (A) and (B), strain CZY11 was used, and 3,4-dihydroxybenzoic acid and 3,4dihydroxybenzyl alcohol were fed into medium, respectively. (C): The initial in vivo activities of COMT towards 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzyl alcohol. For (D), strain CZY12 was used and 3,4-dihydroxybenzoic acid was fed into medium. For (E), strain CZY13 was used and 3,4-dihydroxybenzoic acid was fed into medium. For (F), strain CZY14 was used and 3,4-dihydroxybenzoic acid was fed into medium. Three independent experiments were conducted to generate the data. 668x329mm (300 x 300 DPI)


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Figure 4. Plasmid constructs for de novo production of vanillyl alcohol, including plasmids pZE-CUP, pCS-CS, pZE-CUP-APTA, pZE-C-U, pSA-PobA and pZE-CU-APTA. 445x277mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Microbial production of vanillyl alcohol. For (A), strain CZY15 was used. For (B), strain CZY16 was used. For (C), strain CZY17 was used. For (D), strain CZY18 was used. Three independent experiments were conducted to generate the data. 425x313mm (300 x 300 DPI)


Page 38 of 38

Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl

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